Litoria Caerulea Research Articles

How Membrane-Active Peptides Get into Lipid Membranes.

The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular mechanism by which these membrane-active peptides lyse membranes. The last class of membrane-active peptides discussed are the CPPs, which translocate across the lipid bilayer without inducing severe disruption and have potential as drug vehicles. CPPs are typically highly charged and can show antimicrobial activity by targeting an intracellular target rather than via a direct membrane lytic mechanism. A critical aspect in the structure-function relationship of membrane-active peptides is their specific activity relative to the lipid membrane composition of the cell target. Cell membranes have a wide diversity of lipids, and those of eukaryotic and prokaryotic species differ greatly in composition and structure. The activity of AMPs from Australian tree frogs, toxins, and CPPs has been investigated within various lipid systems to assess whether a relationship between peptide and membrane composition could be identified. NMR spectroscopy techniques are being used to gain atomistic details of how these membrane-active peptides interact with model membranes and cells, and in particular, competitive assays demonstrate the difference between affinity and activity for a specific lipid environment. Overall, the interactions between these relatively small sized peptides and various lipid bilayers give insight into how these peptides function at the membrane interface.

Nanoscale friction and adhesion of tree frog toe pads

Tree frogs have become an object of interest in biomimetics due to their ability to cling to wet and slippery surfaces. In this study, we have investigated the adhesion and friction behavior of toe pads of White’s tree frog (Litoria caerulea) using atomic force microscopy (AFM) in an aqueous medium. Facilitating special types of AFM probes with radii of ∼400 nm and ∼13 μm, we were able to sense the frictional response without damaging the delicate nanopillar structures of the epithelial cells. While we observed no significant adhesion between both types of probes and toe pads in wet conditions, frictional forces under such conditions were very pronounced and friction coefficients amounted between 0.3 and 1.1 for the sliding friction between probes and the epithelial cell surfaces.

A Theoretical Characterization of Curvature Controlled Adhesive Properties of Bio-Inspired Membranes

Some biological systems, such as the tree frog, Litoria caerulea, and the bush-cricket, Tettigonia viridissima, have developed the ability to control adhesion by changing the curvature of their pads. Active control systems of adhesion inspired by these biological models can be very attractive for the development of devices with controllable adhesive properties. In this paper, we present a theory describing the adhesive behavior of an artificial system consisting of an inflatable membrane clamped to a metallic cylinder and filled with air. In such a system, by controlling the internal pressure acting on the membrane, it is possible to modulate the adhesive strength. In particular, an increase of the internal pressure and, hence, the curvature of the membrane, results in a decrease of the pull-off force. Results predicted by the theoretical model are in good agreement with experimental data. The model explains the apparent contradictory results observed for the thick membrane with zero curvature. In fact, in this case, large pull-off forces should be expected, but zero values are measured due to an initial small misalignment between indenter and membrane, which is not possible to control with precision during the experiments. The present model might help to achieve a better understanding of the adhesion behavior of biological systems and of the fingertips that, in a broad sense, may be regarded as shell-like structures.

Controls and constrains of the membrane disrupting action of Aurein 1.2.

Aurein 1.2 is a 13 residue antimicrobial peptide secreted by the Australian tree frog Litoria Aurea. It is a surface-acting membrane disrupting peptide that permeabilizes bacterial membranes via the carpet mechanism; the molecular details of this process are mostly unknown. Here the mechanism of action of Aurein 1.2 was investigated with an emphasis on the role of membrane charge and C-terminal amidation of the peptide. Using quartz crystal microbalance (QCM) fingerprinting it was found that the membrane charge correlates with membrane affinity of the peptide, however the binding and the membrane disrupting processes are not charge driven; increased membrane charge reduces the membrane disrupting activity. Coarse grain simulations revealed that phenylalanine residues act as membrane anchors. Accordingly Aurein 1.2 has the ability to bind to any membrane. Furthermore, bundling precludes membrane disruption in case of wild type peptides, while non C-terminal amidated peptides form random aggregates leading to detachment from the membrane. Hence C-terminal amidation is crucial for Aurein 1.2 action. Our results suggest that Aurein 1.2 acts via aggregation driven membrane penetration. The concomitant change in the tension of the outer leaflet imposes a spontaneous curvature on the membrane, leading to disintegration.

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μM) than against E. coli (64 μM) and an intermediate cytotoxicity against hRBC (30 μM). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.

Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

Isolation of Brucella from a White’s tree frog (Litoria caerulea)

Introduction: Brucellosis is a zoonotic disease that has a significant economic, social and public health impact in many parts of the world. The causative agents are members of the genus Brucella currently comprising 11 species and with an expanding known host range in recent years. Case presentation: One of a pair of White’s tree frogs (Litoria caerulea) developed skin lesions from which a pure growth of a haemolytic organism was obtained. The isolate was identified as Brucella melitensis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, although the colony morphology was inconsistent with this identification. Applying the classical biotyping approach used to subdivide members of the genus Brucella, the isolate did not correspond to any known Brucella sp. However, PCR targeting of genes specific for members of the genus Brucella was strongly positive and 16S rRNA gene sequencing revealed a close relationship with extant Brucella spp. In order to place the isolate more accurately, a multilocus sequencing approach was applied, which confirmed that the isolate represented a novel member of the emerging ‘atypical’ Brucella group, which includes isolates from human disease, from rodents and, more recently, reported isolations from frogs in Germany. Conclusion: This case represents the first report of isolation of a Brucella sp. from frogs outside Germany and suggests that these isolates may be widespread. Whilst there is no evidence to date that these isolates represent a zoonotic threat, the association of other ‘atypical’ Brucella sp. with human disease suggests that appropriate measures should be taken to avoid unnecessary contact with potentially infected amphibians until the zoonotic potential of this group is better understood.

Pathology of a Bohle-like Virus Infection in Two Australian Frog Species (Litoria splendida and Litoria caerulea)

Gross and histopathological examination was performed on seven captive magnificent tree frogs (Litoria splendida) and one green tree frog (Litoria caerulea) that had died or been humanely destroyed while naturally infected with Mahaffey Road virus, a Bohle iridovirus-like ranavirus. Necropsy examination revealed skin lesions consisting of multiple small pale or haemorrhagic papules and ulcers in most frogs. Other common gross findings were perineural haemorrhage affecting the spinal nerves, hydrocoelom, hepatomegaly and splenomegaly with pinpoint pale foci throughout the parenchyma. On histological examination, vasculitis with prominent endothelial necrosis was found in a wide range of tissues. Widespread lymphoid necrosis and fibroblast necrosis were usual findings. Multifocal epithelial cell necrosis in the epidermis, liver and pancreas was found commonly. Non-suppurative meningoencephalitis, myelitis and ganglioneuritis were present variably. Intracytoplasmic basophilic inclusion bodies were found variably in hepatocytes, renal tubular epithelium and keratinocytes. Immunohistochemistry demonstrated ranavirus antigen in endothelial cells, fibroblasts, macrophages, lymphocytes and epithelial cells in a wide range of tissues. The finding of widespread venous and lymphatic endothelial necrosis and demonstration of abundant endothelial antigen suggests that endothelial tropism of the virus plays a significant role in the pathogenesis of the infection.

Reproductive and immune effects of chronic corticosterone treatment in male White's treefrogs, Litoria caerulea.

Amphibian populations are declining globally. The potential contribution of glucocorticoid hormones to these declines has received little attention, but chronic elevation of glucocorticoids has been linked to a suite of negative outcomes across vertebrate taxa. Recently, chronic environmental stress has been associated with precipitous declines in sperm count and sperm viability in White's treefrogs (Litoria caerulea), but the mechanism remains unknown. In order to determine whether corticosterone is responsible for suppressing reproductive and immune function in this species, we elevated circulating concentrations of corticosterone in 10 male captive-bred frogs via transdermal application for 7 days. We compared sperm count, sperm viability, splenic cell count and circulating leucocyte counts in corticosterone-treated frogs with those in untreated control frogs. Chronic application of exogenous corticosterone led to supraphysiological circulating concentrations of corticosterone, but had no effect on sperm count or viability. However, corticosterone-treated frogs demonstrated a significant decrease in circulating eosinophils, which are immune cells implicated in fighting a variety of pathogens, including extracellular parasites. These findings suggest that although chronic elevation of circulating corticosterone is not necessarily associated with reproductive suppression in this species, it may cause immunosuppression. Thus, chronic glucocorticoid elevations in amphibians might enhance susceptibility to infection with pathogens and parasites, and their potential contributions to global population declines warrant further study.

Effects of anthropogenic noise on endocrine and reproductive function in White's treefrog, Litoria caerulea.

Urbanization is a major driver of ecological change and comes with a suite of habitat modifications, including alterations to the local temperature, precipitation, light and noise regimes. Although many recent studies have investigated the behavioural and ecological ramifications of urbanization, physiological work in this area has lagged. We tested the hypothesis that anthropogenic noise is a stressor for amphibians and that chronic exposure to such noise leads to reproductive suppression. In the laboratory, we exposed male White's treefrogs, Litoria caerulea, to conspecific chorus noise either alone or coupled with pre-recorded traffic noise nightly for 1 week. Frogs presented with anthropogenic noise had significantly higher circulating concentrations of corticosterone and significantly decreased sperm count and sperm viability than did control frogs. These results suggest that in addition to having behavioural and ecological effects, anthropogenic change might alter physiology and Darwinian fitness. Future work should integrate disparate fields such as behaviour, ecology and physiology to elucidate fully organisms' responses to habitat change.

Skin sloughing rate increases with chytrid fungus infection load in a susceptible amphibian

Summary Amphibian chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for the greatest disease‐driven loss of vertebrate biodiversity in recorded history. Understanding drivers of host susceptibility to this cutaneous disease is hindered by gaps in our knowledge of the host–pathogen relationship. One such overlooked aspect of susceptibility is variation in skin maintenance processes, particularly skin turnover via routine sloughing. It has been suggested that sloughing plays a role in immune defence, by removing skin‐associated microbes. Thus, skin sloughing may play an important role in the pathogenesis of chytridiomycosis. To determine the relationship between skin sloughing and disease progression, we exposed adult Australian green tree frogs (Litoria caerulea) to a local Bd strain and monitored sloughing rates and individual infection loads on a naturalistic cycling temperature regime (15–23 °C). We determined sloughing rates in real‐time by using an array of infrared video cameras to film frog behaviour and monitored infection load before and after sloughing by swabbing and analysis with quantitative PCR. We found that sloughing rate increased with Bd infection load in infected frogs, but sloughing itself did not affect Bd load on the ventral skin surface. Furthermore, Bd infection did not affect the duration of characteristic sloughing behaviour, and sloughing retained rhythmicity even at high infection loads. Although an increased sloughing rate might be considered advantageous for Bd‐infected animals, it does not appear to curb the progression of disease and may actually contribute to the loss of physiological homoeostasis seen in terminally ill frogs by further inhibiting water and electrolyte transport across the skin. By measuring sloughing rates directly for the first time, our results shed light on how Bd interacts with the physiological processes of the skin and indicate that variation in skin sloughing frequency may play a role in the observed inter‐ and intraspecific variation in susceptibility to disease.

Quantifying Anuran Microhabitat Use to Infer the Potential for Parasite Transmission between Invasive Cane Toads and Two Species of Australian Native Frogs

Parasites that are carried by invasive species can infect native taxa, with devastating consequences. In Australia, invading cane toads (Rhinella marina) carry lungworm parasites (Rhabdias pseudosphaerocephala) that (based on previous laboratory studies) can infect native treefrogs (Litoria caerulea and L. splendida). To assess the potential of parasite transmission from the invader to the native species (and from one infected native frog to another), we used surveys and radiotelemetry to quantify anuran microhabitat use, and proximity to other anurans, in two sites in tropical Australia. Unsurprisingly, treefrogs spent much of their time off the ground (especially by day, and in undisturbed forests) but terrestrial activity was common at night (especially in anthropogenically modified habitats). Microhabitat overlap between cane toads and frogs was generally low, except at night in disturbed areas, whereas overlap between the two frog species was high. The situations of highest overlap, and hence with the greatest danger of parasite transmission, involve aggregations of frogs within crevices by day, and use of open ground by all three anuran species at night. Overall, microhabitat divergence between toads and frogs should reduce, but not eliminate, the transmission of lungworms from invasive toads to vulnerable native frogs.

Experimental evolution alters the rate and temporal pattern of population growth in Batrachochytrium dendrobatidis, a lethal fungal pathogen of amphibians.

Virulence of infectious pathogens can be unstable and evolve rapidly depending on the evolutionary dynamics of the organism. Experimental evolution can be used to characterize pathogen evolution, often with the underlying objective of understanding evolution of virulence. We used experimental evolution techniques (serial transfer experiments) to investigate differential growth and virulence of Batrachochytrium dendrobatidis (Bd), a fungal pathogen that causes amphibian chytridiomycosis. We tested two lineages of Bd that were derived from a single cryo-archived isolate; one lineage (P10) was passaged 10 times, whereas the second lineage (P50) was passaged 50 times. We quantified time to zoospore release, maximum zoospore densities, and timing of zoospore activity and then modeled population growth rates. We also conducted exposure experiments with a susceptible amphibian species, the common green tree frog (Litoria caerulea) to test the differential pathogenicity. We found that the P50 lineage had shorter time to zoospore production (Tmin), faster rate of sporangia death (ds), and an overall greater intrinsic population growth rate (λ). These patterns of population growth in vitro corresponded with higher prevalence and intensities of infection in exposed Litoria caerulea, although the differences were not significant. Our results corroborate studies that suggest that Bd may be able to evolve relatively rapidly. Our findings also challenge the general assumption that pathogens will always attenuate in culture because shifts in Bd virulence may depend on laboratory culturing practices. These findings have practical implications for the laboratory maintenance of Bd isolates and underscore the importance of understanding the evolution of virulence in amphibian chytridiomycosis.

Adhesion control by inflation: implications from biology to artificial attachment device

There is an increasing demand for materials that incorporate advanced adhesion properties, such as an ability to adhere in a reversible and controllable manner. In biological systems, these features are known from adhesive pads of the tree frog, Litoria caerulea, and the bush-cricket, Tettigonia viridissima. These species have convergently developed soft, hemispherically shaped pads that might be able to control their adhesion through active changing the curvature of the pad. Inspired by these biological systems, an artificial model system is developed here. It consists of an inflatable membrane clamped to the metallic cylinder and filled with air. Pull-off force measurements of the membrane surface were conducted in contact with the membrane at five different radii of curvature r (c) with (1) a smooth polyvinylsiloxane membrane and (2) mushroom-shaped adhesive microstructured membrane made of the same polymer. The hypothesis that an increased internal pressure, acting on the membrane, reduces the radius of the membrane curvature, resulting in turn in a lower pull-off force, is verified. Such an active control of adhesion, inspired by biological models, will lead to the development of industrial pick-and-drop devices with controllable adhesive properties.

First line of defence: the role of sloughing in the regulation of cutaneous microbes in frogs.

Amphibian populations worldwide are currently experiencing unprecedented declines due to the combined effects of emerging infectious disease and climate change. The skin is the first line of defence in preventing establishment of pathogens and associated infections. Although amphibians undergo regular sloughing of the outer layer of the skin, the potential for regular sloughing to play a role in influencing cutaneous microbial populations and pathogens has been largely overlooked. In the present study, we assessed the effect of skin sloughing on cultivable cutaneous bacterial abundance in the green tree frog (Litoria caerulea). We also examined the effects of temperature and hydric environment on sloughing frequency and microbial re-establishment rates. Our data showed that cultivable cutaneous bacterial abundance was significantly reduced by sloughing events, and frogs kept at 'summer' temperatures (23-33°C) sloughed almost twice as frequently as those maintained at 'winter' temperatures (13-23°C). No effect of hydric environment on sloughing frequency was observed, but we did find that sloughing in L. caerulea appeared to be linked to ambient light cycles. Examination of the effect of sloughing on microbial recolonization indicated that at cool temperatures, an extended intermoult interval allowed microbial abundance to reach higher levels than at warmer 'summer' temperatures (when the intermoult interval was significantly reduced). Our data suggest that sloughing may significantly influence the establishment and/or maintenance of cutaneous bacterial populations (pathogenic, mutualistic and/or commensal) and this, in turn, may be affected by environmental factors, such as ambient light and temperature. These findings are likely to be important for our understanding of the ecology of skin-based pathogens, such as the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

Evaluation of Intramuscular Alfaxalone in Three Australian Frog Species (Litoria caerulea, Litoria aurea, Litoria booroolongensis)

Abstract The effects of intramuscular (IM) alfaxalone were evaluated in three species of Australian frogs: Australian green tree frogs, (Litoria caerulea); green and golden bell frogs (Litoria aurea); and booroolong frogs (Litoria booroolongensis). Heart rate, respiration rate, responses, reflexes, spontaneous movement, time to effect, and duration of effect were measured. Other observations, including change in color and pupil size, were also noted. Alfaxalone administered IM at 30 mg/kg in Australian green tree frogs and 20 mg/kg in green and golden bell frogs achieved sedation and caused bradypnea. In booroolong frogs, a light plane of anesthesia and bradypnea occurred at 20 mg/kg IM. Apnea was not observed in any of the frogs, and there was no statistically significant bradycardia. No mortalities or adverse effects were noted.

First detection of circovirus-like sequences in amphibians and novel putative circoviruses in fishes

The negative samples of a collection, established originally for seeking new adeno- and herpesviruses in lower vertebrates, were screened for the pres-ence of circoviruses by a consensus nested PCR targeting the gene coding for the replication-associated protein. Six fish samples representing five species, namely asp (Aspius aspius), roach (Rutilus rutilus), common bream (Abramis brama), round goby (Neogobius melanostomus) and monkey goby (Neogobius fluviatilis), as well as three frog samples were found positive for circoviral DNA. Sequence analysis of the amplicons indicated the presence of three novel putative circo-like viruses and a circovirus in Hungarian fishes and one novel circovirus in a common toad (Bufo bufo), and another one in a dead and an alive specimen of green tree frog (Litoria caerulea), respectively. In phylogeny reconstruction, the putative bream circovirus clustered together with circoviruses discovered in other cyprinid fishes recently. Three other piscine circoviral sequences appeared closest to sequences derived from different environmental samples. Surprisingly, the nucleotide sequence derived from two fish samples (a bream and a monkey goby) proved to be from porcine circovirus 2 (PCV2), almost identical to a sequence detected in Sweden previously. This is the first report on the detection of PCV2 in fish and circoviral DNA in amphibian hosts.

English

Restriction enzyme mediated integration (REMI) transgenesis and lentiviral transgenesis are effective methods of introducing transgenes into the genome of frogs. One aquatic amphibian species, Xenopus laevis, and one land dwelling species, Litoria caerulea, were chosen as subjects for transgenesis. REMI produced X. laevis that expressed the fluorescent protein DsRed. REMI was unsuccessful in producing transgenic Litoria. Therefore, lentiviral transgenesis was attempted.  Hatchling Litoria tadpoles were exposed to replication defective lentiviral particles containing the coding sequence for DsRed. Histological evaluation revealed the presence of DsRed in brain, heart, liver, kidney, and muscle tissues. Therefore, lentiviral transgenesis appears to be a viable technique for producing transgenic land-dwelling frogs.   Key words: DsRed, Xenopus laevis, Litoria caerulea, restriction enzyme mediated integration (REMI), Lentivirus.

Comparative Cryo-SEM and AFM studies of hylid and rhacophorid tree frog toe pads

Cryo-scanning electron microscopy (cryo-SEM) and atomic force microscopy (AFM) offer new avenues for the study of the morphology of tree frog adhesive toe pads. Using these techniques, we compare toe pad microstructure in two distantly related species of tree frog, Litoria caerulea, White (Hylidae) and Rhacophorus prominanus, Smith (Rhacophoridae), in which the toe pads are considered to be convergent. AFM demonstrates the extraordinary similarity of both surface microstructures (largely hexagonal epithelial cells surrounded by deep channels) and nanostructures (an array of nanopillars, ca. 350 nm in diameter, all with a small dimple at the apex). The cryo-SEM studies examined the distribution of the fibrillar cytoskeleton within the different layers of the stratified toe pad epithelium, demonstrating that the cytoskeletal elements (keratin tonofilaments) that lie at an angle to the surface are relatively poorly developed in L. caerulea, clearly so in comparison to R. prominanus. Cryo-SEM also enabled the visualization of the fluid layer that is critical to a toe pad's adhesive function. This was achieved by examination of the frozen fluid residues left behind after removal of a toe within the cryo-SEM's experimental chamber. Such 'toeprints' demonstrated the presence of a wedge of fluid surrounding each toe pad, as well as fluid filling the channels that surround each epithelial cell. Cryo-SEM was used to examine epithelial cell shape. In a sample of 582 cells, 59.5% were hexagonal, the remainder being mainly pentagonal (23.1%) or heptagonal (16.1%). The distribution of differently-shaped cells was not random, but was not associated with either pad curvature or the distribution of mucous pores that provide fluid for the frogs' wet adhesion mechanism. Our main finding, the great similarity of toe pad structure in these two species, has important implications for biomimetics, for such convergent evolution suggests a good starting point for attempts to develop adhesives that will function in wet conditions.

Host Stress Response Is Important for the Pathogenesis of the Deadly Amphibian Disease, Chytridiomycosis, in Litoria caerulea

Chytridiomycosis, a disease caused by Batrachochytrium dendrobatidis, has contributed to worldwide amphibian population declines; however, the pathogenesis of this disease is still somewhat unclear. Previous studies suggest that infection disrupts cutaneous sodium transport, which leads to hyponatremia and cardiac failure. However, infection is also correlated with unexplained effects on appetite, skin shedding, and white blood cell profiles. Glucocorticoid hormones may be the biochemical connection between these disparate effects, because they regulate ion homeostasis and can also influence appetite, skin shedding, and white blood cells. During a laboratory outbreak of B. dendrobatidis in Australian Green Tree Frogs, Litoria caerulea, we compared frogs showing clinical signs of chytridiomycosis to infected frogs showing no signs of disease and determined that diseased frogs had elevated baseline corticosterone, decreased plasma sodium and potassium, and altered WBC profiles. Diseased frogs also showed evidence of poorer body condition and elevated metabolic rates compared with frogs showing no signs of disease. Prior to displaying signs of disease, we also observed changes in appetite, body mass, and the presence of shed skin associated with infected but not yet diseased frogs. Collectively, these results suggest that elevated baseline corticosterone is associated with chytridiomycosis and correlates with some of the deleterious effects observed during disease development.