Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.

The crude skin secretion of the pepper frog leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

A hard slog, not a leap frog : globalization and sustainability transitions in developing Asia

How does globalization influence transitions toward more sustainable socio-technical regimes in the developing world? This paper argues that transformations of regimes, the networks and institutions governing technological and environmental practices in an industry, can be positively influenced by globalization but it depends on how global forces interact with local socio-political landscapes-the political-economic institutions, values, and regulations broadly guiding an economy and its relationship to the environment. We evaluate these relationships through a comparison of two kinds of socio-political landscapes-the neo-liberal export-led development model commonly found in the developing world and the uniquely Asian capitalist developmental state. We first show how the neo-liberal model overemphasizes the power of market forces to facilitate upgrading and more sustainable industrialization. We then argue that capitalist developmental states in East and Southeast Asia have been better able to harness global economic forces for technological and sustainability transitions through an openness to trade and investment and effective public-private institutions able to link cleaner technologies and environmental standards to production activities in firms. We buttress this argument with firm-level evidence showing the evolution of socio-technical regimes in two industries-cement and electronics. The case studies demonstrate how interactions with OECD firms can contribute to environmental technique effects provided the socio-political landscape is amenable to changes in an industry's regime. Ultimately, we find the process of transition to be complex and contingent; a hard slog not a leap frog toward a potentially more sustainable future. We close by considering the limitations on the capitalist developmental state model and with comments about what else needs to be learned about globalization's role in sustainability transitions.

Image processing and classification procedure for the analysis of Australian frog vocalisations

Frog species have been declining worldwide at unprecedented rates in the past decades. There are many reasons for this decline including pollution, habitat loss, and invasive species [1]. To preserve, protect, and restore frog biodiversity, it is important to monitor and assess frog species. In this paper, a novel method using image processing techniques for analyzing Australian frog vocalisations is proposed. An FFT is applied to audio data to produce a spectrogram. Then, acoustic events are detected and isolated into corresponding segments through image processing techniques applied to the spectrogram. For each segment, spectral peak tracks are extracted with selected seeds and a region growing technique is utilised to obtain the contour of each frog vocalisation. Based on spectral peak tracks and the contour of each frog vocalisation, six feature sets are extracted. Principal component analysis reduces each feature set down to six principal components which are tested for classification performance with a k-nearest neighbor classifier. This experiment tests the proposed method of classification on fourteen frog species which are geographically well distributed throughout Queensland, Australia. The experimental results show that the best average classification accuracy for the fourteen frog species can be up to 87%.

Acoustic feature extraction using perceptual wavelet packet decomposition for frog call classification

Frog protection has become increasingly essential due to the rapid decline of its biodiversity. Therefore, it is valuable to develop new methods for studying this biodiversity. In this paper, a novel feature extraction method is proposed based on perceptual wavelet packet decomposition for classifying frog calls in noisy environments. Pre-processing and syllable segmentation are first applied to the frog call. Then, a spectral peak track is extracted from each syllable if possible. Track duration, dominant frequency and oscillation rate are directly extracted from the track. With k-means clustering algorithm, the calculated dominant frequency of all frog species is clustered into k parts, which produce a frequency scale for wavelet packet decomposition. Based on the adaptive frequency scale, wavelet packet decomposition is applied to the frog calls. Using the wavelet packet decomposition coefficients, a new feature set named perceptual wavelet packet decomposition sub-band cepstral coefficients is extracted. Finally, a k-nearest neighbour (k-NN) classifier is used for the classification. The experiment results show that the proposed features can achieve an average classification accuracy of 97.45% which outperforms syllable features (86.87%) and Mel-frequency cepstral coefficients (MFCCs) feature (90.80%).

Application of image processing techniques for frog call classification

Frogs have received increasing attention due to their effectiveness for indicating the environment change. Therefore, it is important to monitor and assess frogs. With the development of sensor techniques, large volumes of audio data (including frog calls) have been collected and need to be analysed. After transforming the audio data into its spectrogram representation using short-time Fourier transform, the visual inspection of this representation motivates us to use image processing techniques for analysing audio data. Applying acoustic event detection (AED) method to spectrograms, acoustic events are firstly detected from which ridges are extracted. Three feature sets, Mel-frequency cepstral coefficients (MFCCs), AED feature set and ridge feature set, are then used for frog call classification with a support vector machine classifier. Fifteen frog species widely spread in Queensland, Australia, are selected to evaluate the proposed method. The experimental results show that ridge feature set can achieve an average classification accuracy of 74.73% which outperforms the MFCCs (38.99%) and AED feature set (67.78%).

Adaptive frequency scaled wavelet packet decomposition for frog call classification

Environmental changes have put great pressure on biological systems leading to the rapid decline of biodiversity. To monitor this change and protect biodiversity, animal vocalizations have been widely explored by the aid of deploying acoustic sensors in the field. Consequently, large volumes of acoustic data are collected. However, traditional manual methods that require ecologists to physically visit sites to collect biodiversity data are both costly and time consuming. Therefore it is essential to develop new semi-automated and automated methods to identify species in automated audio recordings. In this study, a novel feature extraction method based on wavelet packet decomposition is proposed for frog call classification. After syllable segmentation, the advertisement call of each frog syllable is represented by a spectral peak track, from which track duration, dominant frequency and oscillation rate are calculated. Then, a k-means clustering algorithm is applied to the dominant frequency, and the centroids of clustering results are used to generate the frequency scale for wavelet packet decomposition (WPD). Next, a new feature set named adaptive frequency scaled wavelet packet decomposition sub-band cepstral coefficients is extracted by performing WPD on the windowed frog calls. Furthermore, the statistics of all feature vectors over each windowed signal are calculated for producing the final feature set. Finally, two well-known classifiers, a k-nearest neighbour classifier and a support vector machine classifier, are used for classification. In our experiments, we use two different datasets from Queensland, Australia (18 frog species from commercial recordings and field recordings of 8 frog species from James Cook University recordings). The weighted classification accuracy with our proposed method is 99.5% and 97.4% for 18 frog species and 8 frog species respectively, which outperforms all other comparable methods.

AITPM Email Newsletter Volume 0905, June 2009 : President's Message

President’s Message Hello fellow AITPM members, Due to three colliding forces of nature I find myself writing this month’s message from home – today, I am still getting over a persistent virus that seemed to set in just after returning from Singapore a couple of weeks ago, which my diabetes won’t let me get away with too easily (no Kermit the Frog swine flu jokes please). Combine this with a very wet day in Brisbane – in fact the wettest for 20 years (how can we complain, except for flash flooding?). And in Queensland today is a state school teachers’ strike, so one half of our brood is over watching TV. Family snapshots aside, the biggest news for our industry of late is the $8.5 billion announced in the Federal Budget for transport infrastructure projects; many “shovel ready”, but some – and fortunate for our profession – desktop ready. This newsletter provides nationwide coverage on the transport infrastructure aspects of the Federal Budget. We’ll need a bit more time to carefully look at the ensuing State Budgets’ announcements. Regarding the federal budget announcements, I am pleased to see serious attention being paid to upgrading the M1 system – I hope to see a motorway standard facility connecting Adelaide to at least Rockhampton in my lifetime. But some other important roads are of course missing out in this particular budget. Various levels of commitment are being made to urban passenger rail – some project significant while others planning significant. Enhancement of suburban rail is important across the capitals and Australia’s medium sized cities such as the Gold Coast and Newcastle. Not much on road safety initiatives jumped out at me, but I believe it is implied in the large road projects and in some of the detail elsewhere. I do believe it’s about time a ‘Vision Zero’ style policy is adopted at the National level, since any death is unacceptable on the road, just as it is in any other workplace. So, overall some good news on building transport infrastructure to keep the economy purring during this recession, and strongly supporting it during future boom times. The other edge to the sword, of course, is that we tax payers will be paying a considerable amount for borrowings for these projects over a long period of time. I close again in reminding everyone again that AITPM’s flagship event, the 2009 AITPM National Conference, Traffic Beyond Tomorrow, is being held in Adelaide from 5 to 7 August. www.aitpm.com has all of the details about how to register, sponsor a booth, session, etc. Best regards all, Jon Bunker

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

Effects of different surfaces on the perception of prey-generated noise by the Indian false vampire bat Megaderma lyra.

The low- and high-frequency components of a rustling sound, created when prey (freshly killed frog) was jerkily pulled on dry and wet sandy floors and asbestos, were recorded and played back to individual Indian false vampire bats (Megaderma lyra). Megaderma lyra responded with flight toward the speakers and captured dead frogs, that were kept as reward. The spectral peaks were at 8.6, 7.1 and 6.8 kHz for the low-frequency components of the sounds created at the dry, asbestos and wet floors, respectively. The spectral peaks for the high-frequency sounds created on the respective floors were at 36.8,27.2 and 23.3 kHz. The sound from the dry floor was more intense than that of from the other two substrata. Prey movements that generated sonic or ultrasonic sounds were both sufficient and necessary for the bats to detect and capture prey. The number of successful prey captures was significantly greater for the dry floor sound, especially to its high-frequency components. Bat-responses were low to the wet floor and moderate to the asbestos floor sounds. The bats did not respond to the sound of unrecorded parts of the tape. Even though the bats flew toward the speakers when the prey generated sounds were played back and captured the dead frogs we cannot rule out the possibility of M. lyra using echolocation to localize prey. However, the study indicates that prey that move on dry sandy floor are more vulnerable to predation by M. lyra.

Detection of anuran calling activity in long field recordings for bio-acoustic monitoring

This paper presents a system to analyze long field recordings with low signal-to-noise ratio (SNR) for bio-acoustic monitoring. A method based on spectral peak track, Shannon entropy, harmonic structure and oscillation structure is proposed to automatically detect anuran (frog) calling activity. Gaussian mixture model (GMM) is introduced for modelling those features. Four anuran species widespread in Queensland, Australia, are selected to evaluate the proposed system. A visualization method based on extracted indices is employed for detection of anuran calling activity which achieves high accuracy.

Acoustic classification of Australian anurans using syllable features

Acoustic classification of anurans (frogs) has received increasing attention for its promising application in biological and environment studies. In this study, a novel feature extraction method for frog call classification is presented based on the analysis of spectrograms. The frog calls are first automatically segmented into syllables. Then, spectral peak tracks are extracted to separate desired signal (frog calls) from background noise. The spectral peak tracks are used to extract various syllable features, including: syllable duration, dominant frequency, oscillation rate, frequency modulation, and energy modulation. Finally, a k-nearest neighbor classifier is used for classifying frog calls based on the results of principal component analysis. The experiment results show that syllable features can achieve an average classification accuracy of 90.5% which outperforms Mel-frequency cepstral coefficients features (79.0%).

Acoustic features for multi-level classification of Australian frogs

Over past few decades, frog species have been experiencing dramatic decline around the world. The reason for this decline includes habitat loss, invasive species, climate change and so on. To better know the status of frog species, classifying frogs has become increasingly important. In this study, acoustic features are investigated for multi-level classification of Australian frogs: family, genus and species, including three families, eleven genera and eighty five species which are collected from Queensland, Australia. For each frog species, six instances are selected from which ten acoustic features are calculated. Then, the multicollinearity between ten features are studied for selecting non-correlated features for subsequent analysis. A decision tree (DT) classifier is used to visually and explicitly determine which acoustic features are relatively important for classifying family, which for genus, and which for species. Finally, a weighted support vector machines (SVMs) classifier is used for the multi- level classification with three most important acoustic features respectively. Our experiment results indicate that using different acoustic feature sets can successfully classify frogs at different levels and the average classification accuracy can be up to 85.6%, 86.1% and 56.2% for family, genus and species respectively.