Comparison of Thermal and Mechanical Noxious Stimuli for Testing Analgesics in White’s Tree Frogs (Litoria caerulea) and Northern Leopard Frogs (Lithobates pipiens)

Determining the clinical efficacy of analgesic drugs in amphibians can be particularly challenging. The current studyinvestigated whether a thermal nociceptive stimulus is useful for the evaluation of analgesic drugs in 2 amphibian species.The objectives of this study were 2-fold: 1) compare 2 models of nociception (thermal and mechanical) using 2 frog species; White’s Tree Frogs (Litoria caerulea; WTF) and Northern Leopard Frogs (Lithobates pipiens; NLF) after administration of saline or morphine sulfate; and 2) evaluate antinociceptive efficacy of morphine sulfate at 2 doses in a common amphibian research species, the NLF, using a mechanical stimulus. Neither WTF nor NLF displayed consistent drug-dependent changes in withdrawal responses to a noxious thermal stimulus applied using the Hargreaves apparatus, but NLF exposed to the noxiousmechanical stimulus demonstrated a significant dose-dependent antinociceptive response to morphine sulfate. These resultsindicate that morphine is not antinociceptive in WTF, supporting previously reported results, and demonstrate the importanceof using an appropriate experimental antinociceptive test in amphibians. Our data suggest that nociception in amphibianspecies may be best evaluated by using mechanical nociceptive models, although species differences must also be considered.

Analgesic Efficacy of Tramadol and Morphine in White’s Tree Frogs (Litoria caerulea)

Published data are sparse regarding the recognition of clinically relevant pain and appropriate analgesia in amphibians. The amphibian analgesia literature has primarily focused on nociceptive pathways in a single species, the northern leopard frog (Rana pipiens). The objective of the current study was to assess the analgesic efficacy and safety of oral tramadol and subcutaneous morphine in a commonly maintained zoo and pet species, White’s tree frog (Litoria caerulea). We hypothesized that tramadol and morphine would provide dose-dependent antinociception, as measured by significant increases in hindlimb withdrawal latency after exposure to a noxious thermal stimulus. Two randomized, placebo-controlled, complete crossover studies were performed, with tramadol (n = 12) administered at 15, 25, and 40 mg/kg PO and morphine (n = 12) administered at 5 and 10 mg/kg SC. Hindlimb withdrawal latency was measured for a maximum of 72 h. No adverse side effects or signs of sedation were observed with any dose or drug evaluated. No significant difference in withdrawal latency was detectedbetween the control and either tramadol or morphine. These negative results were surprising, suggesting that the thermalnociceptive model may not be biologically relevant in amphibian species.

Multiple trans-Torres Strait colonisations by tree frogs in the Litoria caerulea group, with the description of a new species from New Guinea

Australia and New Guinea (together referred to as Sahul) were linked by land for much of the late Tertiary and share many biotic elements. However, New Guinea is dominated by rainforest, and northern Australia by savannah. Resolving patterns of biotic interchange between these two regions is critical to understanding the expansion and contraction of both habitat types. The green tree frog (Litoria caerulea) has a vast range across northern and eastern Australia and New Guinea. An assessment of mitochondrial and morphological diversity in this nominal taxon in New Guinea reveals two taxa. True Litoria caerulea occurs in disjunct savannahs of the Trans-Fly, Central Province and across northern Australia, with very low genetic divergence, implying late Pleistocene connectivity. A previously unrecognised taxon is endemic to New Guinea and widespread in lowland swampy rainforest. Date estimates for the divergence of the new species suggest Pliocene connectivity across lowland tropical habitats of northern Australia and New Guinea. In contrast, the new species shows shallow phylogeographic structuring across the central mountains of New Guinea, implying recent dispersal between the northern and southern lowlands. These results emphasise that the extent and connectivity of lowland rainforest and savannah environments across northern Australia and southern New Guinea have undergone profound shifts since the late Pliocene. http://zoobank.org/urn:lsid:zoobank.org:pub:A577A415-0B71-4663-B4C1-7271B97298CD

Ancestral chytrid pathogen remains hypervirulent following its long coevolution with amphibian hosts

Many amphibian species around the world, except in Asia, suffer morbidity and mortality when infected by the emerging infectious pathogen Batrachochytrium dendrobatidis (Bd). A lineage of the amphibian chytrid fungus isolated from South Korean amphibians (BdAsia-1) is evolutionarily basal to recombinant global pandemic lineages (BdGPL) associated with worldwide amphibian population declines. In Asia, the Bd pathogen and its amphibian hosts have coevolved over 100 years or more. Thus, resilience of Asian amphibian populations to infection might result from attenuated virulence of endemic Bd lineages, evolved immunity to the pathogen or both. We compared susceptibilities of an Australasian amphibian, Litoria caerulea , known to lack resistance to BdGPL, with those of three Korean species, Bufo gargarizans , Bombina orientalis and Hyla japonica , after inoculation with BdAsia-1, BdGPL or a blank solution. Subjects became infected in all experimental treatments but Korean species rapidly cleared themselves of infection, regardless of Bd lineage. They survived with no apparent secondary effects. By contrast, L. caerulea , after infection by either BdAsia-1 or BdGPL, suffered deteriorating body condition and carried progressively higher Bd loads over time. Subsequently, most subjects died. Comparing their effects on L. caerulea , BdAsia-1 induced more rapid disease progression than BdGPL. The results suggest that genomic recombination with other lineages was not necessary for the ancestral Bd lineage to evolve hypervirulence over its long period of coevolution with amphibian hosts. The pathogen's virulence may have driven strong selection for immune responses in endemic Asian amphibian host species.

Discovery of Novel Caeridins from the Skin Secretion of the Australian White’s Tree Frog, Litoria caerulea

Abundant biologically active peptides have been discovered from frog skin secretions, a rich natural source of bioactive compounds with great potential in drug discovery. In this study, three Caeridin peptides, namely, Caeridin-1, S5-Caeridin-1, and Caeridin-a1, were discovered from the skin secretion of the Australian White’s tree frog, Litoria caerulea, for the first time, by means of combining transcriptomic and peptidomic analyses. It also represents the first report on bioactive Caeridins since this family of peptides was initially studied 20 years ago. Chemically synthetic versions of each natural Caeridin demonstrated promising bioactivities either on rat smooth muscles or against microbial growth. Specifically, Caeridin-1 produced contraction of rat bladder smooth muscle, while S5-Caeridin-1 induced relaxation of rat ileum smooth muscle, both at nanomolar concentrations. Moreover, Caeridin-a1 was shown to potently inhibit the growth of the planktonic Gram-positive bacteria Staphylococcus aureus (S. aureus), methicillin-resistant S. aureus (MRSA), and Enterococcus faecalis (E. faecalis), the Gram-negative bacterium, Escherichia coli (E. coli), and the yeast, Candida albicans (C. albicans). The discovery of these Caeridins may induce further intensive and systematic studies of frog skin peptides to promote the discovery of natural templates as lead compounds for drug discovery and therapeutic application.

Frogs host faecal bacteria typically associated with humans

Tree frogs commonly access drinking water tanks; this may have human health implications. Although amphibians might not be expected to host mammalian faecal indicator bacteria (FIB), it is possible that they may have human FIB on their skin after exposure to human waste. We collected faeces and skin wash from green tree frogs (Litoria caerulea) from a natural environment, a suburban site, and a suburban site near a creek occasionally contaminated with sewage effluent. We used molecular techniques to test for FIB that are routinely used to indicate human faecal contamination. Enterococci colonies were isolated from both faecal and skin wash samples, and specific markers (Enterococcus faecium and Bacteroides thetaiotaomicron) were found in frog faeces, demonstrating that these markers are not human- or mammalian-specific. Bacteroides thetaiotaomicron was detected in frogs from both natural and urban sites, but E. faecium was only associated with the sewage impacted site.