Effects of Environmentally Relevant Concentrations of Poultry Litter Associated Contaminates on the Sexual Development of Xenopus laevis

Poultry litter contains high levels of natural sex hormones, nitrogen, phosphorous, and trace amounts of heavy metals. Poultry litter runoff from poultry and farming operations in the Delmarva region can have serious impacts on frog development in the Chesapeake Bay Watershed. In this study, we investigated potential effects of litter compounds on Xenopus laevis development when exposed to environmental levels (0.35 and 0.70 g/L) of litter solution. We found that despite rapid hormone degradation, poultry litter solution still affected X. laevis development. Hormones were also more persistent in the lower poultry litter concentration, leading to even greater effects. Slowed growth and increased female gonadal abnormalities were observed after exposure to 0.35 g/L but not to 0.70 g/L of litter solution, and increased male gonadal abnormalities were observed after treatment to both litter concentrations. The developmental impacts examined in this study may have greater environmental impacts on frog reproduction and survival.

Time interval from ovulation to extrusion in female bullfrog in different photoperiods

It was analyzed in this work the influence of photoperiod on time interval from ovulation induction period to extrusion of ovocits in female bullfrogs (Lithobates catesbeianus). It was used 54 females reared from metamorphosis to 9 months of age under three photoperiods: dark time (DL 0:24), 16 hours of daylight (DL 16:8) and 12 hours of daylight (DL 12:12). Ovulation was induced by intramuscular application of two doses of LHRHa with 12 hours of interval between the injections. After 10, 25, 28, 31, 34 and 37 hours from the first hormone injection, 10-gram samples (3,000 eggs) were extracted from each female at each time interval and fertilized. Egg hatching rate was checked in each sample 72 hours after fertilization. Analysis of variance showed a significant effect of extrusion delay and the interaction between photoperiod and this delay. Extrusion should be carried out 33, 24 and 26 hours after the first hormone dosage in females reared in environments without light, with 12 hours of daylight and with 16 hours of daylight, respectively, to obtain the maximum fertilization rate.

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.

Carrying progeny on the back: reproduction in the Brazilian aquatic frog Pipa carvalhoi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Getting a kick out of it: multimodal signalling during male–male encounters in the foot-flagging frog Micrixalus aff. saxicola from the Western Ghats of India

Several anuran species use multimodal signals to communicate in diverse social contexts. Our study describes acoustic and visual behaviours of the Small Torrent Frog (Micrixalus aff. saxicola), a diurnal frog endemic to the Western Ghats of India. During agonistic interactions males display advertisement calls, foot-flagging and tapping (foot lifting) behaviours to signal the readiness to defend perching sites in perennial streams. Results from a quantitative video analysis of male–male interactions indicate that footflagging displays were used as directional signals toward the opponent male, but were less abundant than calls. The acoustic and visual signals were not functionally linked. The call of Micrixalus aff. saxicola thereby did not act as an alert signal. Analysis of behavioural transitions revealed that kicking behaviours (physical attacks) significantly elicited kicks from interacting males. We suggest that foot-flagging displays ritualized from this frequently observed fighting technique to reduce physical attacks.

Natural History of the Lutz`s Frog Cycloramphus lutzorum Heyer, 1983 (Anura: Cycloramphidae) in the Brazilian Atlantic Forest: Description of the Advertisement Call, Tadpole, and Karyotype

We describe the advertisement call, tadpole, karyotype, and additional information on the natural history of Cycloramphus lutzorum from southern Brazil. Sonograms were generated from digitally recorded calls. Tadpoles were collected in the field for description in the lab, and an adult was collected for karyotyping. Data on seasonal activity were gathered monthly from November 2005 to November 2007. All tadpoles (N = 21), juveniles (N = 18), and adults (N = 52) were found exclusively in streams. Reproduction, as identified by calling frogs, occurred from July through November. Frogs call all day long, but mostly at dusk, from rock crevices inside the stream edges near the splash zone. The call is short and loud, with 11 pulsed notes, of 491-641 ms, with a dominant frequency of 0.98-1.39 kHz. We describe the exotrophic and semiterrestrial tadpoles, always found in constantly humid vertical rock walls in the stream. Tadpoles of C. lutzorum are recognized by differences in labial tooth row formula, eye diameter, body shape, position of nares, and development of tail. Like congeneric species, the karyotype of C. lutzorum comprises 26 metacentric and submetacentric chromosomes. Cycloramphus lutzorum is restricted to and adapted for living in fast flowing streams, many of which are threatened by deforestation, pollution, and habitat loss. Therefore, we recommend the status of C. lutzorum be changed from its current ""Data Deficient"" to ""Near Threatened (NT)"" in the IUCN species red list.

The inguinal macroglands of the frog Physalaemus nattereri (Leptodactylidae): structure, toxic secretion and relationship with deimatic behaviour

Amphibian skin is characterized by the presence of mucous glands, related to cutaneous breathing, reproduction and water balance, and granular glands, related to the production of toxins used in defence. In some species the granular glands can form accumulations in certain regions of the body. This is the case for inguinal macroglands of the leptodactylid frog Physalaemus nattereri, where these structures form a pair of black discs associated with deimatic behaviour. The morphology of the inguinal macroglands and their secretion were studied in this species and correlated to deimatic behaviour. The inguinal macroglands are formed from elongated granular glands that, in contrast with the granular glands of the rest of the skin, have small spherical granules with a proteinic content. In the dermis of the whole body, except for the inguinal macroglands and the inguinal region, a well-developed calcified dermal layer is observed. During deimatic behaviour these macroglands discourage a potential predator from attacking, but if visual cues are insufficient and the predator persists in the attack, atoxic secretion is eliminated in its mouth. This elimination is favoured by the absence of a calcified dermal layer in the macroglands, which makes the dermal region softer than the rest of the dorsal skin.

Natural History of the Lutz's Frog Cycloramphus lutzorum Heyer, 1983 (Anura: Cycloramphidae) in the Brazilian Atlantic Forest: Description of the Advertisement Call, Tadpole, and Karyotype

We describe the advertisement call, tadpole, karyotype, and additional information on the natural history of Cycloramphus lutzorum from southern Brazil. Sonograms were generated from digitally recorded calls. Tadpoles were collected in the field for description in the lab, and an adult was collected for karyotyping. Data on seasonal activity were gathered monthly from November 2005 to November 2007. All tadpoles (N = 21), juveniles (N = 18), and adults (N = 52) were found exclusively in streams. Reproduction, as identified by calling frogs, occurred from July through November. Frogs call all day long, but mostly at dusk, from rock crevices inside the stream edges near the splash zone. The call is short and loud, with 11 pulsed notes, of 491-641 ms, with a dominant frequency of 0.98-1.39 kHz. We describe the exotrophic and semiterrestrial tadpoles, always found in constantly humid vertical rock walls in the stream. Tadpoles of C. lutzorum are recognized by differences in labial tooth row formula, eye diameter, body shape, position of nares, and development of tail. Like congeneric species, the karyotype of C. lutzorum comprises 26 metacentric and submetacentric chromosomes. Cycloramphus lutzorum is restricted to and adapted for living in fast flowing streams, many of which are threatened by deforestation, pollution, and habitat loss. Therefore, we recommend the status of C. lutzorum be changed from its current "Data Deficient" to "Near Threatened (NT)" in the IUCN species red list.

Kin-bias, breeding site selection and female fitness in a cannibalistic Neotropical frog

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

New records of the harlequin frog Atelopus nahumae RUIZ-CARRANZA, ARDILA-ROBAYO & HERNANDEZ-CAMACHO, 1994, in the Sierra Nevada de Santa Marta (Colombia), with notes of its distribution (Anura: Bufonidae)

The known distribution of the harlequin frog, Atelopus nahumae RUIZ-CARRANZA, ARDILA-ROBAYO & HERNANDEZ-CAMACHO, 1994, is reported along with three new locality records in the northwestern Sierra Nevada de Santa Marta (Colombia). In the new localities, observations were made on the frog's mating behavior. This report adds the Cordoba River Basin, another high valley of the Sierra Nevada de Santa Marta, to the known range of A. nahumae. Nevertheless, the general distribution of the species in this region is largely unknown.

Vocal and territorial behavior in the Smith frog (Hypsiboas faber): Relationships with plasma levels of corticosterone and testosterone

The possible trade-off between the roles of glucocorticoids as facilitators of energy substrate mobilization and neural inhibitors of sexual behavior during breeding season is under debate. We studied the relationship between calling and territorial behavior with plasma levels of corticosterone (CORT) and plasma levels of testosterone (T) across the breeding season of Hypsiboas faber, a large and territorial Neotropical treefrog. We investigated these relationships through focal observations of males calling naturally, followed by blood sampling for hormonal radioimmunoassay. We additionally used an experimental approach, which consisted of broadcasting recorded advertisement calls for 10 min to simulate an invasion in the territory of the focal subjects, followed by behavioral observation and blood sampling for hormonal radioimmunoassay. Results showed a pattern of co-variation between CORT and T across the breeding season. Furthermore, individual variation in CORT and T was related to different aspects of behavior: individuals with higher CORT showed higher calling rates, and individuals with higher steroid levels, mainly T, showed higher responsivity to social stimulation by other males in the chorus. Experimental simulation of territorial intrusion by using playback of advertisement calls of this species did not elicit consistent changes in agonistic behavior and CORT, but decreased T in focal males. (C) 2012 Elsevier Inc. All rights reserved.

Documenting reproduction and inequality : revisiting Jean Anyon’s “Social class and school knowledge”

Jean Anyon’s (1981) “Social class and school knowledge” was a landmark work in North American educational research. It provided a richly detailed qualitative description of differential, social-class-based constructions of knowledge and epistemological stance. This essay situates Anyon’s work in two parallel traditions of critical educational research: the sociology of the curriculum and classroom interaction and discourse analysis. It argues for the renewed importance of both quantitative and qualitative research on social reproduction and equity in the current policy context.

Reproduction, mediation, and experience : virtual reality, motion capture and early modern theatre

This article investigates virtual reality representations of performance in London’s late sixteenth-century Rose Theatre, a venue that, by means of current technology, can once again challenge perceptions of space, performance, and memory. The VR model of The Rose represents a virtual recreation of this venue in as much detail as possible and attempts to recover graphic demonstrations of the trace memories of the performance modes of the day. The VR model is based on accurate archeological and theatre historical records and is easy to navigate. The introduction of human figures onto The Rose’s stage via motion capture allows us to explore the relationships between space, actor and environment. The combination of venue and actors facilitates a new way of thinking about how the work of early modern playwrights can be stored and recalled. This virtual theatre is thus activated to intersect productively with contemporary studies in performance; as such, our paper provides a perspective on and embodiment of the relation between technology, memory and experience. It is, at its simplest, a useful archiving project for theatrical history, but it is directly relevant to contemporary performance practice as well. Further, it reflects upon how technology and ‘re-enactments’ of sorts mediate the way in which knowledge and experience are transferred, and even what may be considered ‘knowledge.’ Our work provides opportunities to begin addressing what such intermedial confrontations might produce for ‘remembering, experiencing, thinking and imagining.’ We contend that these confrontations will enhance live theatre performance rather than impeding or disrupting contemporary performance practice. Our ‘paper’ is in the form of a video which covers the intellectual contribution while also permitting a demonstration of the interventions we are discussing.