Heavy metal pollution negatively correlates with anuran species richness and distribution in south-eastern Australia

Heavy metal pollution has likely played an important role in global biodiversity decline, but there remains a paucity of information concerning the effects of metals on amphibian diversity. This study assessed anuran species richness and distribution in relation to sediment metal content and water chemistry in wetlands located along the Merri Creek corridor in Victoria, south-eastern Australia. Anurans were present in 60% (21/35) of study sites, with a total of six species detected: the eastern common froglet (Crinia signifera), the eastern sign-bearing froglet (Crinia parinsignifera), the southern brown tree frog (Litoria ewingii), the growling grass frog (Litoria raniformis), the eastern banjo frog (Limnodynastes dumerilii) and the spotted marsh frog (Limnodynastes tasmaniensis). Mean species richness was 1.77 ± 0.32 per site, and species richness ranged from zero to six species per site. Across sites, species richness correlated negatively with sediment concentrations of six heavy metals: copper, nickel, lead, zinc, cadmium and mercury. Species richness also correlated negatively with wetland water electrical conductivity (a proxy for salinity) and concentrations of orthophosphate. Distributions of the three most commonly observed frog species (C. signifera, L. tasmaniensis and L. ewingii) were significantly negatively associated with the total level of metal contamination at individual sites. The study is the first to provide evidence for an association between metal contamination and anuran species richness and distribution in the southern hemisphere, adding to a small but growing body of evidence that heavy metal pollution has contributed to global amphibian decline

Hormonal induction of gamete release, and in-vitro fertilisation, in the critically endangered Southern Corroboree Frog, Pseudophryne corroboree

<p>Abstract</p> <p>Background</p> <p>Conservation Breeding Programs (CBP's) are playing an important role in the protection of critically endangered anuran amphibians, but for many species recruitment is not successful enough to maintain captive populations, or provide individuals for release. In response, there has been an increasing focus on the use of Assisted Reproductive Technologies (ART), including the administration of reproductive hormones to induce gamete release followed by <it>in vitro </it>fertilisation. The objective of this study was to test the efficacy of two exogenous hormones to induce gamete release, for the purpose of conducting <it>in vitro </it>fertilisation (IVF), in one of Australia's most critically endangered frog species, <it>Pseudophryne corroboree</it>.</p> <p>Methods</p> <p>Male frogs were administered a single dose of either human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRHa), while female frogs received both a priming and ovulatory dose of LHRHa. Spermiation responses were evaluated at 3, 7, 12, 24, 36, 48, 60 and 72 h post hormone administration (PA), and sperm number and viability were quantified using fluorescent microscopy. Ovulation responses were evaluated by stripping females every 12 h PA for 5 days. Once gametes were obtained, IVF was attempted by combining spermic urine with oocytes in a dilute solution of simplified amphibian ringer (SAR).</p> <p>Results</p> <p>Administration of both hCG and LHRHa induced approximately 80% of males to release sperm over 72 h. Peak sperm release occurred at 12 h PA for hCG treated males and 36 h PA for LHRHa treated males. On average, LHRHa treated males released a significantly higher total number of live sperm, and a higher concentration of sperm, over a longer period. In female frogs, administration of LHRHa induced approximately 30% of individuals to release eggs. On average, eggs were released between 24 and 48 h PA, with a peak in egg release at 36 h PA. IVF resulted in a moderate percentage (54.72%) of eggs being fertilised, however all resultant embryos failed prior to gastrulation.</p> <p>Conclusions</p> <p>Hormone treatment successfully induced spermiation and ovulation in <it>P. corroboree</it>, but refinement of gamete induction and IVF techniques will be required before ART protocols can be used to routinely propagate this species.</p

Effect of a refuge from persistent male courtship in the Drosophila laboratory environment

The Drosophila melanogaster laboratory model has been used extensively in studies of sexual conflict because during the process of courtship and mating, males impose several costs upon females (e.g., reduced fecundity). One important difference between the laboratory and the wild is that females in the laboratory lack a spatial refuge from persistent male courtship. Here, we describe two experiments that examine the potential consequences of a spatial refuge for females. In the first experiment, we examined the influence of a spatial refuge on mating rate of females, and in the second one we examined its influence on females\u27 lifetime fecundity. We found that females mated about 25% less often when a spatial refuge was available, but that the absence of a spatial refuge did not substantially increase the level of male-induced harm to females (i.e., sexual conflict). © The Author 2008. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved

Short- and long- term consequences of developmental saline stress: impacts on anuran respiration and behaviour

Secondary salinization has been identified as a major stressor to amphibians. Exposure to elevated salinity necessitates physiological adjustments and biochemical changes that may be energetically demanding. As such, exposure to non-lethal levels of salinity during development could potentially alter anuran metabolic rates and individual performance in both pre- and post-metamorphic life stages. We investigated the effects of non-lethal levels of salinity on metamorphic traits (time to reach metamorphosis and metamorphic mass), tadpole oxygen consumption, escape response behaviour (pre- and post-metamorphosis) and foraging ability post-metamorphosis in two native Australian frog species, the southern brown tree frog (Litoria ewingii) and the striped marsh frog (Limnodynastes peronii). We found that both Lit. ewingii and Lim. peronii exhibited differences in metamorphic traits in response to elevated salinity. Neither species showed significant change in oxygen consumption during development in response to salinity, relative to freshwater controls. Both species displayed impaired escape response behaviours in response to salinity during larval development, but flow-on effects to adult escape response behaviours and foraging performance were species-specific. Our results show that the influence of stressors during development can have consequences for anuran physiology and behaviour at multiple life stages, and emphasize the need for studies that examine the energetics of anuran responses in order to better understand the responses of biota to stressful environments

Dose and life stage-dependent effects of dietary beta-carotene supplementation on the growth and development of the Booroolong frog

Carotenoids are known for their antioxidant capacity and are considered to play an important role in vertebrate growth and development. However, evidence for their beneficial effects remains limited, possibly because very few studies have tested for dose effects across different life stages. The present study investigated the effect of various doses of dietary beta-carotene supplements on the growth and development of larval and post-metamorphic Booroolong frogs (Litoria booroolongensis). Larval and post-metamorphic basal diets (containing 0.015 and 0.005 mg g−1 total carotenoids, respectively) were supplemented with beta-carotene at one of four concentrations: 0 mg g−1 , 0.1 mg g−1 , 1 mg g−1 and 10 mg g−1 . Each treatment included 72 replicate individuals, and individuals remained on the same diet treatment over both life stages (spanning 53 experimental weeks). Our results show that larvae receiving an intermediate (1 mg g−1 ) beta-carotene supplement dose grew faster than unsupplemented larvae (0 mg g−1 ), and metamorphosed earlier. After metamorphosis, there was no effect of the lowest supplement dose (0.1 mg g−1 ) on growth and development. However, juveniles fed the highest supplement dose (10 mg g−1 ) displayed significantly smaller body mass and lower body condition, compared to all other supplement doses, from 4-months through to sexual maturity (7-months). These findings indicate that beta-carotene supplementation has positive effects on growth and development, but only at intermediate doses, and only in the larval life stage. This knowledge may assist with amphibian conservation by expediting the rate that metamorphs can be generated in captive breeding programmes. More broadly, this is the first study to demonstrate both dose and life stagedependent effects of dietary beta-carotene supplementation on vertebrate growth and development

The importance of quantifying fitness-determining traits throughout life to assess the application of reproductive technologies for amphibian species recovery

The application of reproductive technologies to amphibian conservation breeding programs is gaining momentum and the field is poised to contribute significantly toward amphibian species recovery. We briefly discuss the opportunities for reproductive technologies to enhance conservation breeding outcomes, including their potential to enhance the genetic management, and in turn, the fitness of threatened species. Despite this potential, an important consideration that is not yet well understood is the degree to which specific reproductive technologies might influence (either enhance, or in some instances potentially decrease) individual fitness and lead to shifts in population viability. The development of a standardised approach to monitoring offspring throughout life-stages to detect changes to morphology, behaviour, physiology, survivorship, and developmental trajectories is essential. The primary focus of this review is to provide a ‘best-practise’ framework for quantifying key fitness determining traits expected to contribute to the fitness of individuals and long-term viability of populations, which will ultimately allow us to progress the field of amphibian reproductive technologies and assess the impact of protocol refinement

Characterisation of the urinary microbiome of a frog, and the effect of antibiotics on bacterial abundance and sperm viability during refrigerated storage

Reproductive technologies are increasingly being adopted to improve the conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs

Dietary carotenoid supplementation has long‐term and community‐wide effects on the amphibian skin microbiome

The amphibian skin microbiome plays a crucial role in host immunity and pathogen defence, yet we know little about the environmental drivers of skin microbial variation across host individuals. Inter‐individual variation in the availability of micro‐nutrients such as dietary carotenoids, which are involved in amphibian immunity, may be one factor that influences skin microbial assembly across different life history stages. We compared the effect of four carotenoid supplementation regimes during different life stages on the adult skin microbiome using a captive population of the critically endangered southern corroboree frog, Pseudophryne corroboree. We applied 16S rRNA sequencing paired with joint‐species distribution models to examine the effect of supplementation on taxon abundances. We found that carotenoid supplementation had subtle yet taxonomically widespread effects on the skin microbiome, even 4.5 years post supplementation. Supplementation during any life‐history stage tended to have a positive effect on the number of bacterial taxa detected, although explanatory power was low. Some genera were sensitive to supplementation pre‐metamorphosis, but most demonstrated either additive or dominant effects, whereby supplementation during one life history stage had intermediate or similar effects, respectively, to supplementation across life. Carotenoid supplementation increased abundances of taxa belonging to lactic acid bacteria, including Lactococcus and Enterococcus, a group of bacteria that have previously been linked to protection against the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). While the fitness benefits of these microbial shifts require further study, these results suggest a fundamental relationship between nutrition and the amphibian skin microbiome which may be critical to amphibian health and the development of novel conservation strategies

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

iTRAQ Identification of Candidate Serum Biomarkers Associated with Metastatic Progression of Human Prostate Cancer

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer ‘BPH’, (ii) localised cancer with no evidence of progression, ‘non-progressing’ (iii) localised cancer with evidence of biochemical progression, ‘progressing’, and (iv) bone metastasis at presentation ‘metastatic’. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and ‘panels’ of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation