The Black-tailed Antechinus, Antechinus arktos sp. nov.: a new species of carnivorous marsupial from montane regions of the Tweed Volcano caldera, eastern Australia.

We describe a new species of dasyurid marsupial within the genus Antechinus that was previously known as a northern outlier of Dusky Antechinus (A. swainsonii). The Black-tailed Antechinus, Antechinus arktos sp. nov., is known only from areas of high altitude and high rainfall on the Tweed Volcano caldera of far south-east Queensland and north-east New South Wales, Australia. Antechinus arktos formerly sheltered under the taxonomic umbrella of A. swainsonii mimetes, the widespread mainland form of Dusky Antechinus. With the benefit of genetic hindsight, some striking morphological differences are herein resolved: A. s. mimetes is more uniformly deep brown-black to grizzled grey-brown from head to rump, with brownish (clove brown-raw umber) hair on the upper surface of the hindfoot and tail, whereas A. arktos is more vibrantly coloured, with a marked change from greyish-brown head to orange-brown rump, fuscous black on the upper surface of the hindfoot and dense, short fur on the evenly black tail. Further, A. arktos has marked orange-brown fur on the upper and lower eyelid, cheek and in front of the ear and very long guard hairs all over the body; these characters are more subtle in A. s. mimetes. There are striking genetic differences between the two species: at mtDNA, A. s. mimetes from north-east New South Wales is 10% divergent to A. arktos from its type locality at Springbrook NP, Queensland. In contrast, the Ebor A. s. mimetes clades closely with conspecifics from ACT and Victoria. A. arktos skulls are strikingly different to all subspecies of A. swainsonii. A. arktos are markedly larger than A. s. mimetes and A. s. swainsonii (Tasmania) for a range of craniodental measures. Antechinus arktos were historically found at a few proximate mountainous sites in south-east Queensland, and have only recently been recorded from or near the type locality. Even there, the species is likely in low abundance. The Black-tailed Antechinus has plausibly been detrimentally affected by climate change in recent decades, and will be at further risk with increasing warming trends. 

Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry.

Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein-protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein-peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium-tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI-TOF instruments and has general utility for the label-free analysis of microarray assays.

A new dasyurid marsupial from Kroombit Tops, south-east Queensland, Australia: the Silver-headed Antechinus, Antechinus argentus sp. nov. (Marsupialia: Dasyuridae).

Antechinus argentus sp. nov. is currently only known from the plateau at the eastern escarpment of Kroombit Tops National Park, about 400km NNW of Brisbane and 60km SSW of Gladstone, south-east Queensland, Australia. Antechinus flavipes (Waterhouse) is also known from Kroombit Tops NP, 4.5km W of the nearest known population of A. argentus; A. mysticus Baker, Mutton and Van Dyck has yet to be found within Kroombit Tops, but is known from museum specimens taken at Bulburin NP, just 40km ESE, as well as extant populations about 400km to both the south-east and north-west of Kroombit NP. A. argentus can be easily distinguished in the field, having an overall silvery/grey appearance with much paler silver feet and drabber deep greyish-olive rump than A. flavipes, which has distinctive yellow-orange toned feet, rump and tail-base; A. argentus fur is also less coarse than that of A. flavipes. A. argentus has a striking silver-grey head, neck and shoulders, with pale, slightly broken eye-rings, which distinguish it from A. mysticus which has a more subtle greyish-brown head, pale buff dabs of eyeliner and more colourful brownish-yellow rump. Features of the dentary can also be used for identification: A. argentus differs from A. flavipes in having smaller molar teeth, as well as a narrower and smaller skull and from A. mysticus in having on average a narrower snout, smaller skull and dentary lengths and smaller posterior palatal vacuities in the skull. A. argentus is strongly divergent genetically (at mtDNA) from both A. flavipes (9.0-11.2%) and A. mysticus (7.2-7.5%), and forms a very strongly supported clade to the exclusion of all other antechinus species, in both mtDNA and combined (mtDNA and nDNA) phylogenies inferred here. We are yet to make detailed surveys in search of A. argentus from forested areas to the immediate east and north of Kroombit Tops. However, A. mysticus has only been found at these sites in low densities in decades past and not at all in several recent trapping expeditions conducted by the authors. With similar habitat types in close geographic proximity, it is plausible that A. argentus may be found outside Kroombit. Nevertheless, it is striking that from a range of surveys conducted at Kroombit Tops in the last 15 years and intensive surveys by the authors in the last 3 years, totalling more than 5 080 trap nights, just 13 A. argentus have been captured from two sites less than 6 km apart. If this is even close to the true geographic extent of the species, it would possess one of the smallest distributions of an Australian mammal species. With several threats identified, we tentatively recommend that A. argentus be listed as Endangered, pending an exhaustive trapping survey of Kroombit and surrounds. 

A new treefrog (Hylidae: Litoria) from Kroombit Tops, east Australia, and an assessment of conservation status.

The Litoria phyllochroa species-group are small hylid frogs that occur in wet forests of south-east Australia. This group has had a long history of taxonomic confusion and has received little attention in the last decade. A population of this species-group at Kroombit Tops, several hundred kilometers north of all other populations, has been recognised for some time as being genetically highly distinct. Here we describe this population as a new species, L. kroombitensis sp. nov. This species is most similar to L. barringtonensis and L. pearsoniana but is readily distinguished based on differences in morphology, colour pattern, mating call and genetics. Litoria kroombitensis sp. nov. is restricted to Kroombit Tops, an isolated area of wet forest in south-east Queensland. The species inhabits slow and intermittently flowing streams in rainforest and adjoining wet sclerophyll forest. The tadpole of L. kroombitensis sp. nov., described herein, is similar in morphology and behaviour to the tadpoles of other species within the Litoria phyllochroa species-group, in particular L. pearsoniana. Li toria kroombitensis sp. nov. has a very small distribution, with all records coming from the headwaters of five streams, Extensive surveys since the mid-1990s have revealed population declines, attributable to amphibian chytrid fungus (Batrachochytrium dendrobatidis). Other threats include degradation of riparian habitat due to invasive weeds, feral pigs and livestock, and fire. Further, the extent of wet forest habitats at Kroombit Tops is likely to be reduced by climate change impacts. Litoria kroombitensis sp. nov. meets IUCN Red List criteria for critically endangered CR B lab (i-v) due to its small geographic range, naturally fragmented distribution, and observed and projected decline in populations. In this paper we also assess the validity of the names L. barringtonensis, L. pearsoniana and L. piperata. We conclude that the names L. barringtonensis and L. pearsoniana are valid but the validity of L. piperata requires further investigation.

A new species of barred frog, Mixophyes (Anura: Myobatrachidae) from south-eastern Australia identified by molecular genetic analyses.

Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Several of the species found in mid-eastern and south-eastern Australia are listed as threatened, due largely to declines presumably caused by the amphibian disease chytridiomycosis. Given the wide distribution of several of these species and that their distributions cross well-known biogeographic boundaries that often correspond to deep genetic breaks or species boundaries among closely related vertebrates, we undertook a molecular genetic assessment of population structure across the range of each species to determine the presence of undescribed species. Of the four species of Mixophyes subject to molecular population genetic analyses, one, the Stuttering Frog (Mixophyes balbus), showed a level of diversity consistent with the presence of two species. Morphometric, meristic and bioacoustic analyses corroborate these distinctions, and a new species is described for the populations south of the Macleay River valley in mid-eastern New South Wales to east Gippsland in Victoria. Applying the IUCN Red List threat criteria the new species meets the conservation status assessment criteria for Endangered 2B1a,b because its extent of occupancy and area of occupancy are below the threshold value and it has declined and disappeared from the southern two thirds of its distribution over the past 30 years.

Drought, fire, and rainforest endemics: A case study of two threatened frogs impacted by Australia's "Black Summer".

Deepening droughts and unprecedented wildfires are at the leading edge of climate change. Such events pose an emerging threat to species maladapted to these perturbations, with the potential for steeper declines than may be inferred from the gradual erosion of their climatic niche. This study focused on two species of amphibians-Philoria kundagungan and Philoria richmondensis (Limnodynastidae)-from the Gondwanan rainforests of eastern Australia that were extensively affected by the "Black Summer" megafires of 2019/2020 and the severe drought associated with them. We sought to assess the impact of these perturbations by quantifying the extent of habitat affected by fire, assessing patterns of occurrence and abundance of calling males post-fire, and comparing post-fire occurrence and abundance with that observed pre-fire. Some 30% of potentially suitable habitat for P. kundagungan was fire affected, and 12% for P. richmondensis. Field surveys revealed persistence in some burnt rainforest; however, both species were detected at a higher proportion of unburnt sites. There was a clear negative effect of fire on the probability of site occupancy, abundance and the probability of persistence for P. kundagungan. For P. richmondensis, effects of fire were less evident due to the limited penetration of fire into core habitat; however, occupancy rates and abundance of calling males were depressed during the severe drought that prevailed just prior to the fires, with the reappearance of calling males linked to the degree of rehydration of breeding habitat post-fire. Our results highlight the possibility that severe negative impacts of climate change for montane rainforest endemics may be felt much sooner than commonly anticipated under a scenario of gradual (decadal-scale) changes in mean climatic conditions. Instead, the increased rate of severe stochastic events places these narrow range species at a heightened risk of extinction in the near-term.

Nitric oxide-mediated regulation of ß-amyloid clearance via alterations of MMP-9/TIMP-1.

Fibrillar amyloid plaques are largely composed of amyloid-beta (Aß) peptides that are metabolized into products, including Aß1-16, by proteases including matrix metalloproteinase 9 (MMP-9). The balance between production and degradation of Aß proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP-9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP-9/TIMP-1 balance. We show NO-mediated increased MMP-9/TIMP-1 ratios enhanced the degradation of fibrillar Aß in vitro, which was abolished when silenced for MMP-9 protein translation. The in vivo relationship between MMP-9, NO and Aß degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP-9 mediated changes, we generated an antibody recognizing the Aß1-16 fragment, and used mass spectrometry multi-reaction monitoring assay for detection of immunoprecipitated Aß1-16 peptides. Aß1-16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP-1 increased in the APPSwDI/NOS2(-/-) mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.

A new species of Philoria (Anura: Limnodynastidae) from the uplands of the Gondwana Rainforests World Heritage Area of eastern Australia.

The six species of mountain frogs (Philoria: Limnodynastidae: Anura) are endemic to south-eastern Australia. Five species occur in headwater systems in mountainous north-eastern New South Wales (NSW) and south-eastern Queensland (Qld), centred on the Gondwana Rainforests of Australia World Heritage Area. A previous molecular genetic analysis identified divergent genetic lineages in the central and western McPherson Ranges region of Qld and NSW, but sampling was inadequate to test the species status of these lineages. With more comprehensive geographic sampling and examination of the nuclear genome using SNP analysis, we show that an undescribed species, P. knowlesi sp. nov., occurs in the central and western McPherson Ranges (Levers Plateau and Mount Barney complex). The new species is not phylogenetically closely related to P. loveridgei in the nuclear data but is related to one of two divergent lineages within P. loveridgei in the mtDNA data. We postulate that the discordance between the nuclear and mtDNA outcomes is due to ancient introgression of the mtDNA genome from P. loveridgei into the new species. Male advertisement calls and multivariate morphological analyses do not reliably distinguish P. knowlesi sp. nov. from any of the Philoria species in northeast NSW and southeast Qld. The genetic comparisons also enable us to define further the distributions of P. loveridgei and P. kundagungan. Samples from the Lamington Plateau, Springbrook Plateau, Wollumbin (Mt Warning National Park), and the Nightcap Range, are all P. loveridgei, and its distribution is now defined as the eastern McPherson Ranges and Tweed caldera. Philoria kundagungan is distributed from the Mistake Mountains in south-eastern Qld to the Tooloom Scrub on the Koreelah Range, southwest of Woodenbong, in NSW, with two subpopulations identified by SNP analysis. We therefore assessed the IUCN threat category of P. loveridgei and P. kundagungan and undertook new assessments for each of its two subpopulations and for the new taxon P. knowlesi sp. nov., using IUCN Red List criteria. Philoria loveridgei, P. kundagungan (entire range and northern subpopulation separately) and P. knowlesi sp. nov. each meet criteria for Endangered (EN B2(a)(b)[i, iii]). The southern subpopulation of P. kundagungan, in the Koreelah Range, meets criteria for Critically Endangered (CE B2(a)(b)[i, iii]). These taxa are all highly threatened due to the small number of known locations, the restricted nature of their breeding habitat, and direct and indirect threats from climate change, and the potential impact of the amphibian disease chytridiomycosis. Feral pigs are an emerging threat, with significant impacts now observed in Philoria breeding habitat in the Mistake Mountains.

A new hip-pocket frog from mid-eastern Australia (Anura: Myobatrachidae: Assa).

The hip-pocket frog (Assa darlingtoni), a small terrestrial myobatrachid frog found in mid-eastern Australia, has a highly derived, unusual, reproductive mode involving a unique form of male parental care. Males have subcutaneous pouches that open near the hip, and the developing tadpoles are carried in these pouches to post metamorphosis. It is found on several isolated mountain ranges in closed forest habitats, associated with high rainfall and temperate or sub-tropical climates. We established genetic relationships among specimens sampled across the range using phylogenetic analyses of thousands of single nucleotide polymorphisms (SNPs) from the nuclear genome and mitochondrial ND2 gene nucleotide sequences. These analyses uncovered two lineages that are genetically distinct in both nDNA and mtDNA analyses and that have low levels of divergence in male advertisement calls and are morphologically cryptic. Our data support separate species status for each lineage, based on the molecular genetic data. The first, which we name as a new species, Assa wollumbin sp. nov., is restricted to a single mountain, Wollumbin (= Mount Warning), the eroded cone of an ancient shield volcanothe Tweed Volcano. The second, the nominal species A. darlingtoni, has a wider distribution in five geographically disjunct subpopulations along 430 km of the Great Dividing Range in south-eastern Queensland and north-eastern New South Wales. The distributions of the two species closely approach within 15 km of each other on the central plug and rim of the caldera of the Tweed Volcano. Assa wollumbin sp. nov. meets the conservation criteria for Critically Endangered [A3(e), B2(a,b)]. When all subpopulations of A. darlingtoni are combined the conservation assessment is Endangered [A3(e), B2(a,b)]. Because of the fragmented nature of the distribution of A. darlingtoni, combined with the genetic evidence of concordant sub-structuring, we also conducted a conservation assessment on the five subpopulations. Two were assessed as Critically Endangered (DAguilar Range and Conondale/Blackall Ranges), and the remainder as Endangered (Dorrigo Plateau, McPherson Ranges, and Gibraltar Ranges/Washpool).

A national-scale dataset for threats impacting Australia's imperiled flora and fauna.

Australia is in the midst of an extinction crisis, having already lost 10% of terrestrial mammal fauna since European settlement and with hundreds of other species at high risk of extinction. The decline of the nation's biota is a result of an array of threatening processes; however, a comprehensive taxon-specific understanding of threats and their relative impacts remains undocumented nationally. Using expert consultation, we compile the first complete, validated, and consistent taxon-specific threat and impact dataset for all nationally listed threatened taxa in Australia. We confined our analysis to 1,795 terrestrial and aquatic taxa listed as threatened (Vulnerable, Endangered, or Critically Endangered) under Australian Commonwealth law. We engaged taxonomic experts to generate taxon-specific threat and threat impact information to consistently apply the IUCN Threat Classification Scheme and Threat Impact Scoring System, as well as eight broad-level threats and 51 subcategory threats, for all 1,795 threatened terrestrial and aquatic threatened taxa. This compilation produced 4,877 unique taxon-threat-impact combinations with the most frequently listed threats being Habitat loss, fragmentation, and degradation (n = 1,210 taxa), and Invasive species and disease (n = 966 taxa). Yet when only high-impact threats or medium-impact threats are considered, Invasive species and disease become the most prevalent threats. This dataset provides critical information for conservation action planning, national legislation and policy, and prioritizing investments in threatened species management and recovery.

Application of the survey protocol for chytridiomycosis to Queensland, Australia.

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis, has resulted in the extinction of frogs, but the distribution of Bd is incompletely known. We trialled the survey protocol for Bd by attempting to systematically map its distribution in Queensland, Australia. Bd was easily detected in known infected areas, such as the Wet Tropics and South East Queensland. It was not detected in bioregions adjacent to, but inland from or to the north of, infected regions: Einasleigh Uplands and Cape York adjacent to the infected Wet Tropics; and Brigalow Belt South adjacent to the infected South East Queensland bioregion. These regions where Bd was not detected have bordered infected regions for between 15 yr (in northern Queensland) and 30 yr (in southern Queensland), and so they define the geographical limits of Bd with regard to the long-term environmental conditions in Queensland. The Gulf Plains, a bioregion distant from infected bioregions, was also negative. Bd was confined to rainforest and bordering habitats, such as wet eucalypt forests. Infections were largely confined to permanent water-associated species, consistent with this being an important cause of this group having the greatest declines. Our data supports biogeographic climatic models that show much of inland and northern Australia to be too hot and dry to support Bd. As there is limited opportunity for Bd to spread further in Queensland, the priority for management is reducing the impact of Bd in affected populations and assisting frogs to disperse into their former distributions. Given that the survey protocol has been applied successfully in Australia it may be useful for mapping the distribution of Bd in other parts of the world.

Use of a recombinant fluorescent substrate with cleavage sites for all botulinum neurotoxins in high-throughput screening of natural product extracts for inhibitors of serotypes A, B, and E.

The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

Survey protocol for detecting chytridiomycosis in all Australian frog populations.

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused the decline and extinction of frogs, but the distribution of Bd is not completely known. This information is crucial to implementing appropriate quarantine strategies, preparing for outbreaks of chytridiomycosis due to introduction of Bd, and for directing conservation actions towards affected species. This survey protocol provides a simple and standard method for sampling all frog populations in Australia to maximise the chances of detecting Bd. In order to structure and prioritise the protocol, areas are divided by bioregion and frog species are allocated depending on the water bodies they utilize into 3 groups representing different levels of risk of exposure to Bd. Sixty individuals per population need to be tested to achieve 95% certainty of detecting 1 positive frog, based on the minimum apparent prevalence of > or =5% in infected Australian frog populations and using a quantitative real-time TaqMan PCR test. The appropriate season to sample varies among bioregions and will ideally incorporate temperatures favourable for chytridiomycosis (e.g. maximum air temperatures generally <27 degrees C). Opportunistic collection and testing of sick frogs and tadpoles with abnormal mouth-parts should also be done to increase the probability of detecting Bd. The survey priorities in order are (1) threatened species that may have been exposed to Bd, (2) bioregions surrounding infected bioregions/ecological groups, and (3) species of frogs of unknown infection status in infected bioregions. Within these priority groups, sampling should first target ecological groups and species likely to be exposed to Bd, such as those associated with permanent water, and areas within bioregions that have high risk for Bd as indicated by climatic modelling. This protocol can be adapted for use in other countries and a standard protocol will enable comparison among amphibian populations globally.

Surveillance for batrachochytrium dendrobatidis using Mixophyes (Anura: Myobatrachidae) larvae.

Fourteen populations of anuran larvae (tadpoles), including three populations of the endangered Fleay's Barred Frog (Mixophyes fleayi) and 11 populations of the common Great Barred Frog (Mixophyes fasciolatus), in creek sites in the southeast region of Queensland were selected. Site selection was based on a history (within the district) of adult frog population declines and/or disappearances or records of infection of adult frogs or larvae by Batrachochytrium dendrobatidis. Larvae were collected once from each creek site between October 2002 and October 2004, and were between Gosner developmental stages 25 and 40. Total body length ranged from 18 mm to 100 mm. Mouthparts were examined under a dissecting microscope for grossly visible abnormalities, and then examined for histologic evidence of B. dendrobatidis. The most consistent mouthpart abnormalities found were multifocal depigmentation of the jaw sheaths and loss or shortening of the tooth rows. At the individual larva level, presence of mouthpart abnormalities was strongly associated with histologic diagnosis of B. dendrobatidis (93%). At least one larva with abnormal mouthparts was detected at 12 of the 14 sites and histologic evidence of B. dendrobatidis was detected at 13 of the 14 sites. These findings suggest that larvae of Mixophyes species are suitable for surveillance for B. dendrobatidis. We conclude that surveillance of B. dendrobatidis where individual larva prevalences of mouthpart abnormalities and histologic evidence of B. dendrobatidis are as high as those observed in this study (66% and 78%, respectively), relatively small numbers of larvae are required to detect these infections. Medium to large larvae (body length >30 mm) were much more likely to be affected than small larvae (body length < or =30 mm), suggesting that larger individuals should be targeted for surveillance.

Plant diversity and structure describe the presence of a new, threatened Australian marsupial within its highly restricted, post-fire habitat.

Management of critical habitat for threatened species with small ranges requires location-specific, fine-scale survey data. The silver-headed antechinus (Antechinus argentus) is known from only two isolated, fire-prone locations. At least one of these populations, at Kroombit Tops National Park in central-eastern Queensland, Australia, possesses a very small range. Here, we present detailed vegetation species diversity and structure data from three sites comprising the known habitat of A. argentus at Kroombit Tops and relate it to capture data obtained over two years. We found differences in both vegetation and capture data between burnt and unburnt habitat. Leaf litter and grasstrees (Xanthorrhoea johnsonii) were the strongest vegetative predictors for A. argentus capture. The species declined considerably over the two years of the trapping study, and we raise concern for its survival at Kroombit Tops. We suggest that future work should focus on structural vegetative variables (specifically, the diameter and leaf density of grasstree crowns) and relate them to A. argentus occurrence. We also recommend a survey of invertebrate diversity in grasstrees and leaf litter with a comparison to A. argentus prey. The data presented here illustrates how critical detailed monitoring is for planning habitat management and fire regimes, and highlights the utility of a high-resolution approach to habitat mapping. While a traditional approach to fire management contends that pyrodiversity encourages biodiversity, the present study demonstrates that some species prefer long-unburnt habitat. Additionally, in predicting the distribution of rare species like A. argentus, data quality (i.e., spatial resolution) may prevail over data quantity (i.e., number of data).

Techniques for detecting chytridiomycosis in wild frogs: comparing histology with real-time Taqman PCR.

Chytridiomycosis is a lethal disease of amphibians associated with mass mortalities and population declines worldwide. An accurate, non-invasive technique for detecting chytridiomycosis is urgently needed to determine the current geographical distribution of the disease, and its prevalence in wild amphibian populations. Herein we evaluate a recently devised, rapid, non-invasive, swab-PCR assay. We sampled 101 wild juvenile Mixophyes iteratus by both a skin swab for use in PCR analysis, and a toe-clip for examination by histological methods. The swab-PCR assay detected chytridiomycosis infection in a minimum of 14.9% of frogs, whereas histology detected infection in no more than 6.9% of frogs. We conclude that the swab-PCR technique is the more reliable means of detecting chytridiomycosis in wild amphibians, and that it precludes the need for toe-clipping as a means of sampling for the presence of the disease in future surveys. Further, we document a significant negative relationship between a juvenile frog's snout-vent length and its likelihood of being infected with the disease.

Revision of the water-holding frogs, Cyclorana platycephala (Anura: Hylidae), from arid Australia, including a description of a new species.

The water-holding frog, Cyclorana platycephala, occurs in the Australian arid and semi-arid zones but not in the central Australian deserts. Recent inspection of morphological variation in adults and larvae suggests that the taxon comprises three regional populations: eastern, northern and western that may each represent separate species. To assess the systematic status of these populations, we documented phylogenetic relationships using mitochondrial and nuclear DNA markers, divergence in adult and larval morphology and male advertisement call. Our molecular genetic data demonstrates that the western population of C. platycephala is not the sister taxon of eastern and northern representatives of this nominate species, as the latter two are more closely related to another morphologically distinct species, C. verrucosa. Discriminant Function Analysis of 14 morphological traits in adults and 15 in larvae showed a high degree of morphological differentiation of western versus eastern/northern C. platycephala. Calls of eastern and western populations differed in duration, pulse rate, frequency and especially in amplitude modulation pattern across the call duration. We describe the western population as a new species, whose range is contained entirely within Western Australia. In addition, we redescribe Cyclorana platycephala, quantify morphological and genetic differences between the eastern and northern populations, and conclude that these data support recognition of a single species, Cyclorana platycephala, for populations found in New South Wales, the Barkly Tablelands and south-eastern Northern Territory, Queensland and South Australia.

Characterization of beta-leptinotarsin-h and the effects of calcium flux antagonists on its activity.

beta-Leptinotarsin-h, purified from the hemolymph of the beetle Leptinotarsa haldemani, is a potent ( approximately 1 nM) neuroactive protein that rapidly (few seconds) stimulates Ca(2+) influx and neurotransmitter release. Our goals were to further characterize beta-leptinotarsin-h and to test the hypothesis that it stimulates Ca(2+) influx through presynaptic Ca(2+) channels. Analysis of partial amino acid sequences revealed that beta-leptinotarsin-h is a unique protein with significant similarity to only one other protein, the juvenile hormone esterase of Leptinotarsa decemlineata, commonly known as the Colorado potato beetle. We have examined the effect of beta-leptinotarsin-h on Ca(2+) current, Ca(2+) uptake, Ca(2+) levels, and neurotransmitter release in synaptosomes, cell lines, and neuronal systems. We found that its preferred site of action appears to be mammalian presynaptic nerve terminals. We tested antagonists of Ca(2+) flux for their effects on beta-leptinotarsin-h-stimulated Ca(2+) uptake in rat brain synaptosomes. The non-selective Ca(2+) channel blockers flunarizine, Ni(2+), ruthenium red, high-concentration thapsigargin, and SKF 96365 inhibited beta-leptinotarsin-h's activity, but none of the tested selective blockers of voltage-operated Ca(2+) channels (omega-agatoxin IVA, omega-conotoxin GVIA, omega-conotoxin MVIIC, nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand-operated, store-operated, and transduction-operated channels were also not inhibitory. beta-Leptinotarsin-h did not stimulate Na(+) uptake, ruling out Na(+) channels and many non-selective cation channels as targets. We conclude that beta-leptinotarsin-h stimulated Ca(2+) uptake through presynaptic Ca(2+) channels; which channel is yet to be determined. beta-Leptinotarsin-h may prove to be a useful tool with which to investigate calcium channels and calcium flux.

Role of quorum sensing in the pathogenicity of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of human and animal melioidosis. The role of quorum sensing (QS) in the in vivo pathogenicity of B. pseudomallei via inhalational exposure of BALB/c mice and intraperitoneal challenge of Syrian hamsters has not been reported. This investigation demonstrates that B. pseudomallei encodes a minimum of three luxI and five luxR homologues that are involved in animal pathogenicity. Mass spectrometry analysis of culture supernatants revealed that wild-type B. pseudomallei and the luxI mutants synthesized numerous signalling molecules, including N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxyoctanoyl)-L-homoserine lactone, N-(3-hydroxydecanoyl)-L-homoserine lactone and N-(3-oxotetradecanoyl)-L-homoserine lactone, which was further confirmed by heterologous expression of the B. pseudomallei luxI alleles in Escherichia coli. Mutagenesis of the B. pseudomallei QS system increased the time to death and reduced organ colonization of aerosolized BALB/c mice. Further, intraperitoneal challenge of Syrian hamsters with the B. pseudomallei QS mutants resulted in a significant increase in the LD50. Using semi-quantitative plate assays, preliminary analysis suggests that QS does not affect lipase, protease and phospholipase C biosynthesis/secretion in B. pseudomallei. The findings of the investigation demonstrate that B. pseudomallei encodes multiple luxIR genes, and disruption of the QS alleles reduces animal pathogenicity, but does not affect exoproduct secretion.