Pseudophryne Coriacea Research Articles

Cell culture and karyotypic description of Pseudophrynecoriacea (Keferstein, 1868) (Amphibia, Anura) from the New South Wales Central Coast.

The karyotype of the IUCN least concern red-backed toadlet Pseudophryne (P.) coriacea (Keferstein, 1868) from the New South Wales Central Coast is described following tissue culture of toe clipping macerates and conventional DAPI staining. The diploid number is 2n = 24. The karyotype is represented by six large and five small chromosomal pairs and one very small chromosomal pair. The very small chromosome 12 is 12% the size of chromosome 1. One of the large chromosomes is subtelocentric, two of the large chromosomes are submetacentric and the remaining chromosomes are metacentric. The putative nucleolus organiser region (NOR) is observed on chromosome 4. The diploid number and location of the putative NOR correlates to that of the previously published IUCN critically endangered P.corroboree (Moore 1953) and unpublished descriptions of the P.coriacea karyotype. This is the first described cell culture of a species from the genus Pseudophryne Fitzinger, 1843, first published analysis of the P.coriacea karyotype and the first published analysis of centromeric allocation of this genus. Globally there exists a large inventory of tissue samples in cryobanks that are not associated with known recovery mechanisms such as basic cell culture techniques. Detailed cytogenetic analyses of these cryobanked samples are therefore not possible. This work therefore enables: (i) a comparison of the P.coriacea karyotype with that of the critically endangered P.corroboree and (ii) a benchmark for repeat and future cytogenetic and genomic analyses of cryostored samples of this genus.

Sex differences in response to environmental and social breeding cues in an amphibian

Abstract The relative influence of climatic and social factors on sex-specific variation in reproductive behaviour remains poorly understood. Here, we examine the influence of multiple climatic cues in combination with a social cue on the reproductive behaviours of males and females in a terrestrial breeding toadlet (Pseudophryne coriacea). Over a 115-day breeding season, arrival patterns of each sex, and male calling activity, were recorded daily, while climatic variables were logged continuously. Multivariate analysis showed that arrival of males at the breeding site, as well as male nightly calling activity, were most strongly influenced by a climatic variable (rainfall). By contrast, female arrival was strongly correlated with a social variable (male calling activity), with abiotic conditions having no influence, other than a moderate influence of lunar phase (lunar illumination). These results suggest that cues used for breeding are sex specific and provide new evidence that combinations of climatic and social cues can be integrated into breeding decisions.

Evidence that genetic compatibility underpins female mate choice in a monandrous amphibian.

Mate choice for genetic benefits remains controversial, largely because few studies have estimated the relative contributions of additive and non-additive sources of genetic variation to offspring fitness. Moreover, there remains a deficit of these estimates for species where female-mate preferences have been quantified in the wild, especially species characterized by monandry or monogamy. Here, we use artificial fertilization techniques combined with a cross-classified breeding design to simultaneously test for "good genes" and "compatible genes" benefits of mate choice in the monandrous red backed toadlet (Pseudophryne coriacea). In addition, we used a genomic approach to estimate effects of parental-genetic relatedness (assessed using 27, 768 single nucleotide polymorphisms) on offspring fitness. Our results revealed no significant additive genetic effects (sire effects), but highly significant non-additive genetic effects (sire × dam interaction effects), on fertilization success, survival during embryonic development, and hatching success. We also found significant associations between parental genetic similarity and offspring survival (whereby survival was higher when parents were more related), and significant positive relationships between fertilization success and embryo survival through to hatching. These results indicate that offspring viability is significantly influenced by the genetic compatibility of parental genotypes, that more related parents are more genetically compatible, and that gametes with greater compatibility at fertilization produce more viable offspring. More broadly, our findings provide new quantitative genetic evidence that genetic incompatibility underpins female mate preferences. Continued quantitative genetic assessment of the relative importance of good genes versus compatible genes is needed to ascertain the general importance of genetic benefits as a driver of female mate choice.

Hormone-induced ovulation and artificial fertilisation in four terrestrial-breeding anurans.

The development and application of reproductive technologies has great potential to enhance the conservation management of threatened amphibians globally. The present study quantified the efficacy of protocols previously developed for Pseudophryne guentheri for hormonally inducing egg release and artificial fertilisation in three additional terrestrial-breeding species of Australian ground frog; namely Pseudophryne bibronii , Pseudophryne coriacea and Heleioporus eyrei . Females of each species were administered a priming dose of 0.4µg g-1 of gonadotropin-releasing hormone analogue (GnRHa), followed by an ovulatory dose of 2µg g-1 GnRHa. Egg release was achieved in all species (response 94.7-100%), but early embryo survival differed significantly, ranging from 23.8% to 89.3%. Overall, the protocols were considered effective in P. guentheri , P. bibronii and P. coriacea , but further protocol refinement is required for H. eyrei .

Nest site selection in a terrestrial breeding frog: interrelationships between nest moisture, pH and male advertisement

In resource defence mating systems where males control nest sites, complex interrelationships between nest characteristics and nest advertisement are likely to influence female mate choice. Surprisingly, however, we know little about the influence of such relationships on female mating decisions. In terrestrial-breeding oviparous species, one character likely to have a major influence on female nest site selection is nest moisture. In addition to influencing the susceptibility of eggs to desiccation, nest moisture also stands to influence nest pH and offspring fitness, as well as the hydration state of resident males, and their potential to advertise. Together, such interrelationships could strongly influence female mating decisions. The aim of this study was to use a long-term field study combined with a multivariate approach to investigate the combined influence of nest moisture, nest pH and male acoustic advertisement on female nest site selection in a wild population of red-backed toadlets, Pseudophryne coriacea. We found that females deposited more eggs in wetter and less acidic nests, and that these variables were positively correlated. Moreover, nest moisture and pH in combination were positively related to the calling potential of resident males, and nests that were advertised more often received more eggs. These results suggest that nest site selection in red-backed toadlets is influenced by a complex interplay between covarying nest characteristics that together influence male sexual signalling and nest advertisement. More broadly our findings highlight the potential for nesting behaviour to be mechanistically intertwined with mate choice.

Female choice for related males in wild red-backed toadlets (Pseudophryne coriacea)

AbstractMate choice for genetic benefits is assumed to be widespread in nature, yet very few studies have comprehensively examined relationships between female mate choice and male genetic quality in wild populations. Here, we use exhaustive sampling and single nucleotide polymorphisms to provide a partial test of the “good genes as heterozygosity” hypothesis and the “genetic compatibility” hypothesis in an entire population of terrestrial breeding red-backed toadlets, Pseudophryne coriacea. We found that successful males did not display higher heterozygosity, despite a positive relationship between male heterozygosity and offspring heterozygosity. Rather, in the larger of 2 breeding events, we found that successful males were more genetically similar to their mate than expected under random mating, indicating that females can use pre- or post-copulatory mate choice mechanisms to bias paternity toward more related males. These findings provide no support for the good genes as heterozygosity hypothesis but lend support to the genetic compatibility hypothesis. A complete test of this hypothesis will now require evaluating how parental genetic similarity impacts offspring fitness. Terrestrial toadlets show a high degree of site fidelity, high levels of genetic structuring between populations, and frequently hybridize with sister species. As such, female mate choice for related males may be an adaptive strategy to reduce outbreeding depression. Our findings provide the first population-wide evidence for non-random preferential inbreeding in a wild amphibian. We argue that such reproductive patterns may be common in amphibians because extreme genetic differentiation within meta-populations creates an inherently high risk of outbreeding depression.

The unexpected genetic mating system of the red-backed toadlet (Pseudophryne coriacea): A species with prolonged terrestrial breeding and cryptic reproductive behaviour.

Molecular technologies have revolutionized our classification of animal mating systems, yet we still know very little about the genetic mating systems of many vertebrate groups. It is widely believed that anuran amphibians have the highest reproductive diversity of all vertebrates, yet genetic mating systems have been studied in <1% of all described species. Here, we use single nucleotide polymorphisms to quantify the genetic mating system of the terrestrial breeding red-backed toadlet Pseudophryne coriacea. In this species, breeding is prolonged (approximately 5months), and males construct subterranean nests in which females deposit eggs. We predicted that females would display extreme sequential polyandry because this mating system has been reported in a closely related species (P.bibronii). Parentage analysis revealed that mating success was heavily skewed towards a subset of males (30.6% of potential sires) and that nearly all females (92.6%) mated with one male. In a high percentage of occupied nests (37.1%), the resident male was not the genetic sire, and very few nests (4.3%) contained clutches with multiple paternity. Unexpectedly, these results show that sequential polyandry is rare. They also show that there is a high frequency of nest takeover and extreme competition between males for nest sites, but that males rarely sneak matings. Genetic analysis also revealed introgressive hybridization between P.coriacea and the red-crowned toadlet (Pseudophryne australis). Our study demonstrates a high level of mating system complexity, and it shows that closely related anurans can vary dramatically in their genetic mating system.

Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans.

Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used successfully in screening for the presence of BIV nucleic acid in digested formalin-fixed, paraffin-embedded amphibian tissues. Juvenile frogs were more susceptible to BIV than adults. In experimental challenges and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopathological changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. alboguttata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to > 100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy. Ranaviruses could be having an impact on Australian herpetofauna.

The chemistry of cyclic tautomers of tryptophan: Total synthesis of (+)-(ent)-pseudophrynaminol

A diastereoselective synthesis of the unnatural (+)-enantiomer of the hexahydropyrroloindole alkaloid (-)-pseudophrynaminol ( 3), isolated from the skin of the Australian frog Pseudophryne coriacea, from a cyclic tautomer ( 1) of l-tryptophan, is described. The CD spectrum of the synthetic 3 is opposite in sign to that of the natural material enabling the configuration of the natural product to be established as 3aS,8aR.

Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea

1. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryine coriacea (PsC) have been studied in rat superior cervical ganglia at 37°C. 2. PsC (50 mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at 10–20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical ISSD sequence. 3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms. 4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage-dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced. 5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.

Epileptogenic effects of skin extracts from the Australian frog Pseudophryne coriacea after intracerebral microinfusion in rats

The behavioural and electrocortical (ECoG) effects induced by a methanol extract of the skin of the Australian frog Pseudophryne coriacea, directly microinjected into several areas of the brain, were studied in freely moving rats. Administration of the P. coriacea extract (5, 10, 15 and 20 μg) into the dorsal hippocampus produced a dose-dependent and reversible behavioural stimulation and ECoG spikes lasting 20–140 min. Similar but less intense effects were elicited in rats receiving injections into the III cerebral ventricle, amygdala and caudate nucleus. In conclusion, the present experiments show that the skin extract of P. coriacea produces behavioural stimulation and ECoG spikes when injected into the rat brain, the most sensitive area being the hippocampus.

Electrophysiological effects of a pumiliotoxin-B-like alkaloid derived from the skin of the Australian frog Pseudophryne coriacea

Skin extracts of the Australian frog Pseudophryne coriacea (PsC) potentiate and prolong the bioelectric activity of various excitable tissues. When studied by electrophysiological means in a preparation of mouse diaphragm, PsC skin extracts did not affect the spontaneous acetylcholine (ACh) release. However, the indirect stimulation of the preparation in the presence of PsC skin extracts gave rise to a different profile of rhythmic activity showing afterpotentials and variable oscillatory activity. The action potential and the total sodium current recorded in the muscle fibre with the loose patch clamp method were not modified significantly by the alkaloid. Concentrations of PsC skin extract that did not cause repetitive activity, seemed to reduce slightly the quantal content of the evoked release of ACh. The resting potential of muscle fibres was not modified even by the highest PsC skin extract concentrations. These results suggest that the facilitation effects of PsC skin extracts could be due to intracellular mechanisms probably related to the control of the cytosolic calcium concentrations or to an increased excitability of the presynaptic biomembranes.

Alkaloids from Australian Frogs (Myobatrachidae): Pseudophrynamines and Pumiliotoxins

Australian frogs of the genus Pseudophryne contain two distinct classes of alkaloids. The pseudophrynamine class (3a-prenyl pyrrolo[2,3-b]indoles) are unique to this genus of frogs of the family Myobatrachidae, while the pumiliotoxin-A class (8-hydroxy-8-methyl-6-alkylidene-1-azabicyclo[4.3.0]nonanes) also occur in dendrobatid frogs of the genera Dendrobates, Epipedobates, and Minyobates, in ranid frogs of the genus Mantella, and in bufonid toads of the genus Melanophryniscus. All seven species of Pseudophryne examined contain both classes of alkaloids. The pseudophrynamines were the predominant class in both species (Pseudophryne guentheri and Pseudophryne occidentalis) from Western Australia, while all of the eastern species (Pseudophryne australis, Pseudophryne bibronii, Pseudophryne coriacea, Pseudophryne corroboree, and Pseudophryne semimarmorata) contained significant amounts of both pseudophrynamines and pumiliotoxins. Pumiliotoxins, in particular pumiliotoxin B, were predominant in two eastern species (P. australis and a southern population of P. corroboree), while pseudophrynamines were dominant in P. bibronii, four of six populations of P. coriacea, one population of P. semimarmorata, and a northern population of P. corroboree. Structures are proposed for several new alkaloids of the pseudophrynamine class.

Total synthesis of (±) pseudophrynamine A

The first total synthesis of (±) Pseudophrynamine A, a prenyl pyrrolo [2,3-b]indole ester recently isolated from the Australian frog “Pseudophryne coriacea”, is reported.

Total synthesis of ( [formula omitted]) pseudophrynaminol

The first synthesis of ( + ) pseudophrynaminol, a unique pyrrolo [2,3b] indole recently isolated from the Australian frog Pseudophryne coriacea, is described.

Facilitation of [ 3H]acetylcholine and [ 3H]5-hydroxytryptamine release from rat cerebral cortex synaptosomes by a factor extracted from the skin of the Australian frog Pseudophryne coriacea

A partly purified extract of the skin of the Australian frog Pseudophryne coriacea (PsC) evoked the release of [ 3H]acetylcholine ([ 3H]ACh) and of [ 3H]5-hydroxytryptamine ([ 3H]5-HT) from superfused rat cerebral cortex synaptosomes prelabeled with [ 3H]choline or [ 3H]5-HT, respectively. The PsC-evoked release of both transmitters was sensitive to tetrodotoxin and was strictly Ca 2+-dependent. The release of [ 3H]5-HT caused by PsC was unaffected by the 5-HT uptake inhibitor citalopram. Activation of muscarinic autoreceptors by ACh or of serotonin autoreceptors by 5-HT depressed the PsC-evoked release of [ 3H]ACh or of [ 3H]5-HT, respectively. It is concluded that PsC elicits a Ca 2+-dependent exocytotic-like transmitter release, possibly by opening Na + channels in the presynaptic membrane.

Pumiliotoxin B-like alkaloid in extracts of the skin of the Australian myobatrachid frog Pseudophryne coriacea: effects on the systemic blood pressure of experimental animals and the rat heart.

Semi-purified extracts of the skin of the Australian myobatrachid frog Pseudophryne coriacea (PS)displayed striking, reversible and, in part, dose-dependent effects on the systemic blood pressure of the rat and other experimental animals, as well as on the rat heart. The blood pressure response in the rat consisted typically in an abrupt, short-lasting fall, followed by a conspicuous, more persistent rise. The fall in pressure was abolished by atropine and potentiated by physostigmine, indicating a cholinergic mechanism; rise was abolished by prazosin and guanethidine, suggesting a release of catecholamines from adrenergic nerve terminals in the vasculature. Tachyphylaxis was the obvious result of exhaustion of stores of catecholamines. On the heart, as shown in the electrocardiographic tracings, PS produced a variety of rhythm disorders, attributable both to the release of aminergic transmitters and to a direct effect on the myocardium. Whereas the pressure and electrocardiographic responses were virtually unaffected by calcium channel antagonists and agonists, all the effects of PS were sharply reduced or completely abolished by the sodium channel blocker tetrodotoxin. This suggests that activation of sodium channels may play a role in the mechanism of action of PS. However, the exact nature of ionic flux(es)influenced by the extract of frog skin remains to be established.

ChemInform Abstract: A Naturally Occurring Erythro Diastereomer (Ic) of Pumiliotoxin B (Ia).

AbstractSix different populations of the myobatrachid frog Pseudophryne coriacea collected in Australia in 1987 are examined.

A naturally occurring erythro diastereomer of pumiliotoxin b

The 15R,16S erythro diastereomer of pumiliotoxin B has been discovered in the skin of an Australian myobatrachid frog, Pseudophryne coriacea .

A potent factor in extracts of the skin of the Australian frog, PSeudophryne coriacea—III : Potentiation of contractions elicited in avian and mammalian isolated skeletal muscle preparations by direct and indirect electrical stimulation

Extracts of the skin of the Australian frog Pseudophryne coriacea displayed a striking potentiating effect on contractions evoked in isolated skeletal muscle preparations of mammals (phrenic nerve diaphragm) and birds (chick biventer cervicis and semispinalis muscles) by indirect and direct electrical stimulation. There was both a conspicuous increase in the amplitude of the twitch, up to 10-fold, and a remarkable prolongation of the duration of the twitch. The effect was dose- and frequency-dependent. In the presence of the extract, fusion of twitches after tetanic stimulation occurred earlier. No tachyphylaxis upon repeated stimulation by the extract was observed and the response to large doses persisted, declining slowly, for hours. These effects must be ascribed to an alkaloid related in structure to pumiliotoxin B. Response to the extract of Pseudophryne coriacea by indirectly-stimulated preparations was potentiated by physostigmine and blocked by tubocurarine and α-bungarotoxin, demonstrating that in these preparations the extract acted pre-synaptically to facilitate the release of acetylcholine from motor nerve endings. However, the extract of Pseudophryne coriacea displayed equally potent effects in directly stimulated preparations, insensitive to physostigmine and to blockers of nicotinic acetylcholine receptors, indicating a direct action on the skeletal muscle. It is suggested that, like pumiliotoxin B, the Pseudophryne coriacea alkaloid may interfere in the regulation of calcium channels in both nerve and muscle fibres.