The thermal ecology and physiology of reptiles and amphibians: A user's guide.

Research on the thermal ecology and physiology of free-living organisms is accelerating as scientists and managers recognize the urgency of the global biodiversity crisis brought on by climate change. As ectotherms, temperature fundamentally affects most aspects of the lives of amphibians and reptiles, making them excellent models for studying how animals are impacted by changing temperatures. As research on this group of organisms accelerates, it is essential to maintain consistent and optimal methodology so that results can be compared across groups and over time. This review addresses the utility of reptiles and amphibians as model organisms for thermal studies by reviewing the best practices for research on their thermal ecology and physiology, and by highlighting key studies that have advanced the field with new and improved methods. We end by presenting several areas where reptiles and amphibians show great promise for further advancing our understanding of how temperature relations between organisms and their environments are impacted by global climate change.

[The saga of neural induction: almost a century of research]. / La saga de l'induction neurale : presque un siècle de recherche.

Neural induction is a developmental process that allows cells from the ectoderm (the target tissue) to acquire a neural fate in response to signals coming from a specific adjacent embryonic region, the dorsal mesoderm (the inducing tissue). This process described in 1924 in amphibian embryos has not received a molecular explanation until the mid-1990s. Most of the work on neural induction has been carried out in amphibians. At these times, although the role played by the membrane of the target tissue had been suggested, no definitive work had been performed on the transduction of the neuralizing signal. Between 1990 and 2019 our aim was to decipher this transduction. We have underlined the necessary and sufficient role played by calcium signaling to induce ectoderm cells towards a neural fate from the activation of calcium channels to the direct transcription of early neural genes by calcium.

TITLE: La saga de l'induction neurale : presque un siècle de recherche. ABSTRACT: La formation du système nerveux débute par l'induction neurale, un processus qui permet aux cellules de l'ectoderme (tissu cible) d'acquérir un destin neural en réponse à des signaux provenant du mésoderme dorsal (tissu inducteur). Ce processus, décrit en 1924 sur l'amphibien, n'a reçu une explication moléculaire qu'au milieu des années 1990. Pendant cette période, plusieurs auteurs se sont intéressés au rôle joué par la membrane du tissu cible mais peu de travaux décisifs ont décrit la transduction du signal neuralisant. Entre 1990 et 2019, nous avons disséqué la transduction du signal neuralisant, un sujet très peu abordé alors. Nous avons souligné le rôle nécessaire et suffisant du calcium pour orienter les cellules de l'ectoderme vers un destin neural et établi la cascade moléculaire allant de l'activation de canaux membranaires à la transcription de gènes.

Introduction of Developmental Biology at Utkal University, (Odisha, India).

The paper deals with the background and the establishment of a Developmental Biology Laboratory in Utkal University in Odisha state. It describes the process from a humble beginning with limited facilities into a leading research centre, initially for amphibians and later for the endangered olive ridley (Lepidochelys olivacea) turtle. Starting from the biology, reproduction and development in many anurans, the laboratory took up research on regeneration, especially on super-regeneration in tadpoles under the influence of morphogens such as vitamin A (retinoids). Treatment with vitamin A after amputation of the tail inhibited tail regeneration but unexpectedly induced homeotic transformation of tails into limbs in many anurans, starting with the marbled balloon frog Uperodon systoma. This was the first observation of homeotic transformation in any vertebrate. The laboratory continues research on histological and molecular aspects of this phenomenon. In addition, taking advantage of the largest rookery of olive ridley sea turtles in Gahirmatha, in the same state the laboratory has contributed significantly to the biology, breeding patterns, development and especially the temperature-dependent sex determination phenomenon (TSD). This research was extended to biochemical and ultrastructural aspects during development for the first time for any sea turtle. The laboratory has contributed significantly to the conservation of olive ridleys as well as the saltwater crocodile (Crocodylus porosus). Recognition and awards for the laboratory have been received from both national and international bodies.

The power of amphibians to elucidate mechanisms of size control and scaling.

Size is a fundamental feature of biology that affects physiology at all levels, from the organism to organs and tissues to cells and subcellular structures. How size is determined at these different levels, and how biological structures scale to fit together and function properly are important open questions. Historically, amphibian systems have been extremely valuable to describe scaling phenomena, as they occupy some of the extremes in biological size and are amenable to manipulations that alter genome and cell size. More recently, the application of biochemical, biophysical, and embryological techniques to amphibians has provided insight into the molecular mechanisms underlying scaling of subcellular structures to cell size, as well as how perturbation of normal size scaling impacts other aspects of cell and organism physiology.

The development of cell senescence.

Cellular senescence was traditionally considered a stress response that protected the organism by limiting the proliferation of damaged and unwanted cells. However, the recent identification of developmentally-programmed cellular senescence during embryo development has changed our view of the process. There are now a number of examples of developmental senescence in evolutionary distant organisms ranging from mammals to fish, showing senescence at various sites during specific time windows of development. Developmental senescence shares many features with stress-induced senescence but also present some specific characteristics. The different examples of developmental senescence provide evidence of the diverse functions contributed by senescence and represent an opportunity to learn more about this process. Also, the existence of senescence during embryogenesis opens the possibility of identifying human developmental syndromes caused by alterations in this response. Studying in more detail this process will expand our understanding of cellular senescence and could offer new insights into the cause of human pathologies.

Effects of chemical pollutants on reproductive and developmental processes in Italian amphibians.

It is a widely held belief that environmental contaminants contribute to the decline of amphibian populations. By spending most of their early life in water and later stages on the land, amphibians face a constant risk of exposure to pesticides and other chemical pollutants in both aquatic and terrestrial environments. This review presents an overview of the studies carried out in Italian amphibians to highlight hazardous effects of bioaccumulation of chemical pollutants in juveniles and adults in various contaminated environments. Further, the studies in the laboratory setting assessing the effects of chemical pollutants on reproductive and developmental processes are reported. These studies and their relative references have been summarized in a tabular form. Three prominent contaminant groups were identified: herbicides, insecticides, and fungicides; and only a few works reported the effects of other chemical pollutants. Each pollutant group has been delegated to a section. All through the literature survey, it is seen that interest in this topic in Italy is very recent and sparse, where only a few anuran and caudata species and only some chemical pollutants have been studied.

Lineage tracing of sclerotome cells in amphibian reveals that multipotent somitic cells originate from lateral somitic frontier.

The two somite compartments, dorso-lateral dermomyotome and medio-ventral sclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution.

Exposures to chemical contaminants: What can we learn from reproduction and development endpoints in the amphibian toxicology literature?

Environmental contamination is one of the major factors or cofactors affecting amphibian populations. Since 2000, the number of studies conducted in laboratory conditions to understand impacts of chemical exposures increased. They aimed to characterize biological effects on amphibians. This review proposes an overview of biological responses reported after exposures to metals, phytopharmaceuticals or emerging organic contaminants and focuses on endpoints relating to reproduction and development. Due to amphibian peculiar features, these periods of their life cycle are especially critical to pollutant exposures. Despite the large range of tested compounds, the same model species are often used as biological models and morphological alterations are the most studied observations. From the results, the laboratory-to-field extrapolation remained uneasy and exposure designs have to be more elaborated to be closer to environmental conditions. Few studies proposed such experimental approaches. Lastly, gametes, embryos and larvae constitute key stages of amphibian life cycle that can be harmed by exposures to freshwater pollutants. Specific efforts have to be intensified on the earliest stages and notably germ cells.

Morphomechanical reactions and mechanically stressed structures in amphibian embryos, as related to gastrulation and axial organs formation.

Reactions of embryonic tissues to a distributed and concentrated stretching are described and compared with the mechanics of the normal gastrulation movements. A role of mechanically stressed dynamic cell structures in the gastrulation, demarcation of notochord borders and in providing proportionality of the axial rudiments is demonstrated. A morphomechanical scheme of amphibian gastrulation is presented.

Role of Thyroid Hormone Receptor in Amphibian Development.

The amphibian Xenopus laevis has long been used as a model for studying vertebrate cell and developmental biology largely due to the easiness to manipulate this system in vivo and in vitro. While most of the developmental studies have been on Xenopus embryogenesis, considerable efforts have been made to understand its metamorphosis, a process mimicking postembryonic development in mammals when many organs mature into their adult forms in the presence of high levels of thyroid hormone (T3). Amphibian metamorphosis is totally dependent on T3 and offers a number of advantages for experimental analyses compared to the late stage, uterus-enclosed mammalian embryos. Earlier studies on metamorphosis in Xenopus laevis have revealed dual functions of T3 receptors (TR) during premetamorphic development and metamorphosis as well as important roles of TR-interacting corepressors and coactivators during these two periods, respectively. The development of gene-editing technologies that functions in amphibians in recent years has made possible for the first time to study function of endogenous TRs, especially in the highly related diploid anuran species Xenopus tropicalis. Here, we first review the current mechanistic understanding of the regulation of metamorphosis by T3 and TR, and then describe a detailed method to use TALEN to knock out TRα for studying its role in gene regulation by T3 in vivo and Xenopus development.

On the nature and function of organizers.

Organizers, which comprise groups of cells with the ability to instruct adjacent cells into specific states, represent a key principle in developmental biology. The concept was first introduced by Spemann and Mangold, who showed that there is a cellular population in the newt embryo that elicits the development of a secondary axis from adjacent cells. Similar experiments in chicken and rabbit embryos subsequently revealed groups of cells with similar instructive potential. In birds and mammals, organizer activity is often associated with a structure known as the node, which has thus been considered a functional homologue of Spemann's organizer. Here, we take an in-depth look at the structure and function of organizers across species and note that, whereas the amphibian organizer is a contingent collection of elements, each performing a specific function, the elements of organizers in other species are dispersed in time and space. This observation urges us to reconsider the universality and meaning of the organizer concept.

The extraordinary biology and development of marsupial frogs (Hemiphractidae) in comparison with fish, mammals, birds, amphibians and other animals.

The study of oogenesis and early development of frogs belonging to the family Hemiphractidae provide important comparison to the aquatic development of other frogs, such as Xenopus laevis, because reproduction on land characterizes the Hemiphractidae. In this review, the multinucleated oogenesis of the marsupial frog Flectonotus pygmaeus (Hemiphractidae) is analyzed and interpreted. In addition, the adaptations associated with the incubation of embryos in the pouch of the female marsupial frog Gastrotheca riobambae (Hemiphractidae) and the embryonic development of this frog are summarized. Moreover, G. riobambae gastrulation is compared with the gastrulation modes of Engystomops randi and Engystomops coloradorum (Leptodactylidae); Ceratophrys stolzmanni (Ceratophryidae); Hyalinobatrachium fleischmanni and Espadarana callistomma (Centrolenidae); Ameerega bilinguis, Dendrobates auratus, Epipedobates anthonyi, Epipedobates machalilla, Epipedobates tricolor, and Hyloxalus vertebralis (Dendrobatidae); Eleutherodactylus coqui (Terrarana: Eleutherodactylidae), and X. laevis (Pipidae). The comparison indicated two modes of frog gastrulation. In X. laevis and in frogs with aquatic reproduction, convergent extension begins during gastrulation. In contrast, convergent extension occurs in the post-gastrula of frogs with terrestrial reproduction. These two modes of gastrulation resemble the transitions toward meroblastic cleavage found in ray-finned fishes (Actinopterygii). In spite of this difference, the genes that guide early development seem to be highly conserved in frogs. I conclude that the shift of convergent extension to the post-gastrula accompanied the diversification of frog egg size and terrestrial reproductive modes.

Neuroendocrine disruption of organizational and activational hormone programming in poikilothermic vertebrates.

In vertebrates, sexual differentiation of the reproductive system and brain is tightly orchestrated by organizational and activational effects of endogenous hormones. In mammals and birds, the organizational period is typified by a surge of sex hormones during differentiation of specific neural circuits; whereas activational effects are dependent upon later increases in these same hormones at sexual maturation. Depending on the reproductive organ or brain region, initial programming events may be modulated by androgens or require conversion of androgens to estrogens. The prevailing notion based upon findings in mammalian models is that male brain is sculpted to undergo masculinization and defeminization. In absence of these responses, the female brain develops. While timing of organizational and activational events vary across taxa, there are shared features. Further, exposure of different animal models to environmental chemicals such as xenoestrogens such as bisphenol A-BPA and ethinylestradiol-EE2, gestagens, and thyroid hormone disruptors, broadly classified as neuroendocrine disrupting chemicals (NED), during these critical periods may result in similar alterations in brain structure, function, and consequently, behaviors. Organizational effects of neuroendocrine systems in mammals and birds appear to be permanent, whereas teleost fish neuroendocrine systems exhibit plasticity. While there are fewer NED studies in amphibians and reptiles, data suggest that NED disrupt normal organizational-activational effects of endogenous hormones, although it remains to be determined if these disturbances are reversible. The aim of this review is to examine how various environmental chemicals may interrupt normal organizational and activational events in poikilothermic vertebrates. By altering such processes, these chemicals may affect reproductive health of an animal and result in compromised populations and ecosystem-level effects.

Exploitation of Non-mammalian Model Organisms in Epigenetic Research.

Model organisms are widely used in research that is ultimately aimed at understanding the causes and consequences of human disease. It may seem counterintuitive to expect clinically useful information to be obtained from species as diverse as fishes and insects, but because fundamental biological mechanisms share evolutionary origins they transcend species barriers. Epigenetic mechanisms fulfil this expectation admirably as more and more is discovered about the basic operational rules of inheritance, which are much more elaborate than formerly thought. Only a few decades ago, although the complex interplay between genes, inheritance and the environment was recognized, it was difficult to explain. Recent discoveries about the controlling influences of gene silencing through DNA and histone methylation, the roles of so-called non-coding DNA and microRNA, and the way in which these factors respond to environmental conditions have started to shed light on these basic mechanisms. Diverse model species allow epigenetic mechanisms to be studied from different perspectives; for example, some are better suited to studies of sex determination while others may be more convenient for studying the earliest stages of organ development, growth and the influence of nutrition on future wellbeing. The rationale for including this chapter in a book that is focused on uncovering relationships between periconception nutrition in humans is to highlight the opportunities and insights that may be gained by focusing attention on studies in non-mammalian model species.

Biological responses to phenylurea herbicides in fish and amphibians: New directions for characterizing mechanisms of toxicity.

Urea-based herbicides are applied in agriculture to control broadleaf and grassy weeds, acting to either inhibit photosynthesis at photosystem II (phenylureas) or to inhibit acetolactate synthase acetohydroxyacid synthase (sulfonylureas). While there are different chemical formulas for urea-based herbicides, the phenylureas are a widely used class in North America and have been detected in aquatic environments due to agricultural run-off. Here, we summarize the current state of the literature, synthesizing data on phenylureas and their biological effects in two non-target animals, fish and amphibians, with a primary focus on diuron and linuron. In fish, although the acutely lethal effects of diuron in early life stages appear to be >1mg/L, recent studies measuring sub-lethal behavioural and developmental endpoints suggest that diuron causes adverse effects at lower concentrations (i.e. <0.1mg/L). Considerably less toxicity data exist for amphibians, and this is a knowledge gap in the literature. In terms of sub-lethal effects and mode of action (MOA), linuron is well documented to have anti-androgenic effects in vertebrates, including fish. However, there are other MOAs that are not adequately assessed in toxicology studies. In order to identify additional potential MOAs, we conducted in silico analyses for linuron and diuron that were based upon transcriptome studies and chemical structure-function relationships (i.e. ToxCast™, Prediction of Activity Spectra of Substances). Based upon these analyses, we suggest that steroid biosynthesis, cholesterol metabolism and pregnane X receptor activation are common targets, and offer some new endpoints for future investigations of phenylurea herbicides in non-target animals.

Conserved and novel functions of programmed cellular senescence during vertebrate development.

Cellular senescence, a form of stable cell cycle arrest that is traditionally associated with tumour suppression, has been recently found to occur during mammalian development. Here, we show that cell senescence is an intrinsic part of the developmental programme in amphibians. Programmed senescence occurs in specific structures during defined time windows during amphibian development. It contributes to the physiological degeneration of the amphibian pronephros and to the development of the cement gland and oral cavity. In both contexts, senescence depends on TGFß but is independent of ERK/MAPK activation. Furthermore, elimination of senescent cells through temporary TGFß inhibition leads to developmental defects. Our findings uncover conserved and new roles of senescence in vertebrate organogenesis and support the view that cellular senescence may have arisen in evolution as a developmental mechanism.

[BEHAVIORAL AND FUNCTIONAL VESTIBULAR DISTURBANCES AFTER SPACE FLIGHT. 2. FISHES, AMPHIBIANS AND BIRDS].

The review contains data on functional shifts in fishes, amphibians and birds caused by changes in the otolith system operation after stay under weightlessness conditions. These data are of theoretical and practical significance and are important to resolve some fundamental problems of vestibulogy. The analysis of the results of space experiments has shown that weightlessness conditions do not exert a substantial impact on formation and functional state of the otolith system in embryonic fishes, amphibians and birds developed during space flight. Weightlessness conditions do pot inhibit embryonic development of lower vertebrates but even have rather beneficial effect on it. This is consistent with conclusions concerning development of mammalian fetuses. The experimental results show that weightlessness can cause similar functional and behavioral vestibular shifts both in lower vertebrates and in mammals. For example, immediately after an orbital flight the vestibuloocular reflex in fish larvae and tadpoles (without lordosis) was stronger than in control individuals. A similar shift of the otolith reflex was observed in the majority of cosmonauts after short-term orbital flights. Immediately after landing adult terrestrial vertebrates, as well as human beings, exhibit lower activity levels, worse equilibrium and coordination of movements. Another interesting finding observed after landing of the cosmic apparatus was an unusual looping character of tadpole swimming. It is supposed that the unusual motor activity of animals as well as appearance of illusions in cosmonauts and astronauts after switching from 1 to 0 g have the same nature and are related to the change in character of otolith organs stimulation. Considering this similarity of vestibular reactions, using animals seems rather perspective. Besides it allows applying in experiments various invasive techniques.

Environmental levels of Zn do not protect embryos from Cu toxicity in three species of amphibians.

Contaminants often occur as mixtures in the environment, but investigations into toxicity usually employ a single chemical. Metal contaminant mixtures from anthropogenic activities such as mining and coal combustion energy are widespread, yet relatively little research has been performed on effects of these mixtures on amphibians. Considering that amphibians tend to be highly sensitive to copper (Cu) and that metal contaminants often occur as mixtures in the environment, it is important to understand the interactive effects that may result from multiple metals. Interactive effects of Cu and zinc (Zn) on amphibians have been reported as antagonistic and, conversely, synergistic. The goal of our study was to investigate the role of Zn in Cu toxicity to amphibians throughout the embryonic developmental period. We also considered maternal effects and population differences by collecting multiple egg masses from contaminated and reference areas for use in four experiments across three species. We performed acute toxicity experiments with Cu concentrations that cause toxicity (10-200 µg/L) in the absence of other contaminants combined with sublethal concentrations of Zn (100 and 1000 µg/L). Our results suggest very few effects of Zn on Cu toxicity at these concentrations of Zn. As has been previously reported, we found that maternal effects and population history had significant influence on Cu toxicity. The explanation for a lack of interaction between Cu and Zn in this experiment is unknown but may be due to the use of sublethal Zn concentrations when previous experiments have used Zn concentrations associated with acute toxicity. Understanding the inconsistency of amphibian Cu/Zn mixture toxicity studies is an important research direction in order to create generalities that can be used to understand risk of contaminant mixtures in the environment.