Chlamydia pneumoniae is genetically diverse in animals and appears to have crossed the host barrier to humans on (at least) two occasions

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non- Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials.

Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.

Parametric and generative methods with building information modelling : connecting BIM with explorative design modelling

Parametric and generative modelling methods are ways in which computer models are made more flexible, and of formalising domain-specific knowledge. At present, no open standard exists for the interchange of parametric and generative information. The Industry Foundation Classes (IFC) which are an open standard for interoperability in building information models is presented as the base for an open standard in parametric modelling. The advantage of allowing parametric and generative representations are that the early design process can allow for more iteration and changes can be implemented quicker than with traditional models. This paper begins with a formal definition of what constitutes to be parametric and generative modelling methods and then proceeds to describe an open standard in which the interchange of components could be implemented. As an illustrative example of generative design, Frazer’s ‘Reptiles’ project from 1968 is reinterpreted.

Thorough assessment of DNA preservation from fossil bone and sediments excavated from a late Pleistocene–Holocene cave deposit on Kangaroo Island, South Australia

Fossils and sediments preserved in caves are an excellent source of information for investigating impacts of past environmental changes on biodiversity. Until recently studies have relied on morphology-based palaeontological approaches, but recent advances in molecular analytical methods offer excellent potential for extracting a greater array of biological information from these sites. This study presents a thorough assessment of DNA preservation from late Pleistocene–Holocene vertebrate fossils and sediments from Kelly Hill Cave Kangaroo Island, South Australia. Using a combination of extraction techniques and sequencing technologies, ancient DNA was characterised from over 70 bones and 20 sediment samples from 15 stratigraphic layers ranging in age from >20 ka to ∼6.8 ka. A combination of primers targeting marsupial and placental mammals, reptiles and two universal plant primers were used to reveal genetic biodiversity for comparison with the mainland and with the morphological fossil record for Kelly Hill Cave. We demonstrate that Kelly Hill Cave has excellent long-term DNA preservation, back to at least 20 ka. This contrasts with the majority of Australian cave sites thus far explored for ancient DNA preservation, and highlights the great promise Kangaroo Island caves hold for yielding the hitherto-elusive DNA of extinct Australian Pleistocene species.

Benthic shift in a Solomon Islands' lagoon : corals to cyanobacteria

In June 2011 a large phytoplankton bloom resulted in a catastrophic mortality event that affected a large coastal embayment in the Solomon Islands. This consisted of an area in excess of 20 km2 of reef and soft sandy habitats in Marovo Lagoon, the largest double barrier lagoon in the world. This embayment is home to over 1200 people leading largely subsistence lifestyles depending on the impacted reefs for majority of their protein needs. A toxic diatom (Psuedo-nitzchia spp.) and toxic dinoflagellate (Pyrodinium bahamense var. compressum) reached concentrations of millions of cells per litre. The senescent phytoplankton bloom led to complete de-oxygenation of the water column that reportedly caused substantial mortality of marine animal life in the immediate area within a rapid timeframe (24 h). Groups affected included holothurians, crabs and reef and pelagic fish species. Dolphins, reptiles and birds were also found dead within the area, indicating algal toxin accumulation in the food chain. Deep reefs and sediments, whilst initially unaffected, have now been blanketed in large cyanobacterial mats which have negatively impacted live coral cover especially within the deep reef zone (> 6 m depth). Reef recovery within the deep zone has been extremely slow and may indicate an alternative state for the system.

The 10 Australian ecosystems most vulnerable to tipping points

We identify the 10 major terrestrial and marine ecosystems in Australia most vulnerable to tipping points, in which modest environmental changes can cause disproportionately large changes in ecosystem properties. To accomplish this we independently surveyed the coauthors of this paper to produce a list of candidate ecosystems, and then refined this list during a 2-day workshop. The list includes (1) elevationally restricted mountain ecosystems, (2) tropical savannas, (3) coastal floodplains and wetlands, (4) coral reefs, (5) drier rainforests, (6) wetlands and floodplains in the Murray-Darling Basin, (7) the Mediterranean ecosystems of southwestern Australia, (8) offshore islands, (9) temperate eucalypt forests, and (10) salt marshes and mangroves. Some of these ecosystems are vulnerable to widespread phase-changes that could fundamentally alter ecosystem properties such as habitat structure, species composition, fire regimes, or carbon storage. Others appear susceptible to major changes across only part of their geographic range, whereas yet others are susceptible to a large-scale decline of key biotic components, such as small mammals or stream-dwelling amphibians. For each ecosystem we consider the intrinsic features and external drivers that render it susceptible to tipping points, and identify subtypes of the ecosystem that we deem to be especially vulnerable. © 2011 Elsevier Ltd.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Patterned fen formation and development from the Great Sandy Region, south-east Queensland, Australia

The Great Sandy Region (incorporating Fraser Island and the Cooloola sand-mass), south-east Queensland, contains a significant area of Ramsar-listed coastal wetlands, including the globally important patterned fen complexes. These mires form an elaborate network of pools surrounded by vegetated peat ridges and are the only known subtropical, Southern Hemisphere examples, with wetlands of this type typically located in high northern latitudes. Sedimentological, palynological and charcoal analysis from the Wathumba and Moon Point complexes on Fraser Island indicate two periods of swamp formation (that may contain patterned fens), one commencing at 12 000 years ago (Moon Point) and the other ~4300 years ago (Wathumba). Wetland formation and development is thought to be related to a combination of biological and hydrological processes with the dominant peat-forming rush, Empodisma minus, being an important component of both patterned and non-patterned mires within the region. In contrast to Northern Hemisphere paludifying systems, the patterning appears to initiate at the start of wetland development or as part of an infilling process. The wetlands dominated by E. minus are highly resilient to disturbance, particularly burning and sea level alterations, and appear to form important refuge areas for amphibians, fish and birds (both non-migratory and migratory) over thousands of years.

Connecting soundscape to landscape: Which acoustic index best describes landscape configuration?

Soundscape assessment has been proposed as a remote ecological monitoring tool for measuring biodiversity, but few studies have examined how soundscape patterns vary with landscape configuration and condition. The goal of our study was to examine a suite of published acoustic indices to determine whether they provide comparable results relative to varying levels of landscape fragmentation and ecological condition in nineteen forest sites in eastern Australia. Our comparison of six acoustic indices according to time of day revealed that two indices, the acoustic complexity and the bioacoustic index, presented a similar pattern that was linked to avian song intensity, but was not related to landscape and biodiversity attributes. The diversity indices, acoustic entropy and acoustic diversity, and the normalized difference soundscape index revealed high nighttime sound, as well as a dawn and dusk chorus. These indices appear to be sensitive to nocturnal biodiversity which is abundant at night in warm, subtropical environments. We argue that there is need to better understand temporal partitioning of the soundscape by specific taxonomic groups, and this should involve integrated research on amphibians, insects and birds during a 24 h cycle. The three indices that best connected the soundscape with landscape characteristics, ecological condition and bird species richness were acoustic entropy, acoustic evenness and the normalized difference soundscape index. This study has demonstrated that remote soundscape assessment can be implemented as an ecological monitoring tool in fragmented Australian forest landscapes. However, further investigation should be dedicated to refining and/or combining existing acoustic indices and also to determine if these indices are appropriate in other landscapes and for other survey purposes.

Scat contents of the spotted-tailed quoll Dasyurus maculatus in the New England gorges, north-eastern New South Wales

In 1313 scats of the spotted-tailed quoll Dasyurus maculatus, collected over 5 years from the gorge country of north-eastern New South Wales, the most frequent and abundant items were derived from mammals and a restricted set of insect orders. These quolls also ate river-associated items: waterbirds, eels, crayfish, aquatic molluscs and even frogs. Macropods contributed most of the mammal items, with possums, gliders and rodents also being common. Some food, particularly from macropods and lagomorphs, had been scavenged (as shown by fly larvae). The most frequent invertebrates were three orders of generally large insects Coleoptera, Hemiptera and Orthoptera, which were most frequent in summer and almost absent in winter scats. Monthly mean numbers of rodent and small dasyurid items per scat were inversely related to these large insects in scats. The numbers of reptile items were inversely related to the numbers of mammal (especially arboreal and small terrestrial mammal) items per scat, thus types of items interacted in their occurrences in monthly scat samples. Frequencies of most vertebrate items showed no seasonal, but much year-to-year, variation. This quoll population ate four main types of items, each requiring different skills to obtain: they hunted arboreal marsupials (possibly up trees), terrestrial small mammals and reptiles (on the ground), and seasonally available large insects (on trees or the ground), and scavenged carcases, mostly of large mammals but also birds and fishes (wherever they could find them).

Salmonella Virchow and Salmonella Weltevreden in a Random Survey of the Asian House Gecko, Hemidactylus frenatus, in Houses in Northern Australia.

Background: Salmonella enterica serotype Virchow is the most common cause of invasive nontyphoid salmonellosis in North Queensland, particularly in infants, but the zoonotic source is unknown. This study aimed at determining (i) the prevalence of the introduced Asian house gecko, Hemidactylus frenatus, in houses in North Queensland and (ii) whether they were a potential source of Salmonella Virchow. Methods: Asian house geckos were collected in a random survey of houses in Townsville, North Queensland. Gut contents underwent microbiological analysis within 2 h of removal using both direct plating and enrichment broth methods. Any organism found to be a presumptive Salmonella spp. was then sent to a reference lab for confirmation of genus/species, serotyping, and phage typing if indicated. Results: One hundred Asian house geckos were collected from 57 houses. Geckos were present in 100% of houses surveyed, and prevalence of Salmonella in large intestinal contents was 7% (95% confidence interval 2, 12%). Three serotypes were found: Virchow (phage type 8), Weltevreden, and an untypable subspecies 1 serotype 11:-:1,7. Conclusion: Since Salmonella Virchow (phage type 8) is associated with invasive disease, the introduced Asian house gecko may play a significant role in the epidemiology of sporadic salmonellosis in places invaded by these peridomestic reptiles. These results justify more detailed epidemiological studies on the role of the Asian house gecko in sporadic salmonellosis and development of evidence-based strategies to decrease this potential zoonotic hazard.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Spatial and temporal effects of grazing management and rainfall on the vertebrate fauna of a tropical savanna

Grazing by domestic livestock is one of the most widespread uses of the rangelands of Australia. There is limited information on the effects of grazing by domestic livestock on the vertebrate fauna of Australia and the establishment of a long-term grazing experiment in north-eastern Queensland at Wambiana provided an opportunity to attempt an examination of the changes in vertebrate fauna as a consequence of the manipulation of stocking rates. The aim was to identify what the relative effects of vegetation type, stocking rate and other landscape-scale environmental factors were on the patterns recorded. Sixteen 1-ha sites were established within three replicated treatments (moderate, heavy and variable stocking rates). The sites were sampled in the wet and dry seasons in 1999-2000 (T-0) and again in 2003-04 (T-1). All paddocks of the treatments were burnt in 1999. Average annual rainfall declined markedly between the two sampling periods, which made interpretation of the data difficult. A total of 127 species of vertebrate fauna comprising five amphibian, 83 bird, 27 reptile and 12 mammal species were recorded. There was strong separation in faunal composition from T-0 to T-1 although changes in mean compositional dissimilarity between the grazing stocking rate treatments were less well defined. There was a relative change in abundance of 24 bird, four mammal and five reptile species from T-0 to T-1. The generalised linear modelling identified that, in the T-1 data, there was significant variation in the abundance of 16 species explained by the grazing and vegetation factors. This study demonstrated that vertebrate fauna assemblage did change and that these changes were attributable to the interplay between the stocking rates, the vegetation types on the sites surveyed, the burning of the experimental paddocks and the decrease in rainfall over the course of the two surveys. It is recommended that the experiment is sampled again but that the focus should be on a rapid survey of abundant taxa (i.e. birds and reptiles) to allow an increase in the frequency of sampling and replication of the data. This would help to articulate more clearly the trajectory of vertebrate change due to the relative effects of stocking rates compared with wider landscape environmental changes. Given the increasing focus on pastoral development in northern Australia, any opportunity to incorporate the collection of data on biodiversity into grazing manipulation experiments should be taken for the assessment of the effects of land management on faunal species.

Impacts on nontarget avian species from aerial meat baiting for feral pigs

Bait containing sodium fluoroacetate (1080) is widely used for the routine control of feral pigs in Australia. In Queensland, meat baits are popular in western and northern pastoral areas where they are readily accepted by feral pigs and can be distributed aerially. Field studies have indicated some levels of interference and consumption of baits by nontarget species and, based on toxicity data and the 1080 content of baits, many nontarget species (particularly birds and varanids) are potentially at risk through primary poisoning. While occasional deaths of species have been recorded, it remains unclear whether the level of mortality is sufficient to threaten the viability or ecological function of species. A series of field trials at Culgoa National Park in south-western Queensland was conducted to determine the effect of broadscale aerial baiting (1.7 baits per km2) on the density of nontarget avian species that may consume baits. Counts of susceptible bird species were conducted prior to and following aerial baiting, and on three nearby unbaited properties, in May and November 2011, and May 2012. A sample of baits was monitored with remote cameras in the November 2011 and May 2012 trials. Over the three baiting campaigns, there was no evidence of a population-level decline among the seven avian nontarget species that were monitored. Thirty per cent and 15% of baits monitored by remote cameras in the November 2011 and May 2012 trials were sampled by birds, varanids or other reptiles. These results support the continued use of 1080 meat baits for feral pig management in western Queensland and similar environs.