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.

The publications of embryologist Lev V. Beloussov.

A list of papers and books of the late Lev V. Beloussov was compiled and is available in Word and EndNote Supplements. The breadth of his work is briefly described.

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.

Effects of the herbicides linuron and S-metolachlor on Perez's frog embryos.

Presence of pesticides in the environment and their possible effects on aquatic organisms are of great concern worldwide. The extensive use of herbicides in agricultural areas are one of the factors contributing to the known decline of amphibian populations. Thus, as non-target species, amphibians can be exposed in early life stages to herbicides in aquatic systems. In this context, this study aims to evaluate effects of increasing concentrations of two maize herbicides, linuron and S-metolachlor on embryos of the Perez' frog (Pelophylax perezi) during 192 h. Apical endpoints were determined for each herbicide: mortality, hatching rate, malformations and length. Frog embryos presented a LC50 of 21 mg/l linuron and 37.5 mg/l S-metolachlor. Furthermore, sub-lethal concentrations of both herbicides affected normal embryonic development, delaying hatching, decreasing larvae length and causing several malformations. Length of larvae decreased with increasing concentrations of each herbicide, even at the lower concentrations tested. Malformations observed in larvae exposed to both herbicides were oedemas, spinal curvature and deformation, blistering and microphtalmia. Overall, these results highlight the need to assess adverse effects of xenobiotics to early life stages of amphibians regarding beside mortality the embryonic development, which could result in impairments at later stages. However, to unravel mechanisms involved in toxicity of these herbicides further studies regarding lower levels of biological organisation such as biochemical and genomic level should be performed.

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.

Toxic effects of NH4+-N on embryonic development of Bufo gargarizans and Rana chensinensis.

Although nitrogen fertilizer is commonly used worldwide, little information is currently available about NH4+-N toxicity on amphibians. This study determined the acute and chronic toxic effects of NH4+-N on two native Chinese amphibian species (Bufo gargarizans and Rana chensinensis), and compared the negative sensitivity of different embryos to NH4+-N. Static renewal aqueous exposures were performed using B. gargarizans and R. chensinensis embryos at Gosner stage 2 over 96 h. In terms of 96 h-LC50, B. gargarizans and R. chensinensis embryos had significantly different responses to NH4+-N, and the latter was more sensitive to NH4+-N than the former. In the chronic toxicity test, exposure to 10 mg L-1NH4+-N or higher significantly decreased the hatching rate of embryos in both species. Significant increases in the abnormality rate of embryos at 50 mg L-1NH4+-N or higher were observed and morphological abnormalities were characterized by axial flexures, yolk sac edema, and hyperplasia in both species. Additionally, the total length of embryos decreased in a dose-dependent manner after exposure to NH4+-N. The results indicate that NH4+-N exposure can increase abnormality and inhibit the hatching and development of embryos in B. gargarizans and R. chensinensis.

Effects of 4-MBC and triclosan in embryos of the frog Pelophylax perezi.

The widespread and increasing use of personal care products (PCPs) have led to environmental contamination by substances included in these products. These substances have been detected in aquatic compartments and shown to cause adverse effects on non-target aquatic organisms. In this work toxicity of the antimicrobial triclosan (TCS) and of the UV-filter 3-(4-methylbenzylidene) camphor (4-MBC) was assessed in the embryos of Perez' frog Pelophylax perezi. Lethal and sub-lethal parameters were evaluated in embryos in Gosner stage 8-9 exposed to 0.00013-1.3 mg/l of 4-MBC and 0.25-2.50 mg/l of TCS during 144 h. Survival, malformations, length and hatching were evaluated as apical endpoints. Biomarkers of neurotransmission, oxidative stress, energy metabolism and estrogenicity were determined at the biochemical level through the activities of cholinesterase (ChE), catalase (CAT), glutathione S-transferase (GST), lactate dehydrogenase (LDH) and levels of lipid peroxidation (LPO) and vitellogenin (Vtg). Embryo exposure to 4-MBC led to few developmental malformations (up to 3%) and a GST induction at 0.013 mg/l. Triclosan exposure reduced survival, delayed hatching (at 72 h) and development and induced malformations. In addiction ChE was inhibited in the highest concentrations tested and GST and LDH were induced at 0.79 mg/l, the LOEC registered for TCS in Perez' frogs. Overall, our study showed that TCS might exert adverse effects on P. perezi early life stages, but only at four orders of magnitude above the concentrations found in environment. Furthermore, our results highlight the need to assess PCPs toxicity at different levels of biological organization.

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.

Acid stress mediated adaptive divergence in ion channel function during embryogenesis in Rana arvalis.

Ion channels and pumps are responsible for ion flux in cells, and are key mechanisms mediating cellular function. Many environmental stressors, such as salinity and acidification, are known to severely disrupt ionic balance of organisms thereby challenging fitness of natural populations. Although ion channels can have several vital functions during early life-stages (e.g. embryogenesis), it is currently not known i) how developing embryos maintain proper intracellular conditions when exposed to environmental stress and ii) to what extent environmental stress can drive intra-specific divergence in ion channels. Here we studied the moor frog, Rana arvalis, from three divergent populations to investigate the role of different ion channels and pumps for embryonic survival under acid stress (pH 4 vs 7.5) and whether populations adapted to contrasting acidities differ in the relative role of different ion channel/pumps. We found that ion channels that mediate Ca(2+) influx are essential for embryonic survival under acidic pH, and, intriguingly, that populations differ in calcium channel function. Our results suggest that adaptive divergence in embryonic acid stress tolerance of amphibians may in part be mediated by Ca(2+) balance. We suggest that ion flux may mediate adaptive divergence of natural populations at early life-stages in the face of environmental stress.

Role of sediments in modifying the toxicity of two Roundup formulations to six species of larval anurans.

The role of sediment in modifying the toxicity of the original formulation of Roundup® and Roundup WeatherMAX® was examined in aqueous laboratory tests. Six species of anurans (Bufo fowleri, Hyla chrysoscelis, Rana catesbeiana, Rana clamitans, Rana sphenocephala, and Rana pipiens) were exposed at Gosner stage 25 to concentrations of the 2 herbicide formulations in 96-h, static, nonrenewal experiments in the presence and absence of sediment. All species tested had lower median lethal concentration values in water-only exposures of both formulations compared with exposures with sediment. Sediment significantly altered the potency slopes in all tests with the exceptions of H. chrysoscelis and R. clamitans when exposed to the original formulation of Roundup and H. chrysoscelis and R. sphenocephala when exposed to Roundup WeatherMAX. Thresholds were significantly different in all tests, including those in which potency slopes did not differ. Based on water-sediment exposures of the original formulation of Roundup, all 6 species tested had a margin of safety when compared with the predicted environmental concentration of the highest label application rate. Of the 6 species, 5 had a margin of safety when exposed to Roundup WeatherMAX. During incidental exposures in the field, sediments and organic matter present in aquatic systems provide significant sources of environmental ligands. If used according to label instructions, both herbicides should pose minimal risk to anuran amphibians in actual field applications. Environ Toxicol Chem 2014;33:2616-2620. © 2014 SETAC.

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.

Comparison of embryo toxicity using two classes of aquatic vertebrates.

Toxicity tests of musk ketone (MK) and tetrabromobisphenol-A (TBBPA) on embryos were conducted in two amphibian species, Xenopus (Silurana) tropicalis and the Swedish native species Rana arvalis. TBBPA was also tested on fish embryos of Danio rerio. All species were tested in similar experimental setup. Musk ketone caused decreased heart rates at concentrations from 10 and 100 µg/L in R. arvalis and X. tropicalis, respectively. TBBPA caused effects at 1000 µg/L in all three species. The responses were comparable between all three species which supports the relevance for using data from non-native species in national risk assessment.

A view of amphibian embryology during the last century.

Having started working in the field of amphibian embryology over 50 years ago, I make some comments about the changes that seem to me to have taken place in this field over this period. Over the period 1885 to 1960, much of the highly regarded experimental embryology was conducted on amphibian eggs and embryos. Indeed, much of this work was conducted in Germany and Switzerland using eggs and embryos of European newts (salamanders) and frogs of the Rana group. Xenopus started to be used extensively after the 1950s because eggs and embryos could be obtained throughout the year by hormone injection and because sexually mature animals could be raised from an egg within one year. Since the 1960s, publications using Xenopus have exceeded those using other amphibian species by 100-fold. This short commentary highlights some of the major advances attributable to embryological work with Amphibia and exemplifies these advances by reference to those who have made conspicuous contributions in this area.

Variations in lethal and sublethal effects of cypermethrin among aquatic stages and species of anuran amphibians.

Despite the use of model species to predict the effects of chemicals in the environment, unpredicted variation in levels of risk to organisms from xenobiotics can be observed. Physiological and morphological differences between species and life stages may lead to differences in sensitivity, while seasonal and spatial variation in pesticide concentrations may affect the level of risk faced by organisms in the environment. Because anurans breed in aquatic habitats subject to contamination by runoff and spraying, they are particularly vulnerable to pesticides. In the present study, embryos, newly hatched larvae, and larvae with limb buds of 3 anuran amphibian species--Pseudacris regilla, Rana cascadae, and Rana aurora--were exposed for 48 h to either 0.5 µg/L or 5.0 µg/L cypermethrin under laboratory conditions. The authors monitored hatching success, larval survival, and measured growth. Additionally, they assayed avoidance behavior 2 wk after exposure or 2 wk after hatching for individuals exposed as embryos. Hatching and survival were not affected in animals of any species exposed as embryos. After exposure as embryos and as newly hatched larvae, however, P. regilla displayed behavioral abnormalities in response to prodding. Cypermethrin increased mortality in P. regilla exposed in both larval stages. Cypermethrin also increased mortality in larval R. cascadae when exposed at the early stage. These results indicate variation in sensitivity to environmentally relevant concentrations of cypermethrin among anuran species and life stages.

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.

Accumulation and pharmacokinetics of estrogenic chemicals in the pre- and post-hatch embryos of the frog Rana rugosa.

Amphibian eggs spawned in water are exposed immediately to various chemicals present in their water. The present study aimed to investigate the accumulation and pharmacokinetics of 17α-ethynylestradiol (EE(2)), bisphenol A (BPA), and nonylphenol (NP), as well as 17ß-estradiol (E(2)), in the pre-hatch and post-hatch embryos of the frog Rana rugosa. Fertilized eggs were exposed to chemicals at a final concentration of 500 nM in breeding water for two days, then the embryos with jelly coats were reared in fresh-breeding water without supplementation of the xenoestrogens for six more days. All exogenous chemicals were concentrated in the embryo body at two days after fertilization, whereas their concentrations in the jelly coat were the same as those in the breeding water. The bioconcentration factors for E(2), EE(2), BPA, and NP were 217.9, 170.2, 382.3, and 289.1, respectively, suggesting that the estrogenic chemicals were concentrated in the embryo body through the jelly coat.

[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.

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.