Enzymes, embryos, and ancestors.

In the 1950s, cellular regulatory mechanisms were newly recognized; with Arthur Pardee I investigated the initial enzyme of pyrimidine biosynthesis, which he discovered is controlled by feedback inhibition. The protein proved unusual in having separate but interacting sites for substrates and regulators. Howard Schachman and I dissociated the protein into different subunits, one binding regulators and one substrates. The enzyme became an early prime example of allostery. In developmental biology I studied the egg of the frog, Xenopus laevis, characterizing early processes of axis formation. My excellent students and I described cortical rotation, a 30° movement of the egg's cortex over tracks of parallel microtubules anchored to the underlying cytoplasmic core, and we perturbed it to alter Spemann's organizer and effect spectacular phenotypes. The entire sequence of events has been elucidated by others at the molecular level, making Xenopus a prime example of vertebrate axis formation. Marc Kirschner, Christopher Lowe, and I then compared hemichordate (half-chordate) and chordate early development. Despite anatomical-physiological differences, these groups share numerous steps of axis formation, ones that were probably already in use in their pre-Cambrian ancestor. I've thoroughly enjoyed exploring these areas during a 50-year period of great advances in biological sciences by the worldwide research community.

How to disinfect anuran eggs? Sensitivity of anuran embryos to chemicals widely used for the disinfection of larval and post-metamorphic amphibians.

Emerging infectious diseases are major drivers of global and local amphibian biodiversity loss. Therefore, developing effective disinfection methods to manage the impact of diseases in wild and captive "ark" populations are an important goal in amphibian conservation. While chemical disinfectants have been used safely and effectively in larval and adult amphibians infected with pathogenic microbes, their applicability to amphibian egg masses has remained untested. To bridge this gap, we exposed embryos of the common toad (Bufo bufo) and agile frog (Rana dalmatina) experimentally to three widely used disinfectants: voriconazole, chloramphenicol and chlorogen-sesquihydrate. For 3 days we exposed portions of egg masses to these disinfectants at 1×, 2×, 5× and 10× the concentration recommended for the disinfection of tadpoles and adults. Subsequently, we recorded embryonic and larval survival, as well as larval body mass and the incidence of abnormalities 12 days after hatching. Application of voriconazole had species- and concentration-dependent negative impacts on survival and body mass, and caused marked malformations in the viscerocranial structure of B. bufo tadpoles. Exposure to chlorogen-sesquihydrate also resulted in significant mortality in B. bufo embryos and negatively affected body mass of R. dalmatina larvae. Chloramphenicol had little negative effects on embryos or larvae in either species. Based on these results, the application of voriconazole and chlorogen-sesquihydrate cannot be recommended for the disinfection of amphibian eggs, whereas treatment with chloramphenicol appears to be a safe method for eliminating potential pathogens from anuran egg masses and their immediate aquatic environment.

Effect-based environmental monitoring for thyroid disruption in Swedish amphibian tadpoles.

It is well-known that the metamorphosis process in amphibians is dependent on thyroid hormones. Laboratory studies have shown that several environmental contaminants can affect the function of thyroid hormones leading to alterations in the amphibian metamorphosis. The basic idea of the present study was to elucidate if the amphibian metamorphosis might be a useful tool as biomarker for effect-based environmental monitoring, examining wild tadpoles for potential thyroid hormone disruption. A laboratory test was performed to identify the responses from exposure to 6-propylthiouracil (PTU), which has a well-known mechanism on the thyroid system, on Swedish tadpoles from the Rana genus. This was followed by an environmental monitoring study where tadpoles of Rana arvalis, R. temporaria, and Bufo bufo were sampled from various sites in Sweden. Morphological data such as body weight, histopathological measurements of the thyroid glands, and environmental parameters were recorded. The results revealed that Rana tadpoles respond similar as other amphibians to PTU exposure, with interrupted development and increased size relative to the developmental stage. Data on some wild tadpoles showed similar features as the PTU exposed, such as high body weight, thus suggesting potential thyroid disrupting effects. However, histological evaluation of thyroid glands and pesticide analyses of the water revealed no clear evidence of chemical interactions. To a minor degree, the changes in body weight may be explained by natural circumstances such as pH, forest cover, and temperature. The present study cannot fully explain whether the high body weights recorded in some tadpoles have natural or chemical explanations. However, the study reveals that it is clearly achievable to catch tadpoles in suitable stages for the use in this type of biomonitoring and that the use of these biomarkers for assessment of thyroid disruption seems to be highly relevant.

Nanos3 of the frog Rana rugosa: Molecular cloning and characterization.

Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3-deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT-PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3-knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild-type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.

Transcriptome analyses of immune tissues from three Japanese frogs (genus Rana ) reveals their utility in characterizing major histocompatibility complex class II.

BACKGROUND: In Japan and East Asia, endemic frogs appear to be tolerant or not susceptible to chytridiomycosis, a deadly amphibian disease caused by the chytrid fungus Batrachochytridium dendrobatidis (Bd). Japanese frogs may have evolved mechanisms of immune resistance to pathogens such as Bd. This study characterizes immune genes expressed in various tissues of healthy Japanese Rana frogs. RESULTS: We generated transcriptome data sets of skin, spleen and blood from three adult Japanese Ranidae frogs (Japanese brown frog Rana japonica, the montane brown frog Rana ornativentris, and Tago's brown frog Rana tagoi tagoi) as well as whole body of R. japonica and R. ornativentris tadpoles. From this, we identified tissue- and stage-specific differentially expressed genes; in particular, the spleen was most enriched for immune-related genes. A specific immune gene, major histocompatibility complex class IIB (MHC-IIB), was further characterized due to its role in pathogen recognition. We identified a total of 33 MHC-IIB variants from the three focal species (n = 7 individuals each), which displayed evolutionary signatures related to increased MHC variation, including balancing selection. Our supertyping analyses of MHC-IIB variants from Japanese frogs and previously studied frog species identified potential physiochemical properties of MHC-II that may be important for recognizing and binding chytrid-related antigens. CONCLUSIONS: This is one of the first studies to generate transcriptomic resources for Japanese frogs, and contributes to further understanding the immunogenetic factors associated with resistance to infectious diseases in amphibians such as chytridiomycosis. Notably, MHC-IIB supertyping analyses identified unique functional properties of specific MHC-IIB alleles that may partially contribute to Bd resistance, and such properties provide a springboard for future experimental validation.

Analysis of Cell Size in the Gastrula of Ten Frog Species Reveals a Correlation of Egg with Cell Sizes, and a Conserved Pattern of Small Cells in the Marginal Zone.

We investigated the relationship between egg and cell sizes in the early gastrula of ten species of frogs with eggs of 1,100-3,500 µm diameters. We asked whether differences in cell size of the vegetal region, blastocoel roof, and marginal zone of the early gastrula were associated with egg size. Alternatively, we proposed that cell size differences may associate with gastrulation characteristics. The analyzed species were as follows: Xenopus laevis, Engystomops randi, Engystomops coloradorum, Espadarana callistomma, Epipedobates machalilla, Epipedobates anthonyi, Epipedobates tricolor, Dendrobates auratus, Gastrotheca riobambae, and Eleutherodactylus coqui. A positive correlation between egg and cell size was detected in the three regions of the gastrula. The correlation was strong in the vegetal region and blastocoel roof, and weak in the marginal zone. Large eggs allowed the evolution of frog terrestrial reproductive modes by storing nourishment for the developing embryos. Large cells, laden with yolk, occur in the vegetal region. However, small cell size characterized the marginal zone and blastocoel roof. We proposed that small cells of the marginal zone are required for involution and blastopore formation. The evolution pressure toward small cells in the marginal zone contributed to maintain the blastopore as a universal feature of frog gastrulation in eggs of different sizes and gastrulation modes. Our comparative analysis revealed two fundamental and conserved properties of the frog early gastrula, the correlation of egg with cell sizes, and the general small size of cells in the marginal zone.

Fear is the mother of invention: anuran embryos exposed to predator cues alter life-history traits, post-hatching behaviour and neuronal activity patterns.

Neurophysiological modifications associated to phenotypic plasticity in response to predators are largely unexplored, and there is a gap of knowledge on how the information encoded in predator cues is processed by prey sensory systems. To explore these issues, we exposed Rana dalmatina embryos to dragonfly chemical cues (kairomones) up to hatching. At different times after hatching (up to 40 days), we recorded morphology and anti-predator behaviour of tadpoles from control and kairomone-treated embryo groups as well as their neural olfactory responses, by recording the activity of their mitral neurons before and after exposure to a kairomone solution. Treated embryos hatched later and hatchlings were smaller than control siblings. In addition, the tadpoles from the treated group showed a stronger anti-predator response than controls at 10 days (but not at 30 days) post-hatching, though the intensity of the contextual response to the kairomone stimulus did not differ between the two groups. Baseline neuronal activity at 30 days post-hatching, as assessed by the frequency of spontaneous excitatory postsynaptic events and by the firing rate of mitral cells, was higher among tadpoles from the treated versus the control embryo groups. At the same time, neuronal activity showed a stronger increase among tadpoles from the treated versus the control group after a local kairomone perfusion. Hence, a different contextual plasticity between treatments at the neuronal level was not mirrored by the anti-predator behavioural response. In conclusion, our experiments demonstrate ontogenetic plasticity in tadpole neuronal activity after embryonic exposure to predator cues, corroborating the evidence that early-life experience contributes to shaping the phenotype at later life stages.

Induced tolerance from a sublethal insecticide leads to cross-tolerance to other insecticides.

As global pesticide use increases, the ability to rapidly respond to pesticides by increasing tolerance has important implications for the persistence of nontarget organisms. A recent study of larval amphibians discovered that increased tolerance can be induced by an early exposure to low concentrations of a pesticide. Since natural systems are often exposed to a variety of pesticides that vary in mode of action, we need to know whether the induction of increased tolerance to one pesticide confers increased tolerance to other pesticides. Using larval wood frogs (Lithobates sylvaticus), we investigated whether induction of increased tolerance to the insecticide carbaryl (AChE-inhibitor) can induce increased tolerance to other insecticides that have the same mode of action (chlorpyrifos, malathion) or a different mode of action (Na(+)channel-interfering insecticides; permethrin, cypermethrin). We found that embryonic exposure to sublethal concentrations of carbaryl induced higher tolerance to carbaryl and increased cross-tolerance to malathion and cypermethrin but not to chlorpyrifos or permethrin. In one case, the embryonic exposure to carbaryl induced tolerance in a nonlinear pattern (hormesis). These results demonstrate that that the newly discovered phenomenon of induced tolerance also provides induced cross-tolerance that is not restricted to pesticides with the same mode of action.

Independent degeneration of W and Y sex chromosomes in frog Rana rugosa.

The frog Rana rugosa uniquely possesses two different sex-determining systems of XX/XY and ZZ/ZW, separately in the geographic populations. The sex chromosomes of both types share the same origin at chromosome 7, and the structural differences between X and Y or Z and W were evolved through two inversions. In order to ascertain the mechanisms of degeneration of W and Y chromosomes, we gynogenetically produced homozygous diploids WW and YY and examined their viability. Tadpoles from geographic group N (W(N)W(N)) containing three populations died of edema at an early developmental stage within 10 days after hatching, while tadpoles from the geographic group K (W(K)W(K)) that contained two populations died of underdeveloped growth at a much later stage, 40-50 days after fertilization. On the contrary, W(N)W(K) and W(K)W(N) hybrid embryos were viable, successfully passed the two lethal stages, and survived till the attainment of adulthood. The observed survival implies that the lethal genes of the W chromosomes are not shared by the two groups and thus demonstrates their independent degeneration histories between the local groups. In sharp contrast, a sex-linked gene of androgen receptor gene (AR) from the W chromosome was down-regulated in expression in both the groups, suggesting that inactivation of the W-AR allele preceded divergence of the two groups and appearance of the lethal genes. Besides, the YY embryos died of cardiac edema immediately after hatching. The symptom of lethality and the stage of developmental arrest differed from those for either of WW lethal embryos. We therefore conclude that the W and Y chromosomes involve no evolutionary common scenario for degeneration.

The gastrocoel roof plate in embryos of different frogs.

The morphology of the gastrocoel roof plate and the presence of cilia in this structure were examined in embryos of four species of frogs. Embryos of Ceratophrys stolzmanni (Ceratophryidae) and Engystomops randi (Leiuperidae) develop rapidly, provide comparison for the analysis of gastrocoel roof plate development in the slow-developing embryos of Epipedobates machalilla (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Embryos of the analyzed frogs develop from eggs of different sizes, and display different reproductive and developmental strategies. In particular, dorsal convergence and extension and archenteron elongation begin during gastrulation in embryos of rapidly developing frogs, as in Xenopus laevis. In contrast, cells that involute during gastrulation are stored in the large circumblastoporal collar that develops around the closed blastopore in embryos of slow-developing frogs. Dorsal convergence and extension only start after blastopore closure in slow-developing frog embryos. However, in the neurulae, a gastrocoel roof plate develops, despite the accumulation of superficial mesodermal cells in the circumblastoporal collar. Embryos of all four species develop a ciliated gastrocoel roof plate at the beginning of neurulation. Accordingly, fluid-flow across the gastrocoel roof plate is likely the mechanism of left-right asymmetry patterning in these frogs, as in X. laevis and other vertebrates. A ciliated gastrocoel roof plate, with a likely origin as superficial mesoderm, is conserved in frogs belonging to four different families and with different modes of gastrulation.

Larval morphology of dart-poison frogs (Anura: Dendrobatoidea: Aromobatidae and Dendrobatidae).

Tadpoles in the superfamily Dendrobatoidea (families Aromobatidae and Dendrobatidae), housed in zoological collections or illustrated in publications, were studied. For the most part, tadpoles of species within the family Aromobatidae, the subfamilies Colostethinae and Hyloxalinae (of the family Dendrobatidae), and those of the genus Phyllobates, Dendrobatinae (Dendrobatidae) have slender anterior jaw sheaths with a medial notch and slender lateral processes, triangular fleshy projections on the inner margin of the nostrils and digestive tube with constant diameter and color and its axis sinistrally directed, concealing the liver and other organs. These morphologies are different from the ones observed in tadpoles of species included in the Dendrobatinae (minus Phyllobates). Exceptions to these morphological arrangements are noted, being the digestive system arrangement and the nostril ornamentation more plastic than the shape of the upperjaw sheath. Tadpoles of all species of the Dendrobatoidea have similar disposition of digestive organs in early stages, but differentiate in late stages of development. Classifying the upper jaw sheath into the two recognized states is possible from very early stages of development, but gut disposition and nostril ornamentation cannot be determined until late in development, making classification and taxonomic assignment of tadpoles based on these morphological features challenging.

Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla.

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.

[Crucial stages of embryogenesis of Rana arvalis: Part 2. Development of head structures].

Analysis of the dynamics of variation in developing head structures of moor frog (R. arvalis) tadpoles has made it possible to reveal periods in which the limits of variation in relevant traits are narrowed. In the course of individual development, these so-called crucial periods for certain traits are followed by such periods for other traits. However, crucial stages for some head structures have not been revealed, which can be explained by a relatively short period of development considered in the study. Analysis of individual variation at later stages may provide the possibility to identify crucial periods for these structures.

Molecular characterization and expression of a heat-shock cognate 70 (Hsc70) and a heat-shock protein 70 (Hsp70) cDNAs in Rana (Pelophylax) lessonae embryos.

Heat-Shock Protein 70 (HSP70) is a class of highly conserved proteins which is involved in essential functions as molecular chaperones and in the acquired tolerance processes. In this work, two cDNAs encoding a constitutive Hsc70 and an inducible Hsp70 from the water frog Rana (Pelophylax) lessonae, belonging to the Rana (P.) esculenta complex of central Italy, have been isolated and characterized. The two cDNA clones, named Rl-Hsc70 and Rl-Hsp70, encode 646 and 640 amino acid proteins respectively, which present extremely conserved functional domains characteristic of cytosolic members of the HSP70 family. Comparative studies of the amino acid sequences showed that Rl-Hsc70 and Rl-Hsp70 had the highest homology with constitutive and inducible HSP70 members of other amphibian species. The phylogenetic analysis also demonstrated a separate clustering of the Rl-Hsc70 and Rl-Hsp70 with constitutive and inducible members from other vertebrate species. Heat-inducibility assays performed during embryogenesis showed that the two isolated mRNAs displayed different expression profiles. Rl-Hsp70 was induced only in heat shock-treated embryos, whereas Rl-Hsc70 transcript levels, which were constitutively modulated in non-stressed embryos, did not increase following the heat treatment. In situ hybridization experiments demonstrated that both transcripts showed a tissue-specific enrichment in the central nervous system and in the somites.

Effect of road deicing salt on the susceptibility of amphibian embryos to infection by water molds.

Some causative agents of amphibian declines act synergistically to impact individual amphibians and their populations. In particular, pathogenic water molds (aquatic oomycetes) interact with environmental stressors and increase mortality in amphibian embryos. We documented colonization of eggs of three amphibian species, the wood frog (Rana sylvatica), the green frog (Rana clamitans), and the spotted salamander (Ambystoma maculatum), by water molds in the field and examined the interactive effects of road deicing salt and water molds, two known sources of mortality for amphibian embryos, on two species, R. clamitans and A. maculatum in the laboratory. We found that exposure to water molds did not affect embryonic survivorship in either A. maculatum or R. clamitans, regardless of the concentration of road salt to which their eggs were exposed. Road salt decreased survivorship of A. maculatum, but not R. clamitans, and frequency of malformations increased significantly in both species at the highest salinity concentration. The lack of an effect of water molds on survival of embryos and no interaction between road salt and water molds indicates that observations of colonization of these eggs by water molds in the field probably represent a secondary invasion of unfertilized eggs or of embryos that had died of other causes. Given increasing salinization of freshwater habitats on several continents and the global distribution of water molds, our results suggest that some amphibian species may not be susceptible to the combined effects of these factors, permitting amphibian decline researchers to devote their attention to other potential causes.

Toxic effects of 1-methyl-3-octylimidazolium bromide on the early embryonic development of the frog Rana nigromaculata.

Toxic effects of 1-methyl-3-octylimidazolium bromide ([C8mim]Br) on the early embryonic development of the frog Rana nigromaculata were evaluated. Frog embryos in different developmental stages (early cleavage, early gastrula, or neural plate) were exposed to 0, 45, 63, or 88.2 mg/L of the ionic liquid [C8mim]Br for 96 h. The 96-h median lethal concentration values at the early cleavage, early gastrula, and neural plate stages of development were 85.1, 43.4, and 42.4 mg/L, respectively. In embryos exposed to [C8mim]Br, the duration of embryo dechorionation was prolonged in the early cleavage and neural plate, but not the early gastrula, stages of development compared with control embryos. Embryos in the neural plate developmental stage were found to have the highest mortality rate following [C8mim]Br exposure. These results suggest that [C8mim]Br has toxic effects on the early embryonic development of the frog.

Application of metamorphosis assay to a native Japanese amphibian species, Rana rugosa, for assessing effects of thyroid system affecting chemicals.

The aims of this study were to assess the utility of a metamorphosis assay for detecting thyroid hormone-disrupting chemicals using Rana rugosa, a domestic frog species in Japan, and to compare species differences in sensitivity to thyroxine (T(4)) and propylthiouracil (PTU) among R. rugosa, Xenopus laevis and Xenopus (Silurana) tropicalis. Tadpoles of R. rugosa (TK stages III/IV) were exposed to standard test chemicals for acceleration (T(4)) and inhibition (PTU) of metamorphosis for 28 days in semi-static condition and total body length and developmental stage (TK stage) were recorded every week. T(4) (0.61 and 2.24 microg/L in actual concentrations) and PTU (19.73, 76.83, and 155.67 mg/L in actual concentrations) induced significant acceleration and inhibition of metamorphosis, respectively. The present results indicate that the metamorphosis assay is successfully applied to the domestic frog species, R. rugosa, suggesting this assay can be used for the assessment of chemicals on ecological impacts in wild frog species.

Learning by embryos and the ghost of predation future.

Most research on the effects of exposure to stressful stimuli during embryonic development has focused on post-embryonic behaviour that appears to be abnormal or maladaptive. Here, we tested whether exposure to some stressful stimuli (predatory cues) can lead to post-embryonic behaviour that is adaptive. When eggs of ringed salamanders (Ambystoma annulatum) were exposed to chemical cues from predators, post-hatching larvae showed reduced activity and greater shelter-seeking behaviour; larvae that had been exposed to control cues did not show these behaviours. In addition, wood frog (Rana sylvatica)tadpoles learned to respond to chemical cues from unfamiliar predators with danger based on embryonic conditioning. Therefore, if embryonic experience is a good predictor of future risk, learning associated with exposure to negative stimuli during development may be adaptive.

Immunohistochemical detection and biological activities of CYP17 (P450c17) in the indifferent gonad of the frog Rana rugosa.

Sex steroids play a crucial role in the gonad differentiation in various species of vertebrates. However, little is known regarding the localization and biological activity of steroid-metabolizing enzymes during gonadal sex differentiation in amphibians. In the present study, we showed by real-time RT-PCR analysis that the expression of CYP17, one of the key steroidogenic enzymes, was higher in the indifferent gonad during sex differentiation in male than in female tadpoles of Rana rugosa but that there was no difference detected in the 3betaHSD mRNA level between the male and female gonads. We next examined the localization of CYP17, 3betaHSD and 17betaHSD in the indifferent and differentiating gonads by using three kinds of antibodies specific for CYP17, 3betaHSD and 17betaHSD, respectively. Positive signals for CYP17, 3betaHSD and 17betaHSD were observed in somatic cells of the indifferent gonad of males and in the interstitial cell of the testis. The enzymatic activity of CYP17 was also examined in the gonad during sex differentiation in this species. [(3)H]Progesterone (Prog) was converted to [(3)H]androstenedione (AE) in the indifferent gonad in males and females, but the rate of its conversion was higher in males than in females. Moreover, fluorescence in situ hybridization (FISH) analysis revealed that the CYP17 gene was located on the q arm of chromosome 9, indicating that CYP17 was autosomal in R. rugosa. Taken together, the results demonstrate that the CYP17 protein is synthesized in somatic cells of the indifferent gonad during gonadal sex differentiation in R. rugosa and that it is more active in converting Prog to AE in males than in females. The data suggest that CYP17 may be involved in testicular formation during sex differentiation in this species.