Some aspects of the hypothalamic and pituitary development, metamorphosis, and reproductive behavior as studied in amphibians.

In this review article, information about the development of the hypothalamo-hypophyseal axis, endocrine control of metamorphosis, and hormonal and pheromonal involvements in reproductive behavior in some amphibian species is assembled from the works conducted mainly by our research group. The hypothalamic and pituitary development was studied using Bufo embryos and larvae. The primordium of the epithelial hypophysis originates at the anterior neural ridge and migrates underneath the brain to form a Rathke's pouch-like structure. The hypothalamo-hypophyseal axis develops under the influence of thyroid hormone (TH). For the morphological and functional development of the median eminence, which is a key structure in the transport of regulatory hormones to the pituitary, contact of the adenohypophysis with the undeveloped median eminence is necessary. For the development of proopiomelanocortin-producing cells, contact of the pituitary primordium with the infundibulum is required. The significance of avascularization in terms of the function of the intermediate lobe of the pituitary was evidenced with transgenic Xenopus frogs expressing a vascular endothelial growth factor in melanotropes. Metamorphosis progresses via the interaction of TH, adrenal corticosteroids, and prolactin (PRL). We emphasize that PRL has a dual role: modulation of the speed of metamorphic changes and functional development of organs for adult life. A brief description about a novel type of PRL (1B) that was detected was made. A possible reason why the main hypothalamic factor that stimulates the release of thyrotropin is not thyrotropin-releasing hormone, but corticotropin-releasing factor is considered in light of the fact that amphibians are poikilotherms. As regards the reproductive behavior in amphibians, studies were focused on the courtship behavior of the newt, Cynops pyrrhogaster. Male newts exhibit a unique courtship behavior toward sexually developed conspecific females. Hormonal interactions eliciting this behavior and hormonal control of the courtship pheromone secretion are discussed on the basis of our experimental results.

Inter-amphibian predation in the Early Cretaceous of China.

For most fossil taxa, dietary inference relies primarily on indirect evidence from jaw morphology and the dentition. In rare cases, however, preserved gut contents provide direct evidence of feeding strategy and species interaction. This is important in the reconstruction of food webs and energy flow through ancient ecosystems. The Early Cretaceous Chinese Jehol Biota has yielded several such examples, with lizards, birds, small dinosaurs, and mammals as both predator and prey. Here we describe an Early Cretaceous fossil frog specimen, genus Genibatrachus, that contains an adult salamander within its body cavity. The salamander is attributed to the hynobiid-like genus Nuominerpeton. The salamander skeleton is complete and articulated, suggesting it was caught and swallowed shortly before the frog itself died and was buried.

How the Human Brain Represents Perceived Dangerousness or "Predacity" of Animals.

UNLABELLED: Common or folk knowledge about animals is dominated by three dimensions: (1) level of cognitive complexity or "animacy;" (2) dangerousness or "predacity;" and (3) size. We investigated the neural basis of the perceived dangerousness or aggressiveness of animals, which we refer to more generally as "perception of threat." Using functional magnetic resonance imaging (fMRI), we analyzed neural activity evoked by viewing images of animal categories that spanned the dissociable semantic dimensions of threat and taxonomic class. The results reveal a distributed network for perception of threat extending along the right superior temporal sulcus. We compared neural representational spaces with target representational spaces based on behavioral judgments and a computational model of early vision and found a processing pathway in which perceived threat emerges as a dominant dimension: whereas visual features predominate in early visual cortex and taxonomy in lateral occipital and ventral temporal cortices, these dimensions fall away progressively from posterior to anterior temporal cortices, leaving threat as the dominant explanatory variable. Our results suggest that the perception of threat in the human brain is associated with neural structures that underlie perception and cognition of social actions and intentions, suggesting a broader role for these regions than has been thought previously, one that includes the perception of potential threat from agents independent of their biological class. SIGNIFICANCE STATEMENT: For centuries, philosophers have wondered how the human mind organizes the world into meaningful categories and concepts. Today this question is at the core of cognitive science, but our focus has shifted to understanding how knowledge manifests in dynamic activity of neural systems in the human brain. This study advances the young field of empirical neuroepistemology by characterizing the neural systems engaged by an important dimension in our cognitive representation of the animal kingdom ontological subdomain: how the brain represents the perceived threat, dangerousness, or "predacity" of animals. Our findings reveal how activity for domain-specific knowledge of animals overlaps the social perception networks of the brain, suggesting domain-general mechanisms underlying the representation of conspecifics and other animals.

Measuring and modelling the radiological impact of a phosphogypsum deposition site on the surrounding environment.

Phosphogypsum (PG) is a waste product (residue) from the production of phosphoric acid characterized by technologically enhanced natural radioactivity. Croatia's largest PG deposition site is situated at the edge of Lonjsko Polje Nature Park, a sensitive ecosystem possibly endangered by PG particles. This field study investigates two aspects relevant for the general radiological impact of PG: risk assessment for the environment and risk assessment for occupationally exposed workers and local inhabitants. Activity concentrations of natural radionuclides ((238)U, (235)U, (232)Th, (226)Ra, (210)Pb, and (40)K) were measured in the PG (at the deposition site), soil, and grass samples (in the vicinity of the site). The ERICA Assessment Tool was used to estimate the radiological impact of PG particles on non-human biota of the Lonjsko Polje Nature Park. The average annual effective dose for occupationally exposed workers was 0.4 mSv which was within the worldwide range.

Challenges with effective nutrient supplementation for amphibians: A review of cricket studies.

Over the last 25 years, numerous studies have investigated the impact of insect supplementation on insect nutrient content. In light of recent nutrition related challenges with regards to zoo amphibians fed an insect based diet, this review attempts to comprehensively compile both anecdotal and published data in the context of practical application on this topic. Insects, primarily crickets, used for amphibian diets historically demonstrate low concentrations of key nutrients including calcium and vitamin A. Commonly used practices for supplementation involving powder dusting or gut loading have been shown to improve delivery of calcium and vitamin A, though often not reaching desired nutrient concentrations. The large variety of factors influencing insect nutrient content are difficult to control, making study design, and results often inconsistent. Formulation and availability of more effective gut loading diets, combined with a standardized protocol for insect husbandry and dietary management may be the most effective way to supplement insects for use in amphibian feeding programs. Ideally, the nutritional improvement of feeder insects would begin at the breeder level; however, until this becomes a viable choice, we confirm that supplementation of crickets through both gut-loading and dusting appear necessary to support the nutritional health of amphibians and other insectivores in managed collections.

Updates on amphibian nutrition and nutritive value of common feeder insects.

The study of amphibian nutrition requires a detailed review of species-specific natural prey analysis. Invertebrate nutrient composition has been formally studied for more than 60 years and presents the following conclusions: (1) in general, insects are poor in overall calcium content; (2) larval insects have high fat and protein components; and (3) altering the gut contents of some insects can improve their overall nutritive quality. The fat-soluble vitamin profile for most inverts is lacking. There are new guidelines for calcium and vitamin A supplementation that can help augment invertebrate nutrient profiles to match the minimum NRC requirements established for rats.

Interacting symbionts and immunity in the amphibian skin mucosome predict disease risk and probiotic effectiveness.

Pathogenesis is strongly dependent on microbial context, but development of probiotic therapies has neglected the impact of ecological interactions. Dynamics among microbial communities, host immune responses, and environmental conditions may alter the effect of probiotics in human and veterinary medicine, agriculture and aquaculture, and the proposed treatment of emerging wildlife and zoonotic diseases such as those occurring on amphibians or vectored by mosquitoes. Here we use a holistic measure of amphibian mucosal defenses to test the effects of probiotic treatments and to assess disease risk under different ecological contexts. We developed a non-invasive assay for antifungal function of the skin mucosal ecosystem (mucosome function) integrating host immune factors and the microbial community as an alternative to pathogen exposure experiments. From approximately 8500 amphibians sampled across Europe, we compared field infection prevalence with mucosome function against the emerging fungal pathogen Batrachochytrium dendrobatidis. Four species were tested with laboratory exposure experiments, and a highly susceptible species, Alytes obstetricans, was treated with a variety of temperature and microbial conditions to test the effects of probiotic therapies and environmental conditions on mucosome function. We found that antifungal function of the amphibian skin mucosome predicts the prevalence of infection with the fungal pathogen in natural populations, and is linked to survival in laboratory exposure experiments. When altered by probiotic therapy, the mucosome increased antifungal capacity, while previous exposure to the pathogen was suppressive. In culture, antifungal properties of probiotics depended strongly on immunological and environmental context including temperature, competition, and pathogen presence. Functional changes in microbiota with shifts in temperature provide an alternative mechanistic explanation for patterns of disease susceptibility related to climate beyond direct impact on host or pathogen. This nonlethal management tool can be used to optimize and quickly assess the relative benefits of probiotic therapies under different climatic, microbial, or host conditions.

Time domains of the hypoxic ventilatory response in ectothermic vertebrates.

Over a decade has passed since Powell et al. (Respir Physiol 112:123-134, 1998) described and defined the time domains of the hypoxic ventilatory response (HVR) in adult mammals. These time domains, however, have yet to receive much attention in other vertebrate groups. The initial, acute HVR of fish, amphibians and reptiles serves to minimize the imbalance between oxygen supply and demand. If the hypoxia is sustained, a suite of secondary adjustments occur giving rise to a more long-term balance (acclimatization) that allows the behaviors of normal life. These secondary responses can change over time as a function of the nature of the stimulus (the pattern and intensity of the hypoxic exposure). To add to the complexity of this process, hypoxia can also lead to metabolic suppression (the hypoxic metabolic response) and the magnitude of this is also time dependent. Unlike the original review of Powell et al. (Respir Physiol 112:123-134, 1998) that only considered the HVR in adult animals, we also consider relevant developmental time points where information is available. Finally, in amphibians and reptiles with incompletely divided hearts the magnitude of the ventilatory response will be modulated by hypoxia-induced changes in intra-cardiac shunting that also improve the match between O(2) supply and demand, and these too change in a time-dependent fashion. While the current literature on this topic is reviewed here, it is noted that this area has received little attention. We attempt to redefine time domains in a more 'holistic' fashion that better accommodates research on ectotherms. If we are to distinguish between the genetic, developmental and environmental influences underlying the various ventilatory responses to hypoxia, however, we must design future experiments with time domains in mind.

The hypothalamus-pituitary-thyroid axis in teleosts and amphibians: endocrine disruption and its consequences to natural populations.

Teleosts and pond-breeding amphibians may be exposed to a wide variety of anthropogenic, waterborne contaminants that affect the hypothalamus-pituitary-thyroid (HPT) axis. Because thyroid hormone is required for their normal development and reproduction, the potential impact of HPT-disrupting contaminants on natural teleost and amphibian populations raises special concern. There is laboratory evidence indicating that persistent organic pollutants, heavy metals, pharmaceutical and personal care products, agricultural chemicals, and aerospace products may alter HPT activity, development, and reproduction in teleosts and amphibians. However, at present there is no evidence to clearly link contaminant-induced HPT alterations to impairments in teleost or amphibian population health in the field. Also, with the exception of perchlorate for which laboratory studies have shown a direct link between HPT disruption and adverse impacts on development and reproductive physiology, little is known about if or how other HPT-disrupting contaminants affect organismal performance. Future field studies should focus on establishing temporal associations between the presence of HPT-disrupting chemicals, the occurrence of HPT alterations, and adverse effects on development and reproduction in natural populations; as well as determining how complex mixtures of HPT contaminants affect organismal and population health.

Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species.

The survival of hundreds of threatened amphibian species is increasingly dependent on conservation breeding programs (CBPs). However, there is an ongoing loss of genetic variation in CBPs for most amphibians, reptiles, birds, and mammals. Low genetic variation results in the failure of CBPs to provide genetically competent individuals for release in supplementation or rehabitation programs. In contrast, in the aquaculture of fish the perpetuation of genetic variation and the production of large numbers of genetically competent individuals for release is accomplished through the cryopreservation of sperm. Successful protocols for the cryopreservation of amphibian sperm from excised testes, and the use of motile frozen then thawed sperm for fertilisation, have been adapted from those used with fish. However, there have been no protocols published for the cryopreservation of amphibian hormonally induced sperm (HIS) that have achieved fertility. We investigated protocols for the cryopreservation of amphibian HIS with the European common frog (Rana temporaria) as a model research species. We induced spermiation in R. temporaria through the intraperitoneal administration of 50 µg LHRHa and sampled HIS through expression in spermic urine. Highly motile HIS at a concentration of 200 × 10(6)/mL was then mixed 1:1 with cryodiluents to form cryosuspensions. Initial studies showed that; 1) concentrations of ∼15 × 10(6)/mL of HIS achieve maximum fertilisation, 2) TRIS buffer in cryodiluents did not improve the recovery of sperm after cryopreservation, and 3) high concentrations of DMSO (dimethylsulphoxide) cryoprotectant reduce egg and larval survival. We then compared four optimised cryopreservation protocols for HIS with the final concentrations of cryodiluents in cryosuspensions of; 1) DMSO, (½ Ringer Solution (RS), 10% sucrose, 12% DMSO); 2) DMSO/egg yolk, (½ RS, 10% sucrose, 12% DMSO, 10% egg yolk), 3) DMFA, (½ RS, 10% sucrose, 12% dimethylformamide (DMFA)), and 4) MIS/glycerol, (Motility Inhibiting Saline (MIS), 5% glycerol, 2.5% sucrose, 5% egg yolk). Cryosuspensions were frozen in LN(2) vapour, stored in LN(2), thawed in 40° C water bath, and activated by slow equilibration with 1:3 dilutions of cryosuspensions with 20 mM/L NaCl. Protocol efficacies were assessed through the post-thaw percentage of; 1) sperm motility, 2) sperm membrane integrity, 3) fertilisation, 4) fertilised eggs hatching, and 5) larval survival from fertilised eggs to 7 d. The DMFA cryodiluent proved superior to the DMSO based cryodiluents in recovery of sperm motility and fertility after cryopreservation. MIS/glycerol cryodiluent provided low sperm viability and no fertility. Considering the ease of obtaining HIS from many Rana species, the success of our protocols offer the potential for the perpetuation of the genetic variation of the 42 threatened Rana species and the 193 threatened Ranid species in total.

Amphibian biology and husbandry.

Extant amphibians comprise three lineages-- salamanders (Urodela or Caudata), frogs and toads (Anura), and caecilians (Gymnophiona, Apoda, or Caecilia)--which contain more than 6,000 species. Fewer than a dozen species of amphibians are commonly maintained in laboratory colonies, and the husbandry requirements for the vast majority of amphibians are poorly known. For these species, a review of basic characteristics of amphibian biology supplemented by inferences drawn from the morphological and physiological characteristics of the species in question provides a basis for decisions about housing and feeding. Amphibians are ectotherms, and their skin is permeable to water, ions, and respiratory gases. Most species are secretive and, in many cases, nocturnal. The essential characteristics of their environment include appropriate levels of humidity, temperature, and lighting as well as retreat sites. Terrestrial and arboreal species require moist substrates, water dishes, and high relative humidity. Because temperature requirements for most species are poorly known, it is advisable to use a temperature mosaic that will allow an animal to find an appropriate temperature within its cage. Photoperiod may affect physiology and behavior (especially reproduction and hibernation), and although the importance of ultraviolet light for calcium metabolism by amphibians is not yet known, ecological observations suggest that it might be important for some species of frogs. Some amphibians are territorial, and some use olfactory cues to mark their territory and to recognize other individuals of their species. All amphibians are carnivorous as adults, and the feeding response of many species is elicited by the movement of prey. Diets should include a mixture of prey species, and it may be advisable to load prey with vitamins and minerals.

Evolution of the amygdala: new insights from studies in amphibians.

The histology of amphibian brains gives an impression of relative simplicity when compared with that of reptiles or mammals. The amphibian telencephalon is small and contains comparatively few and large neurons, which in most parts constitute a dense periventricular cellular layer. However, the view emerging from the last decade is that the brains of all tetrapods, including amphibians, share a general bauplan resulting from common ancestry and the need to perform similar vital functions. To what extent this common organization also applies to higher brain functions is unknown due to a limited knowledge of the neurobiology of early vertebrates. The amygdala is widely recognized as a brain center critical for basic forms of emotional learning (e.g., fear conditioning) and its structure in amphibians could suggest how this capacity evolved. A functional systems approach is used here to synthesize the results of our anatomical investigations of the amphibian amygdala. It is proposed that the connectivity of the amphibian telencephalon portends a capacity for multi-modal association in a limbic system largely similar to that of amniote vertebrates. One remarkable exception is the presence of new sensory-associative regions of the amygdala in amniotes: the posterior dorsal ventricular ridge plus lateral nuclei in reptiles and the basolateral complex in mammals. These presumably homologous regions apparently are capable of modulating the phylogenetically older central amygdala and allow more complex forms of emotional learning.

Ecosystem remodelling among vertebrates at the Permian-Triassic boundary in Russia.

The mass extinction at the Permian-Triassic boundary, 251 million years (Myr) ago, is accepted as the most profound loss of life on record. Global data compilations indicate a loss of 50% of families or more, both in the sea and on land, and these figures scale to a loss of 80-96% of species, based on rarefaction analyses. This level of loss is confirmed by local and regional-scale studies of marine sections, but the terrestrial record has been harder to analyse in such close detail. Here we document the nature of the event in Russia in a comprehensive survey of 675 specimens of amphibians and reptiles from 289 localities spanning 13 successive geological time zones in the South Urals basin. These changes in diversity and turnover cannot be explained simply by sampling effects. There was a profound loss of genera and families, and simplification of ecosystems, with the loss of small fish-eaters and insect-eaters, medium and large herbivores and large carnivores. Faunal dynamics also changed, from high rates of turnover through the Late Permian period to greater stability at low diversity through the Early Triassic period. Even after 15 Myr of ecosystem rebuilding, some guilds were apparently still absent-small fish-eaters, small insect-eaters, large herbivores and top carnivores.

Neuropeptides and amphibian prey-catching behavior.

In mammals, a number of hypothalamic neuropeptides have been implicated in stress-induced feeding disorders. Recent studies in anurans suggest that stress-related neuropeptides may act on elemental aspects of visuomotor control to regulate feeding. Corticotropin-releasing hormone (CRH) and alpha-melanocyte-stimulating hormone, potent an orexic peptides in mammals, inhibit visually-guided prey-catching in toads. Neuropeptide Y (NPY), an orexic peptide in mammals, may be an important neuromodulator in inhibitory pre-tectal-tectal pathways involved in distinguishing predator and prey. Melanocortin, NPY and CRH neurons project onto key visuomotor structures within the amphibian brain, suggesting physiological roles in the modulation of prey-catching. Thus, neuropeptides involved in feeding behavior in mammals influence the efficacy of a visual stimulus in releasing prey-catching behavior. These neuropeptides may play an important role in how frogs and toads gather and process visual information, particularly during stress.

Environmental estrogens and reproductive biology in amphibians.

In this paper, the effects of an estrogenic compound, 4-nonyl-phenol (NP), on the amphibians Rana esculenta and Triturus carnifex are described together with those on sexual differentiation in Xenopus laevis. NP increased plasma vitellogenin in male frogs and newts in a dose-related manner; moreover, inhibitory effects on gonadotropin and prolactin (PRL) secretion by pituitary were found together with an elevation of plasma androgens. NP treatment also caused a remarkable increase in number of prolactin-immunolabeled cells, suggesting that xenoestrogen might induce, at least in the newt pituitary, a PRL accumulation possibly due to a reduction of the hormone release. In addition, both NP and bisphenol A caused feminization by increasing the percentage of female phenotypes in X. laevis, and the in vivo effects were more pronounced than those of estradiol-17beta.

Sensory pathways linking social and environmental cues to endocrine control regions of amphibian forebrains.

The secretion of gonadotropin releasing hormone (GnRH), and hence sex steroids, is influenced by social signals produced by conspecifics and by environmental cues such as day length. The sensory systems processing these varied signals must therefore connect with the GnRH control centers in the preoptic area and hypothalamus. Forebrain pathways in frogs provide a model for how vertebrates accomplish this. Auditory information, which can transmit the features of the vocal communication signals used by frogs during reproductive social behavior, reaches both the preoptic area and ventral hypothalamus via thalamic and midbrain nuclei. An examination of these auditory pathways suggests that there are subtle differences in their anatomical, physiological, and histochemical characteristics. The suprachiasmatic nucleus, which is important for controlling circadian rhythms in vertebrates, also projects to both the preoptic area and ventral hypothalamus, providing information about environmental characteristics in parallel with the information about social signals. This pattern of connections suggests that the two major GnRH control centers in amphibian brains receive independent, parallel information about the social and environmental cues influencing reproduction.

Parathyroid hormone-like immunoreactivity in brains of tetrapod vertebrates.

Heat-stable nondialyzable immunoreactive (IR) parathyroid hormone (PTH)-like activity, that coelutes with authentic PTH on Sep-Pak C18 columns and reverse-phase high-performance liquid chromatography, was measured in the brain, hypothalamus, and pituitary of amphibian, reptilian, avian, and mammalian species, using two specific antisera raised against the 48-64 region of the intact PTH molecule. In each case the IR PTH concentration was greater than that present in peripheral plasma and in rats was not affected by dietary calcium status. Extracts of muscle, liver, and kidney tissue were without IR PTH activity. These results demonstrate the extraparathyroidal occurrence of PTH-like peptides in nontumorous neuroendocrine tissues of vertebrate species. These findings may have evolutionary significance, since IR PTH was present in the brain and plasma of species that lack encapsulated parathyroid glands.

Suitability of the mammalian model in comparative reproductive endocrinology.

Perhaps one should be impressed by the basic generality of many of the features of the mammalian HHG system. The applicability of this model to other groups argues for the generally conservative nature of the basic organization of the reproductive endocrine system. Even in cases where exceptions are apparent, we must be cognizant of the relatively narrow base of mammalian species on which the classical mammalian model is built. While reference to the mammalian system provides a useful first approach to studies on new groups, in view of the many exceptions in details of this system, it is important that we avoid undue constraints that might be imposed by the the mammalian model. In this connection it would seem advisable to test each principle carefully before assuming that it exists or is as important as in mammals. A particularly significant issue in this regard is the question of temporal organization of endocrine secretions: Both the existence and relative importance of discontinuous, pulsatile, stimulation must be explored in more groups. It must be emphasized that past experience has already shown us that a simple phylogenetic approach is not likely to provide a clear pattern for the similarities and differences in the reproductive endocrine system. Thus, we should not be too hasty in our extrapolations of data, even among closely related species.