Design, Synthesis and Antifungal Activity of Stapled Aurein1.2 Peptides

Aurein1.2 is a 13-residue antimicrobial peptide secreted by the Australian tree frog Litoria aurea. In order to improve its stabilities, the helical contents and corresponding biological activities of Aurein1.2 (a series of stapled analogues) were synthesized, and their potential antifungal activities were evaluated. Not surprisingly, the stapled Aurein1.2 peptides showed higher proteolytic stability and helicity than the linear counterpart. The minimum inhibitory concentration (MIC) of ten stapled peptides against six strains of common pathogenic fungi was determined by the microscale broth dilution method recommended by CLSI. Of them, Sau-1, Sau-2, Sau-5, and Sau-9 exhibited better inhibitory effects on the fungi than the linear peptide. These stapled Aurein1.2 peptides may serve as the leading compounds for further optimization and antifungal therapy.

Novel use of statistics to determine precise relationship assignment to estimate breeding output of a threatened amphibian with tadpole genotypes

Relatedness (rxy) measures are useful in molecular ecology studies as they can provide a means to answer biological hypotheses where pedigree information is valuable. Our understanding of the reproductive ecology of the threatened amphibian Litoria aurea is not complete where applying rxy measurements may provide further elucidation. Here we use SNPs to identify which rxy estimators (or combination of) most precisely assign relationships in L. aurea tadpoles to determine how many breeding pairs contribute to producing propagules in explosive breeding events. We aimed to (1) use simulated L. aurea genotype data to determine the precision of six rxy estimators, (2) compare the precision of relationship assignment thresholds between rxy estimators computed by discriminant function analysis (DFA) and test if using multiple estimators improved precision, and (3) Apply the best performing DFA model to assign relationships in wild tadpoles to quantify how many mating pairs reproduced in explosive breeding events. We hypothesised that each tadpole cohort produced during explosive breeding events would be conceived by >|2| mating pairs. The triadic maximum likelihood estimator had the highest Pearson’s R2 (0.92) and the lowest amount of misclassifications in the DFA (16.00%). A multi-variate DFA that included three rxy estimators further reduced the misclassifications down to 11.88%. There was evidence that more than one mating pair contributed to each explosive breeding event (n = 11). We show that the multi-variate DFA enabled precise relationship classification of free-living tadpoles which improved knowledge on amphibian reproductive ecology. We recommend this method for relationship assignments in SNP genotyped datasets.

Observations of an unexpected abundance of estuarine mosquitoes associated with an urban freshwater wetland

ABSTRACT Urban freshwater wetlands have been identified as a potential source of mosquitoes of pest and public health concern in Australia. Mosquitoes most commonly associated with these habitats include Anopheles annulipes, Culex annulirostris, Culex quinquefasciatus, Coquillettidia linealis and Mansonia uniformis. However, adaptive management of freshwater wetlands for wildlife conservation or vegetation management can alter the local environmental conditions and may provide opportunities for other mosquito species to exploit these habitats. A series of small wetlands at Sydney Olympic Park, NSW, were drained in spring 2016 and allowed to refill via rainfall to improve conditions for the endangered green and golden bell frog Litoria aurea. Below-average rainfall was experienced that summer and the wetlands did not fully refill until early autumn. Surprisingly, immature stages of the estuarine mosquito, Aedes vigilax, were observed in the freshwater wetlands in exceptional abundances during this period. Adjacent to these freshwater wetlands there are extensive saltmarsh and mangrove habitats where highly abundant populations of Ae. vigilax are common. Observations of use of freshwater habitats by this estuarine mosquito have implications for urban freshwater wetland management and rehabilitation strategies, which currently do not consider the potential pest and public health issues associated with estuarine mosquitoes.

Identifying sex-linked markers in Litoria aurea: a novel approach to understanding sex chromosome evolution in an amphibian

Few taxa exhibit the variability of sex-determining modes as amphibians. However, due to the presence of homomorphic sex chromosomes in many species, this phenomenon has been difficult to study. The Australian frog, Litoria aurea, has been relatively well studied over the past 20 years due to widespread declines largely attributable to chytrid fungus. However, it has been subject to few molecular studies and its mode of sex determination remained unknown. We applied DArTseq™ to develop sex-linked single nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA) markers in 44 phenotypically sexed L. aurea individuals from the Molonglo River in NSW, Australia. We conclusively identified a male heterogametic (XX-XY) sex determination mode in this species, identifying 11 perfectly sex-linked SNP and six strongly sex-linked PA markers. We identified a further 47 moderately sex-linked SNP loci, likely serving as evidence indicative of XY recombination. Furthermore, within these 47 loci, a group of nine males were found to have a feminised Y chromosome that significantly differed to all other males. We postulate ancestral sex-reversal as a means for the evolution of this now pseudoautosomal region on the Y chromosome. Our findings present new evidence for the ‘fountain of youth’ hypothesis for the retention of homomorphic sex chromosomes in amphibians and describe a novel approach for the study of sex chromosome evolution in amphibia.

Parallel evolution of frog antimicrobial peptides produces identical conformations but subtly distinct membrane and antibacterial activities

ABSTRACTFrogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Despite the high similarity in physical properties and preference for adopting secondary amphipathic, α-helix conformations in membrane mimicking milieu, their spectrum of activity and potency often varies considerably. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of species specific effective antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes; the greater conformational flexibility and the higher amplitude channel conductance induced offers a rationale for the greater potency and broader spectrum of activity of temporin L over aurein 2.5. Specific contributions from individual residues are identified that define the mechanisms of action of each AMP. Our findings suggest AMPs in frogs are examples of parallel evolution whose utility is based on apparently similar but subtly distinct mechanisms of action.

Differences in microhabitat selection patterns between a remnant and constructed landscape following management intervention

Context Achieving successful conservation outcomes in habitat creation and reintroductions requires an understanding of how species use their habitat and respond to these interventions. However, few initiatives directly compare microhabitat selection between remnant and managed habitats to measure effectiveness and evaluate outcomes. Probability of detection is also rarely included in studies on microhabitat use, which may lead to erroneous conclusions if detectability varies between variables. Methods In this study, we used the endangered green and golden bell frog (Litoria aurea) to compare differences in microhabitat-use patterns in both a remnant and a constructed habitat. A detectability study was also conducted to determine detection probabilities among microhabitats. Key results Aquatic vegetation was used more than expected in both the remnant and constructed habitats, and rock piles were utilised less than expected in the constructed habitat, despite their recommendation in most habitat templates. We found that detection probabilities altered the outcomes of abundance estimates for nearly all the measured microhabitat variables. Conclusions Future management for this species should focus on providing high proportions of aquatic vegetation. Furthermore, although rock piles have been utilised greatly in past L. aurea habitat creation, placing large rocks on a managed site is expensive and time consuming. Future management initiatives may need to focus on providing smaller proportion of rocks, which would be a more appropriate use of resources. Implications With conservation management projects increasing over the next few decades, understanding habitat use before implementing strategies should be a priority as it will provide important insights and inform decision-making for optimum habitat creation and restoration. Furthermore, accounting for detectability in microhabitat use studies is essential to avoid wrong conclusions that may negatively affect the success of ecological management strategies.

No evidence of protracted population decline across 17 years in an unmanaged population of the green and golden bell frog in north-eastern New South Wales

Describing the population trends of threatened species over time is central to their management and conservation. The green and golden bell frog (Litoria aurea) is a formerly common species of south-eastern Australia that has declined to ~40 populations in New South Wales, and experienced a substantial contraction of its geographic range. We aimed to determine whether an unmanaged population at the northern end of its range had declined across a 17-year period. We estimated population size at the beginning and end of this period, using several population models to fully characterise this population. Different modelling approaches gave different population estimates. Based on a similar number of survey occasions the adult male segment of the population was estimated using the Popan model at 112.0 (±13.5, s.e.; 95% CI: 85.5–138.8) in 1998/99 and 95.2 (±17.6; 60.8–129.7) in 2015/16. With the inclusion of maturing subadults following the practice of earlier studies, the population was estimated at 163.6 (±25.9; 112.8–214.5) males in 2015/16. These estimates represent an index of a larger population because the largest wetland was subsampled. Our data provide no evidence of a declining population. Our study highlights the need to understand the implications of using different population models and two age-classes to estimate population parameters.