Transcriptomic profiling of Rana [Lithobates] catesbeiana back skin during natural and thyroid hormone-induced metamorphosis under different temperature regimes with particular emphasis on innate immune system components.

As amphibians undergo thyroid hormone (TH)-dependent metamorphosis from an aquatic tadpole to the terrestrial frog, their innate immune system must adapt to the new environment. Skin is a primary line of defense, yet this organ undergoes extensive remodelling during metamorphosis and how it responds to TH is poorly understood. Temperature modulation, which regulates metamorphic timing, is a unique way to uncover early TH-induced transcriptomic events. Metamorphosis of premetamorphic tadpoles is induced by exogenous TH administration at 24 °C but is paused at 5 °C. However, at 5 °C a "molecular memory" of TH exposure is retained that results in an accelerated metamorphosis upon shifting to 24 °C. We used RNA-sequencing to identify changes in Rana (Lithobates) catesbeiana back skin gene expression during natural and TH-induced metamorphosis. During natural metamorphosis, significant differential expression (DE) was observed in >6500 transcripts including classic TH-responsive transcripts (thrb and thibz), heat shock proteins, and innate immune system components: keratins, mucins, and antimicrobial peptides (AMPs). Premetamorphic tadpoles maintained at 5 °C showed 83 DE transcripts within 48 h after TH administration, including thibz which has previously been identified as a molecular memory component in other tissues. Over 3600 DE transcripts were detected in TH-treated tadpoles at 24 °C or when tadpoles held at 5 °C were shifted to 24 °C. Gene ontology (GO) terms related to transcription, RNA metabolic processes, and translation were enriched in both datasets and immune related GO terms were observed in the temperature-modulated experiment. Our findings have implications on survival as climate change affects amphibia worldwide.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles.

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a "molecular memory" of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3'-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

Analysis of evolutionary and functional features of the bullfrog SULT1 family.

We identified the bullfrog Rana catesbeiana sulfotransferase 1 (SULT1) family from the BLAST search tool of the public databases based on the SULT1 families of Nanorana parkeri, Xenopus laevis, and Xenopus tropicalis as queries, revealing the characteristics of the anuran SULT1 family. The results showed that the anuran SULT1 family comprises six subfamilies, four of which were related to the mammalian SULT1 subfamily. Additionally, the bullfrog has two SULT1Cc subfamily members that are consistent with the characteristics of the expanded Xenopus SULT1C subfamily. Several members of the bullfrog SULT1 family were suggested to play important roles in sulfation during metamorphosis. Among these, cDNAs encoding SULT1Cc1 and SULT1Y1 were cloned, and the sulfation activity was analyzed using recombinant proteins. The affinity for 2-naphthol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the enzymatic reaction rate were higher in SULT1Cc1 than in SULT1Y1. Both the enzymes showed inhibitory effect of many thyroid hormones (THs) analogs on the sulfation of 2-naphthol. The potency of sulfation activities of SULT1Cc1 and SULT1Y1 against T4 indicated their possible role in the intracellular T4 clearance during metamorphosis.

Natural occurrences and characterization of Elizabethkingia miricola infection in cultured bullfrogs (Rana catesbeiana).

Introduction: The bacterium Elizabethkingia miricola is a multispecies pathogen associated with meningitis-like disease that has been isolated from several amphibian species, including the bullfrog, but this is the first isolation in Guangxi. In the present study, the dominant bacteria were isolated from the brains of five bullfrogs with meningitis-like disease on a South China farm in Guangxi. Methods: The NFEM01 isolate was identified by Gram staining; morphological observations; 16S rRNA, rpoB, and mutT-based phylogenetic tree analysis; and physiochemical characterization and was subjected to drug sensitivity and artificial infection testing. Results and discussion: As a result of identification, the NFEM01 strain was found to be E. miricola. An artificial infection experiment revealed that NFEM01 infected bullfrogs and could cause symptoms of typical meningitis-like disease. As a result of the bacterial drug sensitivity test, NFEM01 is highly sensitive to mequindox, rifampicin, enrofloxacin, nitrofural, and oxytetracycline and there was strong resistance to gentamicin, florfenicol, neomycin, penicillin, amoxicillin, doxycycline, and sulfamonomethoxine. This study provides a reference to further study the pathogenesis mechanism of E. miricola-induced bullfrog meningitislike disease and its prevention and treatment.

Models and data of AMPlify: a deep learning tool for antimicrobial peptide prediction.

OBJECTIVES: Antibiotic resistance is a rising global threat to human health and is prompting researchers to seek effective alternatives to conventional antibiotics, which include antimicrobial peptides (AMPs). Recently, we have reported AMPlify, an attentive deep learning model for predicting AMPs in databases of peptide sequences. In our tests, AMPlify outperformed the state-of-the-art. We have illustrated its use on data describing the American bullfrog (Rana [Lithobates] catesbeiana) genome. Here we present the model files and training/test data sets we used in that study. The original model (the balanced model) was trained on a balanced set of AMP and non-AMP sequences curated from public databases. In this data note, we additionally provide a model trained on an imbalanced set, in which non-AMP sequences far outnumber AMP sequences. We note that the balanced and imbalanced models would serve different use cases, and both would serve the research community, facilitating the discovery and development of novel AMPs. DATA DESCRIPTION: This data note provides two sets of models, as well as two AMP and four non-AMP sequence sets for training and testing the balanced and imbalanced models. Each model set includes five single sub-models that form an ensemble model. The first model set corresponds to the original model trained on a balanced training set that has been described in the original AMPlify manuscript, while the second model set was trained on an imbalanced training set.

Genetic structure of American bullfrog populations in Brazil.

Non-native species are a major problem affecting numerous biomes around the globe. Information on their population genetics is crucial for understanding their invasion history and dynamics. We evaluated the population structure of the non-native American bullfrog, Aquarana catesbeiana, in Brazil on the basis of 324 samples collected from feral and captive groups at 38 sites in seven of the nine states where feral populations occur. We genotyped all samples using previously developed, highly polymorphic microsatellite loci and performed a discriminant analysis of principal components together with Jost's D index to quantify pairwise differentiation between populations. We then amplified 1,047 base pairs of the mitochondrial cytochrome b (cytb) gene from the most divergent samples from each genetic population and calculated their pairwise differences. Both the microsatellite and cytb data indicated that bullfrogs comprise two populations. Population grouping 1 is widespread and possesses two cytb haplotypes. Population grouping 2 is restricted to only one state and possesses only one of the haplotypes from Population grouping 1. We show that there were two imports of bullfrogs to Brazil and that there is low genetic exchange between population groupings. Also, we find that there is no genetic divergence among feral and captive populations suggesting continuous releases. The limited genetic variability present in the country is associated to the small number of introductions and founders. Feral bullfrogs are highly associated to leaks from farms, and control measures should focus on preventing escapes using other resources than genetics, as feral and captive populations do not differ.

Invasive Bullfrogs Maintain MHC Polymorphism Including Alleles Associated with Chytrid Fungal Infection.

Maintenance of genetic diversity at adaptive loci may facilitate invasions by non-native species by allowing populations to adapt to novel environments, despite the loss of diversity at neutral loci that typically occurs during founder events. To evaluate this prediction, we compared genetic diversity at major histocompatibility complex (MHC) and cytochrome b (cytb) loci from 20 populations of the American bullfrog (Rana catesbeiana) across theinvasive and native ranges in North America and quantified the presence of the pathogen Batrachochytrium dendrobatidis (Bd). Compared to native populations, invasive populations had significantly higher Bd prevalence and intensity, significantly higher pairwise MHC and cytb FST, and significantly lower cytb diversity, but maintained similar levels of MHC diversity. The two most common MHC alleles (LiCA_B and Rapi_33) were associated with a significant decreased risk of Bd infection, and we detected positive selection acting on four peptide binding residues. Phylogenetic analysis suggested invasive populations likely arose from a single founding population in the American Midwest with a possible subsequent invasion in the northwest. Overall, our study suggests that the maintenance of diversity at adaptive loci may contribute to invasion success and highlights the importance of quantifying diversity at functional loci to assess the evolutionary potential of invasive populations.

Microcystin-leucine arginine exposure induced intestinal lipid accumulation and MC-LR efflux disorder in Lithobates catesbeianus tadpoles.

Few studies exist on the toxic effects of chronic exposure to microcystins (MCs) on amphibian intestines, and the toxicity mechanisms are unclear. Here, we evaluated the impact of subchronic exposure (30 days) to environmentally realistic microcystin-leucine arginine (MC-LR) concentrations (0 µg/L, 0.5 µg/L and 2 µg/L) on tadpole (Lithobates catesbeianus) intestines by analyzing the histopathological and subcellular microstructural damage, the antioxidative and oxidative enzyme activities, and the transcriptome levels. Histopathological results showed severe damage accompanied by inflammation to the intestinal tissues as the MC-LR exposure concentration increased from 0.5 µg/L to 2 µg/L. RNA-sequencing analysis identified 634 and 1,147 differentially expressed genes (DEGs) after exposure to 0.5 µg/L and 2 µg/L MC-LR, respectively, compared with those of the control group (0 µg/L). Biosynthesis of unsaturated fatty acids and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were upregulated in the intestinal tissues of the exposed groups, with many lipid droplets being observed on transmission electron microscopy, implying that MC-LR may induce lipid accumulation in frog intestines. Moreover, 2 µg/L of MC-LR exposure inhibited the xenobiotic and toxicant biodegradation related to detoxification, implying that the tadpoles' intestinal detoxification ability was weakened after exposure to 2 µg/L MC-LR, which may aggravate intestinal toxicity. Lipid accumulation and toxin efflux disorder may be caused by MC-LR-induced endoplasmic reticular stress. This study presents new evidence that MC-LR harms amphibians by impairing intestinal lipid metabolism and toxin efflux, providing a theoretical basis for evaluating the health risks of MC-LR to amphibians.

Relationship between serum thyroid hormones and their associated metabolites, and gene expression bioindicators in the back skin of Rana [Lithobates] catesbeiana tadpoles and frogs during metamorphosis.

Anuran metamorphosis is characterized by profound morphological changes including remodeling of tissues and organs. This transition is initiated by thyroid hormones (THs). However, the current knowledge of changing levels of THs during metamorphosis relies on pooled samples using methods known for high variability with sparse reporting of measured variation. Moreover, establishing a clear linkage between key gene expression bioindicators and TH levels throughout the metamorphic process is needed. Using state-of-the-art ultra-high performance liquid chromatography isotope-dilution tandem mass spectrometry, we targeted 12 THs and metabolites in the serum of Rana [Lithobates] catesbeiana (n=5-10) across seven distinct postembryonic stages beginning with premetamorphic tadpoles (Gosner stage 31-33) and continuing through metamorphosis to a juvenile frog (Gosner stage 46). TH levels were related to TH-relevant gene transcripts (thra, thrb, and thibz) in back skin of the same individual animals. Significant increases from basal levels were observed for thyroxine (T4) and 3,3',5-triiodothyronine (T3) at Gosner stage 41, reaching maximal levels at Gosner stage 44 (28 ± 10 and 2.3 ± 0.5 ng/mL, respectively), and decreasing to basal levels in juvenile frogs. In contrast, 3,5-diiodothyronine (T2) increased significantly at Gosner stage 40 and was maintained elevated until stage 44. While thra transcript levels remained constant and then decreased at the end of metamorphic climax, thrb and thibz were induced to maximal levels at Gosner stage 41, followed by a decrease to basal levels in the froglet. This exemplifies the exquisite timing of events during metamorphosis as classic early response genes are transcribed in anticipation of peak TH concentrations. The distinct T2 concentration profile suggests a biological role of this biomolecule in anuran postembryonic development and an additional aspect that may be a target of anthropogenic chemicals that can disrupt anuran metamorphosis and TH signalling. Hence, as a second aim of the study, we set out to find additional bioindicators of metamorphosis, which can aid future investigations of developmental disruption. Using a sensitive nanoLC-Orbitrap system an untargeted analysis workflow was applied. Among 6,062 endogenous metabolites, 421 showed metamorphosis-dependent concentration dynamics. These potential bioindicators included several carnitines, prostaglandins and some steroid hormones.

Accurate detection and quantification of seasonal abundance of American bullfrog (Lithobates catesbeianus) using ddPCR eDNA assays.

The invasive American bullfrog (Lithobates catesbeianus) imperils freshwater biodiversity worldwide. Effective management hinges on early detection of incipient invasions and subsequent rapid response, as established populations are extremely difficult to eradicate. Although environmental DNA (eDNA) detection methods provide a highly sensitive alternative to conventional surveillance techniques, extensive testing is imperative to generate reliable output. Here, we tested and compared the performance of two primer/probe assays to detect and quantify the abundance of bullfrogs in Western Europe in silico and in situ using digital droplet PCR (ddPCR). Although both assays proved to be equally target-specific and sensitive, one outperformed the other in ddPCR detection resolution (i.e., distinguishing groups of target-positive and target-negative droplets), and hence was selected for further analyses. Mesocosm experiments revealed that tadpole abundance and biomass explained 99% of the variation in eDNA concentration. Because per individual eDNA emission rates did not differ significantly among tadpoles and juveniles, and adults mostly reside out of the water, eDNA concentration can be used as an approximation of local bullfrog abundance in natural populations. Seasonal eDNA patterns in three colonized ponds showed parallel fluctuations in bullfrog eDNA concentration. An increase in eDNA concentration was detected in spring, followed by a strong peak coinciding with the breeding season (August, September or October), and continuously low eDNA concentrations during winter. With this study, we report the validation process required for appropriately implementing eDNA barcoding analyses in lentic systems. We demonstrate that this technique can serve as a solid and reliable tool to detect the early stages of bullfrog invasions and to quantify temporal changes in abundance that will be useful in coordinating large-scale bullfrog eradication programs and evaluating their efficiency.

Analysis of global and gene-specific acetylation of histones in the liver of American bullfrog (Rana catesbeiana) tadpoles acclimated to low temperature.

Severe environmental stressors such as low temperatures can affect gene expression by changing epigenetic states. American bullfrog (Rana catesbeiana) can overwinter as tadpoles, which can be active even in winter. However, the molecular mechanisms of epigenetic controls by which the tadpoles acclimate to low temperature are still unclear. In this study, we aimed to clarify the molecular mechanisms of global and gene-specific epigenetic regulations of low-temperature acclimation. We found that the global acetylation was decreased in the liver of bullfrog tadpoles acclimated to low temperature. The amounts of transcripts for two histone acetyltransferases were higher in the liver of tadpoles acclimated to low temperature than in those acclimated to warm temperature, while we observed no significant differences in the amounts of transcripts for histone deacetylases. We also found that the amounts of transcripts and acetylated histones on the specific temperature-responsive genes scd and cyp7a1 whose transcripts were increased and decreased, respectively, in response to low temperature were positively correlated. Cellular acetyl-CoA levels were higher in the liver of tadpoles acclimated to low temperature than in those acclimated to warm temperature. These results contradicted the states of histone acetylation, suggesting that bullfrog tadpoles have different epigenetic mechanisms to modify the histones when compared with those of other organisms such as reptiles and mammals, even though the relationship between the transcript amount and the states of histone acetylation on temperature-responsive genes was similar to that of mammals.

Genotoxic effects of 4-nonylphenol and Cyproterone Acetate on Rana catesbeiana (anura) tadpoles and juveniles.

Genotoxic analyses are commonly used in ecotoxicological studies as early biomarkers to investigate the potential effects of environmental contaminants on biological models. Several pollutants can induce DNA damage and, therefore, counting micronuclei and other nuclear abnormalities are efficient tools to evaluate genotoxicity. Some pollutants such as 4-nonylphenol (NP), a detergent used mainly in industries, and Cyproterone Acetate (CPA), an antiandrogenic medicine, have already shown genotoxic effects on some vertebrates. However, although amphibians are considered bioindicators of environmental quality and their populations are declining worldwide, the effects of these compounds on anurans are not yet known and, therefore, we believe that it is important to investigate such effects on anurans. Since water contamination is one of the ultimate causes of amphibian decline, ecotoxicological studies are important to discuss the appropriate solutions to avoid species extinction. Thus, this study investigates the genotoxic effects on Rana catesbeiana tadpoles and juveniles after being exposed to 1, 10 and 100 µg/L NP and 0.025, 0.25 and 2.5 ng/L CPA, by counting the nuclear abnormalities after exposure. The laboratory experiments lasted 28 days. The experimental conditions were the same except for the water volume since tadpoles and juveniles exhibit different habits at different developmental stages. Compared to juveniles, tadpoles were more susceptible to both compounds as indicated by the increased nuclear abnormalities observed in the highest NP concentration and all tested CPA concentrations. The juveniles, on the other hand, responded only to the two highest CPA concentrations. We concluded that CPA, even at very low concentrations, is extremely harmful to both anuran developmental stages and, particularly, to tadpoles. The significant effects observed on tadpoles is an important outcome of this study since 100 µg/L or higher NP concentrations are frequently detected in the environment.

Propagule pressure and hunting pressure jointly determine genetic evolution in insular populations of a global frog invader.

Islands are often considered to be more susceptible to biological invasions and to suffer greater impacts from invaders than mainland areas, and this difference is generally attributed to differences in species introductions, ecological factors or human activities between islands and mainland areas. Genetic variation, as a good estimate of evolutionary potential, can influence the invasion process and impacts of alien species. However, few studies have compared the genetic diversity of alien species between islands and a corresponding mainland. Here, we examined the genetic variation and differentiation in feral populations (30 sampled individuals/population) of a globally invasive species (the American bullfrog, Lithobates catesbeianus) that was extensively farmed on 14 islands in the Zhoushan Archipelago of China and in three nearby regions on the mainland. We quantified the relative importance of propagule pressure and hunting pressures on the genetic variation of bullfrog populations and found that insular populations have greater genetic variation than their mainland counterparts. Although genetic differentiation between the populations was observed, no evidence of recent bottlenecks or population expansion in any of the tested population was found. Our results suggest that the propagule pressures of bullfrogs escaping from farms, multiple releases and hunting pressure influence the genetic variation among bullfrog populations. These results might have important implications for understanding the establishment and evolution of alien species on islands and for the management of invasive species.

Seasonal acclimatization and thermal acclimation induce global histone epigenetic changes in liver of bullfrog (Lithobates catesbeianus) tadpole.

The American bullfrog (Lithobates catesbeianus) is a eurythermal amphibian that is naturally distributed from subarctic to subtropical areas. The tadpoles of this species overwinter, in water, in cold environments. Therefore, they may have adapted to a wide range of temperatures in an active state. To understand the adaptation mechanisms to cope with low or high temperatures, we investigated global epigenetic modifications, histone variants, transcript levels of related genes, and the cellular acetyl coenzyme A (acetyl-CoA) and free CoA (CoA-SH) levels, in the livers of tadpoles collected in summer and winter and of those acclimated to 4 °C and 21 °C. Among epigenetic marks tested, the levels of acetylated histones and the histone variant H2A.Z were influenced by different temperature conditions. Histone acetylation levels were higher in summer than in winter and increased within 3 days of warm acclimation, whereas histone H2A.Z levels were higher in winter than in summer and decreased within 2 weeks of warm acclimation. Transcript analysis revealed that decreased expression of histone H2A.Z in warm acclimation was regulated at the transcriptional level. Acetyl-CoA levels were not correlated with those of the acetylated histones, indicating that cellular acetyl-CoA levels may not directly influence the state of histone acetylation in the tadpole liver. Such epigenetic and metabolic changes in the tadpole liver may contribute to the maintenance of energy balance during seasonal acclimatization and thermal acclimation.

Transcriptomics investigation of thyroid hormone disruption in the olfactory system of the Rana [Lithobates] catesbeiana tadpole.

Thyroid hormones (THs) regulate vertebrate growth, development, and metabolism. Despite their importance, there is a need for effective detection of TH-disruption by endocrine disrupting chemicals (EDCs). The frog olfactory system substantially remodels during TH-dependent metamorphosis and the objective of the present study is to examine olfactory system gene expression for TH biomarkers that can evaluate the biological effects of complex mixtures such as municipal wastewater. We first examine classic TH-response gene transcripts using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) in the olfactory epithelium (OE) and olfactory bulb (OB) of premetamorphic Rana (Lithobates) catesbeiana tadpoles after 48 h exposure to biologically-relevant concentrations of the THs, 3,5,3'-triiodothyronine (T3) and L-thyroxine (T4), or 17-beta estradiol (E2); a hormone that can crosstalk with THs. As the OE was particularly sensitive to THs, further RNA-seq analysis found >30,000 TH-responsive contigs. In contrast, E2 affected 267 contigs of which only 57 overlapped with THs suggesting that E2 has limited effect on the OE at this developmental phase. Gene ontology enrichment analyses identified sensory perception and nucleoside diphosphate phosphorylation as the top affected terms for THs and E2, respectively. Using classic and additional RNA-seq-derived TH-response gene transcripts, we queried TH-disrupting activity in municipal wastewater effluent from two different treatment systems: anaerobic membrane bioreactor (AnMBR) and membrane enhanced biological phosphorous removal (MEBPR). While we observed physical EDC removal in both systems, some TH disruption activity was retained in the effluents. This work lays an important foundation for linking TH-dependent gene expression with olfactory system function in amphibians.

Effect of ultra-high pressure on molecular structure and properties of bullfrog skin collagen.

Ultra-high pressure technology has attracted a great deal of attention in recent years, and has been widely used in food science, medicine, and other fields. This study aimed to determine the effect of ultra-high pressure on the structure and properties of collagen. Native collagen extracted from bullfrog skin was processed under different ultra-high pressure treatment conditions (300, 400, and 500MPa). Then systematic analysis of the molecular structures and properties of the samples after ultra-high pressure treatment were performed. It was found that the conformation of collagen molecules could be adjusted by ultra-high pressure treatment, and this regulation was closely related to the level of treatment pressure. A possible mechanism of the impact of ultra-high pressure on the collagen molecular structures was speculated according to the experimental results. At low pressure levels (300-400MPa), the pressure perpendicular to collagen axis dominates and leads to a tightening of the triple helix structure of collagen, while the pressure parallel to collagen axis is dominant and the triple helix tends to dissociate like a zipper at high pressure levels (>400MPa). These structural changes would simultaneously result in various changes to thermal stability, self-assembly properties, and antigenicity of collagen.

Behavioral and molecular analyses of olfaction-mediated avoidance responses of Rana (Lithobates) catesbeiana tadpoles: Sensitivity to thyroid hormones, estrogen, and treated municipal wastewater effluent.

Olfaction is critical for survival, facilitating predator avoidance and food location. The nature of the olfactory system changes during amphibian metamorphosis as the aquatic herbivorous tadpole transitions to a terrestrial, carnivorous frog. Metamorphosis is principally dependent on the action of thyroid hormones (THs), l-thyroxine (T4) and 3,5,3'-triiodothyronine (T3), yet little is known about their influence on olfaction during this phase of postembryonic development. We exposed Taylor Kollros stage I-XIII Rana (Lithobates) catesbeiana tadpoles to physiological concentrations of T4, T3, or 17-beta-estradiol (E2) for 48h and evaluated a predator cue avoidance response. The avoidance response in T3-exposed tadpoles was abolished while T4- or E2-exposed tadpoles were unaffected compared to control tadpoles. qPCR analyses on classic TH-response gene transcripts (thra, thrb, and thibz) in the olfactory epithelium demonstrated that, while both THs produced molecular responses, T3 elicited greater responses than T4. Municipal wastewater feed stock was spiked with a defined pharmaceutical and personal care product (PPCP) cocktail and treated with an anaerobic membrane bioreactor (AnMBR). Despite substantially reduced PPCP levels, exposure to this effluent abolished avoidance behavior relative to AnMBR effluent whose feed stock was spiked with vehicle. Thibz transcript levels increased upon exposure to either effluent indicating TH mimic activity. The present work is the first to demonstrate differential TH responsiveness of the frog tadpole olfactory system with both behavioral and molecular alterations. A systems-based analysis is warranted to further elucidate the mechanism of action on the olfactory epithelium and identify further molecular bioindicators linked to behavioral response disruption.

The North American bullfrog draft genome provides insight into hormonal regulation of long noncoding RNA.

Frogs play important ecological roles, and several species are important model organisms for scientific research. The globally distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Unfortunately, there are currently no genomic resources for the former, important group of amphibians. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines. We report a 5.8 Gbp (NG50 = 69 kbp) genome assembly of a representative North American bullfrog (Rana [Lithobates] catesbeiana). The genome contains over 22,000 predicted protein-coding genes and 6,223 candidate long noncoding RNAs (lncRNAs). RNA-Seq experiments show thyroid hormone causes widespread transcriptional change among protein-coding and putative lncRNA genes. This initial bullfrog draft genome will serve as a key resource with broad utility including amphibian research, developmental biology, and environmental research.

Influence of Temperature on the Thyroidogenic Effects of Diuron and Its Metabolite 3,4-DCA in Tadpoles of the American Bullfrog (Lithobates catesbeianus).

Temperature is a key variable affecting the timing of amphibian metamorphosis from tadpoles to tetrapods, through the production and subsequent function of thyroid hormones (TH). Thyroid function can be impaired by environmental contaminants as well as temperature. Tadpoles can experience large temperature fluctuations in their habitats and many species are distributed in areas that may be impacted by agriculture. Diuron is a widely used herbicide detected in freshwater ecosystems and may impact endocrine function in aquatic organisms. We evaluated the influence of temperature (28 and 34 °C) on the action of diuron and its metabolite 3,4-dichloroaniline (3,4-DCA) on thyroid function and metamorphosis in tadpoles of Lithobates catesbeianus. Exposure to both compounds induced more pronounced changes in gene expression and plasma 3,3',5-triiodothyronine (T3) concentrations in tadpoles treated at higher temperature. T3 concentrations were increased in tadpoles exposed to 200 ng/L of diuron at 34 °C and an acceleration of metamorphosis was observed for the same group. Transcriptomic responses included alteration of thyroid hormone induced bZip protein (thibz), deiodinases (dio2, dio3), thyroid receptors (trα, trß) and Krüppel-like factor 9 (klf9), suggesting regulation by temperature on TH-gene expression. These results suggest that environmental temperature should be considered in risk assessments of environmental contaminants for amphibian species.

RNase activity of sialic acid-binding lectin from bullfrog eggs drives antitumor effect via the activation of p38 MAPK to caspase-3/7 signaling pathway in human breast cancer cells.

Sialic acid-binding lectin obtained from bullfrog eggs (SBL) induces cell death in cancer cells but not in normal cells. This antitumor effect is mediated through its ribonuclease (RNase) activity. However, the underlying molecular mechanisms remain unclear. We found that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated when SBL induced cell death in three human breast cancer cell lines: SK-BR-3, MCF-7, and MDA­MB231. The suppression of p38 MAPK phosphorylation by a p38 MAPK inhibitor as well as short interference RNA knockdown of p38 MAPK expression significantly decreased cell death and increased the cell viability of SBL-treated MDA­MB231 cells. H103A, an SBL mutant lacking in RNase activity, showed decreased SBL-induced cell death compared with native SBL. However, the loss of RNase activity of SBL had no effect on its internalization into cells. The H103A mutant also displayed decreased phosphorylation of p38 MAPK. Moreover, SBL promoted caspase­3/7 activation followed by a cleavage of poly (ADP-ribose)-polymerase, whereas the SBL mutant, H103A, lost this ability. The SBL-induced caspase­3/7 activation was suppressed by the p38 MAPK inhibitor, SB203580, as well as pan-caspase inhibitor, zVAD-fmk. In the presence of zVAD-fmk, the SBL-induced cell death was decreased. In addition, the cell viability of SBL-treated MDA­MB231 cells recovered by zVAD-fmk treatment. Taken together, our results suggest that the RNase activity of SBL leads to breast cancer cell death through the activation of p38 MAPK followed by the activation of caspase­3/7.