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Animals must partition limited resources between their own growth and subsequent reproduction. Endocrine disruptors (ED) may cause maternal metabolic disorders that decrease successful reproduction and might be responsible for multi- and transgenerational effects in amphibians. We found that the frog Silurana (Xenopus) tropicalis, exposed to environmentally relevant concentrations of benzo[a]pyrene and triclosan throughout its life cycle, produced F1 females with delayed sexual maturity and decreased size and weight. These F1 females displayed a marked metabolic syndrome associated with decreased fasting plasma cholesterol and triglyceride concentrations and decreased gonadal development. F1 females from F0 exposed animals also had decreased reproductive investment highlighted by a decrease of oocyte lipid reserves associated with significant F2-tadpole mortality. F2 females from F0 exposed animals also displayed a marked metabolic syndrome but were able to correctly direct liver lipid metabolism to the constitution of fat bodies and oocyte yolk stores. In addition, the F2 females produced progeny that had normal mortality levels at 5 days post hatching compared to the controls suggesting a good reproductive investment. Our data confirmed that these ED, at concentrations often found in natural ponds, can induce multi- and transgenerational metabolic disorders in the progeny of amphibians that are not directly exposed. We present a hypothesis to explain the transmission of the metabolic syndrome across generations through modification of egg reserves. However, when high mortality occurred at the tadpole stage, surviving females were able to cope with metabolic costs and produce viable progeny through sufficient investment in the contents of oocyte reserves.

In order for Alberta's thick bitumen to be transported through pipelines, condensates are added creating a diluted bitumen (dilbit) mixture. Recent pipeline expansion projects have generated concern about potential dilbit spills on aquatic wildlife health. Studies have suggested that polycyclic aromatic compounds (PACs) are toxic to aquatic vertebrates and could potentially also interfere with their endocrine system. The research objectives of this study were to investigate the toxicity of dilbit to developing frog embryos and to identify the molecular mechanisms of action involved. Fertilized embryos of Western clawed frog (Silurana tropicalis) were exposed for 72 h to water accommodated fractions (WAF; 0.7–8.9 μg/L TPACs) and chemically-enhanced WAFs (CEWAF; 0.09–56.7 μg/L TPACs) of Access Western Blend (AWB) and Cold Lake Blend (CLB) dilbits. Both dilbit's CEWAFs significantly increased embryonic mortality and malformation incidence in the highest treatments tested, while WAF treatments led to no visible toxic effects. Increases of the cytochrome P450 1A (cyp1a) mRNA levels were observed for all WAF and CEWAF dilbit treatments suggesting that phase I detoxification is activated in the dilbit-exposed larvae. When exposed to PAC concentrations ranging from 0.09 to 8.9 μg/L, the frogs displayed no observable malformations, but expressed significant increases of cyp1a mRNA levels (2- to 25-fold; indicating a suitable biomarker of exposure); however, when concentrations were of 46.6 μg/L or higher, both malformed frog phenotype and induction of cyp1a mRNA level (>250-fold) were measured (indicating a suitable biomarker of response). The expression of several genes related to cellular detoxification and endocrine disruption were also measured, but were not significantly altered by the treatments. In sum, cyp1a mRNA level is a highly sensitive endpoint to measure subtle molecular changes induced by PAC exposure in the frog embryos and larvae, and data suggest that PAC concentration higher than 46 μg/L would be toxic to the developing S. tropicalis.

Many chemicals in commonly used household and industrial products are being released into the environment, yet their toxicity is poorly understood. The synthetic phenolic antioxidant, 4,4′-thiobis(6-t-butyl-m-cresol) (CAS 96-69-5; TBBC) is present in many common products made of rubber and plastic. Yet, this phenolic antioxidant has not been tested for potential toxicity and developmental disruption in amphibians, a sensitive and susceptible class. We investigated whether acute and chronic exposure to TBBC would interfere with thyroid hormone-dependent developmental processes in the frog Silurana tropicalis and thus affect its early life-stage development. We exposed S. tropicalis embryos at the Nieuwkoop-Faber (NF) 9–10 stage to TBBC at nominal concentrations (0, 25, 50, 75, 100, 200 and 400 μg/L) to determine the 96h lethal concentrations and sublethal effects. We conducted a chronic exposure starting at stage NF47-48 to three sublethal TBBC nominal concentrations (0, 0.002, 0.1 and 5 μg/L) for 48–52 days to evaluate effects on growth and metamorphosis. The 96h lethal and effective (malformations) TBBC concentrations (LC50 and EC50) were 70.5 and 76.5 μg/L, respectively. Acute exposure to all TBBC concentrations affected S. tropicalis growth and was lethal at 200 and 400 μg/L. Chronic exposure to sublethal TBBC concentrations reduced body size by 8% at 5 μg/L and body mass by 17% at 0.002 μg/L when metamorphosis was completed. This study demonstrates that TBBC is toxic, induces malformations and inhibits tadpole growth after acute and chronic exposures. These findings call for further investigations on the mode of actions of TBBC and related antioxidants for developmental disruption in amphibians.

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 μg/L)/linuron (9 or 45 μg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.

A series of ex vivo exposures using testicular and ovarian tissues of sexually mature Western clawed frogs (Silurana tropicalis) were designed to examine molecular mechanisms of thyroid hormone (TH) and androgen crosstalk sans hypophyseal feedback as well as investigate potential sex-specific differences. Tissues were exposed ex vivo to either triiodothyronine (T3), iopanoic acid (IOP), one co-treatment of IOP + 5α-dihydrotestosterone (5α-DHT), 5α-DHT, 5β-dihydrotestosterone (5β-DHT), or testosterone (T). Direct exposure to different androgens led to androgen specific increases in thyroid receptor and deiodinase transcripts in testes (trβ and dio1) but a decrease in expression in ovaries (trβ and dio3), suggesting that male and female frogs can be differently affected by androgenic compounds. Moreover, exposure to select androgens differentially increased estrogen-related transcription (estrogen receptor alpha (erα) and aromatase (cyp19)) and production (estradiol) in ovaries and testes indicating the activation of alternate metabolic pathways yielding estrogenic metabolites. Sex-steroid-related transcription (i.e., steroid 5α-reductase type 2 (srd5α2) and erα) and production (i.e., 5α-DHT) were also differentially regulated by THs. The presence and frequency of transcription factor binding sites in the putative promoter regions of TH- and sex steroid-related genes were also examined in S. tropicalis, rodent, and fish models using in silico analysis. In summary, this study provides an improved mechanistic understanding of TH- and androgen-mediated actions and reveals differential transcriptional effects as a function of sex in frogs.

Environmental gestagens are an emerging class of contaminants that have been recently measured in surface water and can interfere with reproduction in aquatic vertebrates. Gestagens include endogenous progestogens, such as progesterone (P4), which bind P4-receptors and have critically important roles in vertebrate physiology and reproduction. Gestagens also include synthetic progestins, which are components of human and veterinary drugs, such as melengestrol acetate (MGA). Endogenous progestogens are essential in the regulation of reproduction in mammalian species, but the role of P4 in amphibian larval development remains unclear. This project aims to understand the roles and the regulatory mechanisms of P4 in amphibians and to assess the consequences of exposures to environmental gestagens on the P4-receptor signaling pathways in frogs. Here, we established the developmental profiles of the P4 receptors: the intracellular progesterone receptor (ipgr), the membrane progesterone receptor β (mpgrβ), and the progesterone receptor membrane component 1 (pgrmc1) in Western clawed frog (Silurana tropicalis) embryos using real-time qPCR. P4-receptor mRNAs were detected throughout embryogenesis. Transcripts for ipgr and pgrmc1 were detected in embryos at Nieuwkoop and Faber (NF) stage 2 and 7, indicative of maternal transfer of mRNA. We also assessed the effects of P4 and MGA exposure in embryonic and early larval development. Endocrine responses were evaluated through transcript analysis of a suite of gene targets of interest, including: ipgr, mpgrβ, pgrmc1, androgen receptor (ar), estrogen receptor α (erα), follicle stimulating hormone β (fshβ), prolactin (prl), and the steroid 5-alpha reductase family (srd5α1, 2, and 3). Acute exposure (NF 12–46) to P4 caused a 2- to 5-fold change increase of ipgr, mpgrβ, pgrmc1, and ar mRNA levels at the environmentally relevant concentration of 195 ng/L P4. Acute exposure to MGA induced a 56% decrease of srd5α3 at 1140 ng/L MGA. We conclude that environmental exposure to P4 induced multiple endocrine-related transcript responses in amphibians; however, the differential responses of MGA suggest that the effects of MGA are not mediated through the classical P4 signaling pathway in S. tropicalis.

Highlights • There have been few reports about gene cloning and expression of α-amylases from E. fetida. • Ef-Amy I and II were shown to 89% identity of amino acid sequences. • The catalytically important residues of α-amylase of GH family 13 were conserved in Ef-amy I and II. • The substrate specificities of rEf-Amy I and II were dissimilar. • It found that rEf-Amy I and II could be possible use for simultaneous saccharification and fermentation process., We identified the raw-starch-digesting α-amylase genes a earthworm Eisenia fetid α amylase I and II (Ef-Amy I and Ef-Amy II). Each gene consists of 1,530 base pairs (bp) that encode proteins of 510 amino acids, as indicated by the corresponding mRNA sequences. Ef-Amy I and II showed an 89% amino acid identity. The amino acid sequences of Ef-Amy I and II were similar to those of the α-amylases from porcine pancreas, human pancreas, Tenebrio molitor, Oryctolagus cuniculus, and Xenopus (Silurana) tropicalis. Each gene encoding mature Ef-Amy I and II was expressed in the GS115 strain of Pichia pastoris. The molecular masses of the recombinant Ef-Amy I and II were 57 kDa each, and catalytically important residues of α-amylases of the GH family 13 were conserved in both proteins. These amylases exhibited raw-starch-digesting activity at 4 °C. The substrate specificities of rEf-Amy I and II were dissimilar. rEf-Amy I and II were shown to be active even in 40% ethanol, 4 M NaCl, and 4 M KCl., Graphical abstract Image, graphical abstract

The two species of Xenopus most commonly used in biomedical research are the diploid Xenopus (Silurana) tropicalis and the tetraploid Xenopus laevis. The X. tropicalis genome sequence has been available since 2010 and this year the X. laevis, genome from two distinct genetic backgrounds has been published. Multiple genome assemblies available for both species and transcriptomic and epigenetic data sets are growing rapidly, all of which are available from a variety of web resources. This review describes the contents of these resources, how to locate and download genomic data, and also how to view and manipulate these data on various public genome browsers, with an emphasis on Xenbase, the Xenopus model organism database.

Naphthenic acids (NAs) are oil-derived mixtures of carboxylic acids and are considered emerging contaminants with the potential to disrupt development of aquatic species. In the Oil Sands Region of Canada, NAs are components of the water released following processing of the bitumen-containing sand. The aim of this research was to identify potential mechanisms of toxicity of NA mixtures. Silurana (Xenopus) tropicalis embryos were raised in water spiked with commercial oil-derived NA extracts (S1 and S2) at a sub-lethal concentration (2 mg/L). The transcriptomic responses of the whole 4-day old embryos following exposure were assessed using a custom oligonucleotide microarray. Both NA mixtures induced embryonic abnormalities that included edema, and cardiac and gut abnormalities. Exposure to NAs also affected morphometric parameters and decreased total length, tail length, and interorbital distance of the embryos. Gene ontology analysis revealed that 18 biological processes, 5 cellular components, and 19 molecular functions were significantly enriched after both S1 and S2 exposures. Sub-network enrichment analysis revealed pathways that were related to phenotypic abnormalities; these included gut function, edema, and cartilage differentiation. Other notable networks affected by NAs included metabolism and cell membrane integrity. In a separate dose-response experiment, the expression of key genes identified by microarray (cyp4b1, abcg2, slc26a6, eprs, and slc5a1) was determined by Real-Time qPCR in S. tropicalis embryos exposed to the commercial NAs and to acid-extractable organics (AEOs) prepared from Oil Sands Process-Affected Water. In general, the RT-qPCR data agreed with the microarray data. In S. tropicalis embryos exposed to the AEOs, the mRNA levels of eprs (bifunctional glutamate/proline-tRNA ligase) and slcs5a1 (sodium/glucose cotransporter 1) were significantly decreased compared to the controls. Such changes are likely indicative of increased edema and disrupted gut function, respectively. These data suggest that NAs have multiple modes of action to induce developmental toxicity in amphibians. Some modes of action may be shared between commercial NAs and AEOs.

The impact of the brominated flame-retardant mixture, DE-71, on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined. A partial lifecycle study exposing S. tropicalis to varying concentrations of DE-71 (0.0, 0.65, 1.3, 2.5, and 5.0 μg/l [nominal]) was conducted from early gastrula-stage embryo to 150 days postmetamorphosis (dpm). Exposure of S. tropicalis to DE-71 induced liver necrosis and induced abnormal ovary development characterized by previtellogenic oocyte necrosis and arrested development of vitellogenic oocytes in females in a concentration-dependent manner. Decreased mean plasma dihydrotestosterone (DHT) and T, gonad T, and increased mean plasma E2 levels were found in 150 dpm DE-71-treated male S. tropicalis compared to controls. Plasma E2 levels in females were not significantly altered compared to control S. tropicalis, although lower plasma and gonad T were detected. Mean gonadal CYP 19 aromatase activity in both male and female S. tropicalis exposed to DE-71 was not appreciably affected. Decreased mean male 5α-reductase and CYP17 activities in both male and females were observed compared to control frogs. Overall, these studies suggested that PBDE exposure induced liver necrosis and abnormal ovary development; and reduced circulating and gonadal androgens resulting in a phenotypic skew in sex ratio toward the female sex in S. tropicalis.

Phthalates are used worldwide in the manufacturing of plastics, added to cosmetic products, personal care products, pharmaceuticals, medical devices, and paints; and are widely detected in soil, surface water, and organism tissues. Phthalate esters have been previously shown to interfere with the endocrine system in vertebrates. However, few studies have investigated the effects of phthalates on testosterone-converting enzymes that affect hormone levels and reproduction. In the present study, we exposed the Western clawed frog (Silurana tropicalis) to 0.1, 1, and 10 μM diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), and diethyl phthalate (DEP) during early amphibian embryonic development. Additional DBP exposures were conducted ex vivo using mature frog testes. Malformations and mRNA levels of genes associated to reproduction and oxidative stress were evaluated. 0.1 μM DEHP, DBP, and DEP induced an array of malformations, including incomplete gut coiling, edemas, and eye malformations. Moreover, all three phthalates increased the expression of androgen-related genes, such as steroid-5α-reductase 1, 2, 3, steroid-5β-reductase, and androgen receptor at concentrations ranging from 0.1 to 10 μM depending on the phthalate and gene. Data suggest that the phthalate esters tested are teratogens to the amphibian embryo and that these phthalates exhibit an androgenic activity in amphibians.

Perchlorate (ClO4−) contamination has been reported in ground and surface waters across North America. However, few studies have examined the effects of prolonged exposure to this thyroid hormone disrupting chemical, particularly at environmentally relevant concentrations in lower vertebrates, such as amphibians. The aim of this study was to examine the effects of a yearlong chronic exposure to ClO4− in adult male and female Western clawed frogs (Silurana tropicalis). Frogs were spawned and raised from fertilized embryo until sexual maturity in potassium perchlorate (KClO4)-treated water at different concentrations (0, 20, 53, and 107 μg/L). Developmental and reproductive indices – including adult morphology, androgen plasma levels, gonadal thyroid hormone- and sex steroid-related transcript levels, and sperm motility – were evaluated in male and female adult frogs. Female growth (e.g., body mass, snout-vent length, and hind limb length) was significantly reduced following chronic exposure to environmentally relevant concentrations of KClO4 resulting in females with morphometric indices similar to those of control males – indicating potential sex-specific sensitivities to KClO4. Changes to reproductive indices (i.e., plasma androgen levels, gonadal thyroid hormone- and sex steroid-related transcript levels, and sperm motility) were also observed in both sexes and suggest that KClO4 exposure may also have indirect secondary effects on the reproductive axes in male and female adult frogs. These effects were observed at concentrations at or below those reported in surface waters contaminated with ClO4− suggesting that this contaminant may have developmental and reproductive effects post-metamorphosis in natural amphibian populations.

Arsenic compounds are widespread environmental contaminants and exposure elicits serious health issues, including early developmental anomalies. Depending on the oxidation state, the intermediates of arsenic metabolism interfere with a range of subcellular events, but the fundamental molecular events that lead to speciation-dependent arsenic toxicity are not fully elucidated. This study therefore assesses the impact of arsenic exposure on early development by measuring speciation and gene expression profiles in the developing Western clawed frog (Silurana tropicalis) larvae following the environmental relevant 0.5 and 1 ppm arsenate exposure. Using HPLC-ICP-MS, arsenate, dimethylarsenic acid, arsenobetaine, arsenocholine, and tetramethylarsonium ion were detected. Microarray and pathway analyses were utilized to characterize the comprehensive transcriptomic responses to arsenic exposure. Clustering analysis of expression data showed distinct gene expression patterns in arsenate treated groups when compared with the control. Pathway enrichment revealed common biological themes enriched in both treatments, including cell signal transduction, cell survival, and developmental pathways. Moreover, the 0.5 ppm exposure led to the enrichment of pathways and biological processes involved in arsenic intake or efflux, as well as histone remodeling. These compensatory responses are hypothesized to be responsible for maintaining an in-body arsenic level comparable to control animals. With no appreciable changes observed in malformation and mortality between control and exposed larvae, this is the first study to suggest that the underlying transcriptomic regulations related to signal transduction, cell survival, developmental pathways, and histone remodeling may contribute to maintaining ongoing development while coping with the potential arsenic toxicity in S. tropicalis during early development.

Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys–Cys–Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

Xenopus laevis (African clawed frog) has two types of proto-type galectins that are similar to mammalian galectin-1 in amino acid sequence. One type, comprising xgalectin-Ia and -Ib, is regarded as being equivalent to galectin-1, and the other type, comprising xgalectin-Va and -Vb, is expected to be a unique galectin subgroup. The latter is considerably abundant in frog skin; however, its biological function remains unclear. We determined the crystal structures of two proto-type galectins, xgalectin-Ib and -Va. The structures showed that both galectins formed a mammalian galectin-1-like homodimer, and furthermore, xgalectin-Va formed a homotetramer. This tetramer structure has not been reported for other galectins. Gel filtration and other experiments indicated that xgalectin-Va was in a dimer-tetramer equilibrium in solution, and lactose binding enhanced the tetramer formation. The residues involved in the dimer-dimer association were conserved in xgalectin-Va and -Vb, and one of the Xenopus (Silurana) tropicalis proto-type galectins, but not in xgalectin-Ia and -Ib, and other galectin-1-equivalent proteins. Xgalectin-Va preferred Galβ1-3GalNAc and not Galβ1-4GlcNAc, while xgalectin-Ib preferred Galβ1-4GlcNAc as well as human galectin-1. Xgalectin-Va/Vb would have diverged from the galectin-1 group with accompanying acquisition of the higher oligomer formation and altered ligand selectivity.

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Thyroid hormones (THs) and androgens have been shown to be extensively involved in sexual development; however, relatively little is known with regard to TH-related and androgenic actions in sex determination. We first established expression profiles of three sex-determining genes ( sf1 , dax-1 , and sox9 ) during the embryonic development of Western clawed frogs ( Silurana tropicalis ). Transcripts of sf1 and sox9 were detected in embryos before the period in which embryonic transcription commences indicating maternal transfer, whereas dax-1 transcripts were not detected until later in development. To examine whether TH status affects sex-determining gene expression in embryonic S. tropicalis, embryos were exposed to co-treatments of iopanoic acid (IOP), thyroxine (T4), or triiodothyronine (T3) for 96 h. Expression profiles of TH receptors and deiodinases reflect inhibition of peripheral deiodinase activity by IOP and recovery by T3. Relevantly, elevated TH levels significantly increased the expression of sf1 and dax-1 in embryonic S. tropicalis . Further supporting TH-mediated regulation, examination of the presence and frequency of transcription factor binding sites in the putative promoter regions of sex-determining genes in S. tropicalis and rodent and fish models using in silico analysis also identified TH motifs in the putative promoter regions of sf1 and dax-1 . Together these findings advocate that TH actions as early as the period of embryogenesis may affect gonadal fate in frogs. Mechanisms of TH and androgenic crosstalk in relation to the regulation of steroid-related gene expression were also investigated.

Seven 1,3,5- triazine ( s -triazine) herbicides (ametryn, prometryn, dimethametryn, simazine, atrazine, propazine, and cyanazine) were tested using an amphibian ( Silurana tropicalis ) metamorphosis assay focusing on morphometric, gravimetric, and thyroid-histological endpoints. Premetamorphic tadpoles were exposed to each s -triazine at 2 concentrations between 1/1000 and 1/10 of the 96-h acute toxicity values, until all tadpoles in the control group reached either the late prometamorphosic stages or the initial stage of metamorphic climax. All s -triazines tested induced significant retardation in growth and development at the higher concentrations (0.2–1.0 mg/L), and some of them induced similar effects even at the lower concentrations (0.02–0.1 mg/L) while each showing a linear dose-response. Total size of the thyroid glands tended to be reduced corresponding to the delayed development, but without showing histomorphological lesions typical of anti-thyroid chemicals. These consistent results suggest that the s -triazines can act as a chemical stressor inhibiting tadpole growth and development, possibly without disrupting the thyroid axis. In addition, tadpoles exhibiting spinal curvatures appeared in either one or both of the lower and higher concentration groups for each s -triazine tested. The incidence rate in the s -triazine exposure groups where tadpoles with scoliosis were observed ranged from 3.3% to 63.3%, some of which were significantly higher than that in the respective control groups (0–6.7%). It is speculated that the s -triazines may promote to occur axial malformations in developing tadpoles.

Skin secretions from frogs belonging to the genera Xenopus, Silurana, Hymenochirus, and Pseudhymenochirus in the family Pipidae are a rich source of host-defense peptides with varying degrees of antimicrobial activities and cytotoxicities to mammalian cells. Magainin, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragment (CPF), and xenopsin-precursor fragment (XPF) peptides have been isolated from norepinephrine-stimulated skin secretions from several species of Xenopus and Silurana. Hymenochirins and pseudhymenochirins have been isolated from Hymenochirus boettgeri and Pseudhymenochirus merlini. A major obstacle to the development of these peptides as anti-infective agents is their hemolytic activities against human erythrocytes. Analogs of the magainins, CPF peptides and hymenochirin-1B with increased antimicrobial potencies and low cytotoxicities have been developed that are active (MIC < 5 μM) against multidrug-resistant clinical isolates of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Stenotrophomonas maltophilia and Klebsiella pneumoniae. Despite this, the therapeutic potential of frog skin peptides as anti-infective agents has not been realized so that alternative clinical applications as anti-cancer, anti-viral, anti-diabetic, or immunomodulatory drugs are being explored.

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.

Aim The objective of the investigation was to predict the three dimensional structure of ageing related proteins of Silurana tropicalis Gray 1864. Methods The protein sequences were collected from UniProt. Homology modeling of protein structures was generated in SWISS-MODEL workplace. The structures were assessed by ERRAT and RAMPAGE. Results In S. tropicalis Gray 1864, ageing related total 5 proteins sequences namely prohibitin-2 (301 aa), serum response factor-binding protein 1 (535 aa), reactive oxygen species modulator 1 (79 aa), CDGSH iron-sulfur domain-containing protein 2 (135 aa) and an uncharacterized protein (668 aa) were found (UniProt). SWISS-MODEL workspace generated reliable 3D structures of only two proteins prohibitin-2 (301 aa) and CDGSH iron-sulfur domain-containing protein 2 (135 aa), while it failed to give proper structures for other proteins due to lack of templates. In ERRAT assessment, prohibiin-2 and CDGSH iron-sulfur domain-containing protein 2 scored 51.82% and 97.48% respectively. While in RAMPAGE, prohibitin 2 and CDGSH iron-sulfur domain-containing protein 2 scored 90.7% and 97.7% respectively. Conclusion The present study demonstrated the 3D structures of prohibitin-2 and CDGSH iron-sulfur domain-containing protein 2.

Sex chromosome divergence has been documented across phylogenetically diverse species, with amphibians typically having cytologically nondiverged ("homomorphic") sex chromosomes. With an aim of further characterizing sex chromosome divergence of an amphibian, we used "RAD-tags" and Sanger sequencing to examine sex specificity and heterozygosity in the Western clawed frog Silurana tropicalis (also known as Xenopus tropicalis). Our findings based on approximately 20 million genotype calls and approximately 200 polymerase chain reaction-amplified regions across multiple male and female genomes failed to identify a substantially sized genomic region with genotypic hallmarks of sex chromosome divergence, including in regions known to be tightly linked to the sex-determining region. We also found that expression and molecular evolution of genes linked to the sex-determining region did not differ substantially from genes in other parts of the genome. This suggests that the pseudoautosomal region, where recombination occurs, comprises a large portion of the sex chromosomes of S. tropicalis. These results may in part explain why African clawed frogs have such a high incidence of polyploidization, shed light on why amphibians have a high rate of sex chromosome turnover, and raise questions about why homomorphic sex chromosomes are so prevalent in amphibians.

Naphthenic acids (NA) have been identified as harmful environmental contaminants that influence survival, growth and development of wildlife. Amphibian larvae are particularly susceptible to waterborne contaminants, but little information exists regarding exposure of amphibian embryos or tadpoles to NA. Our results demonstrate that embryos of Lithobates pipiens and Silurana tropicalis exposed to 2–4 mg/l of a commercial NA blend suffer significant reductions (32% and 25%, respectively) in growth and development upon hatching. Increased incidences of deformities were observed in exposed individuals of both species, but were only significant in L. pipiens . Embryos suffered 100% mortality following exposure to 6 mg/l NAs, and narcosis at lower concentrations. LC50 estimates were 4.10 mg/l for 72-h exposure in L. pipiens and 4.13, 3.51, and 2.95 mg/l for 24-, 48-, and 72-h exposure in S. tropicalis . These data suggest that exposure to NAs at environmentally realized concentrations may negatively affect tadpole populations.

Nucleocytoplasmic hybrid (cybrid) embryos result from the combination of the nucleus of one species, and the egg cytoplasm of another species. Cybrid embryos can be obtained either in the haploid state by the cross-fertilization or intra-cytoplasmic injection of an enucleated egg with sperm from another species, or in the diploid state by the technique of interspecies somatic cell nuclear transfer (iSCNT). Cybrids that originate from the combination of the nucleus and the cytoplasm of distantly related species commonly expire during early embryonic development, and the cause of this arrest is currently under investigation. Here we show that cells isolated from a Xenopus cybrid (Xenopus (Silurana) tropicalis haploid nucleus combined with Xenopus laevis egg cytoplasm) embryo are unable to proliferate and expand normally in vitro. We also provide evidence that the lack of nuclear donor species maternal poly(A)(+) RNA-dependent factors in the recipient species egg may contribute to the developmental dead-end of distantly-related cybrid embryos. Overall, the data are consistent with the view that the development promoted by one species' nucleus is dependent on the presence of maternally-derived, mRNA encoded, species-specific factors. These results also show that cybrid development can be improved without nuclear species mitochondria supplementation or replacement.

The estimation of phylogenetic relationships is an essential component of understanding evolution. Accurate phylogenetic estimation is difficult, however, when internodes are short and old, when genealogical discordance is com- mon due to large ancestral effective population sizes or ancestral population structure, and when homoplasy is prevalent. Inference of divergence times is also hampered by unknown and uneven rates of evolution, the incomplete fossil record, uncertainty in relationships between fossil and extant lineages, and uncertainty in the age of fossils. Ideally, these challenges can be overcome by developing large "phylogenomic" data sets and by analyzing them with methods that accommodate features of the evolutionary process, such as genealogical discordance, recurrent substitution, recombination, ancestral pop- ulation structure, gene flow after speciation among sampled and unsampled taxa, and variation in evolutionary rates. In some phylogenetic problems, it is possible to use information that is independent of fossils, such as the geological record, to identify putative triggers for diversification whose associated estimated divergence times can then be compared a poste- riori with estimated relationships and ages of fossils. The history of diversification of pipid frog genera Pipa, Hymenochirus, Silurana, and Xenopus, for instance, is characterized by many of these evolutionary and analytical challenges. These frogs diversified dozens of millions of years ago, they have a relatively rich fossil record, their distributions span continental plates with a well characterized geological record of ancient connectivity, and there is considerable disagreement across studies in estimated evolutionary relationships. We used high throughput sequencing and public databases to generate a large phylogenomic data set with which we estimated evolutionary relationships using multilocus coalescence methods. We collected sequence data from Pipa, Hymenochirus, Silurana, and Xenopus and the outgroup taxon Rhinophrynus dorsalis from coding sequence of 113 autosomal regions, averaging � 300 bp in length (range: 102-1695 bp) and also a portion of the mitochondrial genome. Analysis of these data using multiple approaches recovers strong support for the ((Xenopus, Silurana)(Pipa, Hymenochirus)) topology, and geologically calibrated divergence time estimates that are consistent with es- timated ages and phylogenetic affinities of many fossils. These results provide new insights into the biogeography and chronology of pipid diversification during the breakup of Gondwanaland and illustrate how phylogenomic data may be necessary to tackle tough problems in molecular systematics. (Coalescence; gene tree; high-throughout sequencing; lineage sorting; pipid; species tree; Xenopus.)

Most anurans have no identified sex-markers; therefore, alternative methods for identification of early changes in sex ratios are required. In this study, Lithobates sylvaticus and Silurana tropicalis tadpoles were sampled at different developmental stages covering the entire process of sex differentiation. Three candidate genes known to be involved in sex differentiation in other vertebrate species were selected to develop a method to identify phenotypic sex in frogs: cytochrome p450 aromatase (cyp19), forkhead box L2 (foxl2) and the cytochrome 17-alpha-hydroxylase/17,20 lyase (cyp17). Cloning of these genes revealed nucleotide identity values ranging between 75–97% when compared to other amphibian species. Gene expression of cyp17,cyp19 and foxl2 in L. sylvaticus adult gonads and gonad-mesonephros complex (GMC) of tadpoles was analyzed by real-time RT-PCR. Results showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of cyp19 alone can be used as a robust predictor of phenotypic sex in L. sylvaticus tadpoles. In addition, we validated this method measuring cyp19 mRNA levels in S. tropicalis GMCs. We propose measuring cyp19 as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future.

For most frogs, advertisement calls are essential for reproductive success, conveying information on species identity, male quality, sexual state and location. While the evolutionary divergence of call characters has been examined in a number of species, the relative impacts of genetic drift or natural and sexual selection remain unclear. Insights into the evolutionary trajectory of vocal signals can be gained by examining how advertisement calls vary in a phylogenetic context. Evolution by genetic drift would be supported if more closely related species express more similar songs. Conversely, a poor correlation between evolutionary history and song expression would suggest evolution shaped by natural or sexual selection. Here, we measure seven song characters in 20 described and two undescribed species of African clawed frogs (genera Xenopus and Silurana) and four populations of X. laevis. We identify three call types — click, burst and trill — that can be distinguished by click number, call rate and intensity modulation. A fourth type is biphasic, consisting of two of the above. Call types vary in complexity from the simplest, a click, to the most complex, a biphasic call. Maximum parsimony analysis of variation in call type suggests that the ancestral type was of intermediate complexity. Each call type evolved independently more than once and call type is typically not shared by closely related species. These results indicate that call type is homoplasious and has low phylogenetic signal. We conclude that the evolution of call type is not due to genetic drift, but is under selective pressure.

The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3',4,4',5-pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6- and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26- and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 μM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 μM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene- and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions.

Nonclassical MHC class Ib (class Ib) genes are heterogeneous genes encoding molecules that are structurally similar to classical MHC class Ia molecules but with limited tissue distribution and polymorphism. Mammalian class Ib genes have diverse and often uncharacterized functions, and because of their rapid rate of evolution, class Ib phylogeny is difficult to establish. We have conducted an extensive genomic, molecular, and phylogenetic characterization of class Ib genes in two Xenopodinae amphibian species of different genera that diverged from a common ancestor as long ago as primates and rodents (∼65 million years). In contrast with the unsteadiness of mammalian class Ib genes, our results reveal an unusual degree of conservation of most Xenopodinae class Ib gene lineages, including a novel monogenic lineage represented by the divergent Xenopus laevis XNC10 gene and its unequivocal Silurana (Xenopus) tropicalis orthologue, SNC10. The preferential expression of this gene lineage by thymocytes themselves from the onset of thymic organogenesis is consistent with a specialized role of class Ib in early T cell development and suggests such a function is conserved in all tetrapods.

DM-W is a dominant, female-specific, regulator of sex determination in the African clawed frog Xenopus laevis. This gene is derived from partial duplication of DMRT1, a male-related autosomal gene. We set out to better understand sex determination in Xenopus by studying this pair of genes. We found that DM-W evolved in Xenopus after divergence from the sister genus Silurana but before divergence of X. laevis and X. clivii, and that DM-W arose from partial duplication of DMRT1β, which is one of the two DMRT1 paralogs in the tetraploid ancestor of Xenopus. Using the rate ratio of nonsynonymous to synonymous substitutions per site and multilocus polymorphism data, we show that DM-W evolved non-neutrally. By cloning paralogs and using a pyrosequencing assay, we also demonstrate that DMRT1 underwent phylogenetically biased pseudogenization after polyploidization, and that expression of this gene is regulated by mechanisms that vary through development. One explanation for these observations is that the expression domain of DMRT1β was marginalized, which would explain why this paralog is dispensable in Xenopus polyploids and why DM-W has a narrow expression domain. These findings illustrate how evolution of the genetic control of stable phenotypes is facilitated by redundancy, degeneration, and compartmentalized regulation.

I examined the toxicity of rice paddy herbicides simetryn, mefenacet, and thiobencarb to amphibians by conducting acute toxicity tests with tadpoles of Silurana tropicalis, which has attracted as a new model species instead of Xenopus laevis. The tadpoles at stages 49 and 50 were exposed to the herbicides at several concentrations during 96 h, and median lethal concentration (LC50) values were calculated at 24-h intervals. The LC50 values of simetryn, mefenacet, and thiobencarb were 16.9-3.70 mg/L (79.3-17.4 microM), 3.06-2.70 mg/L (10.3-9.04 microM), and 1.77-0.752 mg/L (6.85-2.92 microM), respectively. The most toxic herbicide was thiobencarb followed by mefenacet and simetryn. As for thiobencarb, the sensitivity of S. tropicalis was similar to that of X. laevis reported previously. This suggests that S. tropicalis as well as X. laevis can act as a model species in acute toxicity tests. The LC50 values of the three herbicides were very close to or at most two orders of magnitude higher than the maximum concentrations likely to occur in paddy water. Therefore, the three herbicides can be harmful to amphibian larvae living in paddy water.

Amphibian metamorphosis is an excellent example of hormone-dependent control of development. Thyroid hormones (THs) regulate almost all aspects of metamorphosis, including brain development and larval neuroendocrine function. Sex steroids are also important for early brain function, although little is known about interactions between the two hormonal systems. In the present study, we established brain developmental profiles for thyroid hormone receptors (tralpha and trbeta), deiodinases (dio1, dio2 and dio3), aromatase (cyp19) mRNA and activity, oestrogen receptors (eralpha and erbeta), androgen receptor (ar) and 5α-reductases (srd5alpha1 and srd5alpha2) mRNA during Silurana (Xenopus) tropicalis metamorphosis. Real-time reverse transcriptase-polymerase chain reaction analyses revealed that all of the genes were expressed in the brain and for most of the genes expression increased during development, with the exception of dio2, srd5alpha1 and srd5alpha2. The ability of premetamorphic tadpoles to respond to exogenous THs was used to investigate the regulation of TH- and sex steroid-related genes in the brain during development. Exposure of premetamorphic tadpoles to triiodothyronine (T3; 0, 0.5, 5 and 50 nm) for 48 h resulted in concentration-dependent increases in trbeta, dio2, dio3, eralpha and erbeta. Expression of srd5alpha2 showed large increases (six- to 7.5-fold) for all three concentrations of T3. No changes were detected in dio1, ar and cyp19 transcript levels; however, cyp19 activity increased significantly at 50 nm T3. The results obtained suggest that expression of TH-related genes and er during development could be regulated by rising levels of THs, as previously documented in Lithobates (Rana) pipiens. The positive regulation of srd5alpha by T3 in the brain suggests that endogenous TH levels help maintain or control the rate at which srd5alpha mRNA levels decrease as metamorphosis progresses. Finally, we have identified sex steroid-related genes that are responsive to T3, providing additional evidence of crosstalk between THs and sex steroids in the tadpole brain.

Endocrine disrupting compounds have been shown to alter gonad differentiation in both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species. In some cases, these affected individuals are completely sex reversed and are morphologically indistinguishable from normal individuals of the opposite sex. Detecting shifts in sex ratios following chemical exposure often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with genetic sex determination and homomorphic sex chromosomes (such as amphibians and many fish). The ability to assess the genetic sex of individuals would allow for detection of sex reversal (genotype-phenotype mismatches) that have greater statistical power compared to examining changes in sex ratios. Utilizing amplified fragment length polymorphisms (AFLPs), we developed a method for genotyping sex in the amphibian, Xenopus (Silurana) tropicalis, that can be incorporated into endocrine disruptor screening assays that examine the effects of chemicals on gonad differentiation. AFLPs from 512 primer pairs were assessed in one spawn of X. tropicalis. Each primer pair yielded, on average, 100 fragments. In total 17 sex-linked AFLPs were identified, isolated, and sequenced. A recombination map of these AFLPs was generated using over 300 individuals with four AFLPs having a recombination rate of 0% with regard to sex. A BLASTn search of the X. tropicalis genome using these AFLP sequences resulted in identification of sex-linked scaffolds. Areas of these scaffolds were searched for additional polymorphisms that could be utilized for genotyping sex. Retrospective and prospective strategies for incorporating genotyping sex in endocrine disruptor bioassays with X. tropicalis were developed. A Monte Carlo simulation comparing analyzing data as sex ratio shifts versus assessment of sex reversal using genotyping demonstrates the increase in statistical power that can be obtained by genotyping sex in studies dealing with altered gonad differentiation. This approach to identifying sex-linked markers and developing sex genotyping methods is applicable to other species with genetic sex determination.

Laser microdissection was used for the preparation of whole chromosome painting probes in Silurana (Xenopus) tropicalis. Subsequent cross-species fluorescence in situ hybridization (Zoo-FISH) on its tetraploid relative Xenopus laevis revealed persistence of chromosomal quartets even after 50-65 million years of separate evolution. Their arrangement is in a partial concordance with previous experiments based on similarity of a high-resolution replication banding pattern. Further support for an allotetraploid origin of X. laevis was given by hybridization with a probe derived from the smallest X. tropicalis chromosome (Xt10). Here, pericentric areas of both arms of Xl 14 and 18 were stained, indicating intrachromosomal rearrangements. The positions of signals were not in agreement with the chromosomal quartets revealed by painting probes Xt 8 and 9 (Xl 11 + 14 and Xl 15 + 18, respectively). This suggests that both X. tropicalis chromosomes underwent non-reciprocal translocation of Xt10 separately in at least two different ancient ancestors. In addition, the observed translocation events could explain the origin of individuals with 18 chromosomes in diploid karyotypes, probably extinct after the genesis of the allotetraploid X. laevis (2n = 36).

Xenopus laevis is the model of choice for evolutionary, comparative, and developmental studies of immunity, and invaluable research tools including MHC-defined clones, inbred strains, cell lines, and monoclonal antibodies are available for these studies. Recent efforts to use Silurana (Xenopus) tropicalis for genetic analyses have led to the sequencing of the whole genome. Ongoing genome mapping and mutagenesis studies will provide a new dimension to the study of immunity. Here we review what is known about the immune system of X. laevis integrated with available genomic information from S. tropicalis. This review provides compelling evidence for the high degree of similarity and evolutionary conservation between Xenopus and mammalian immune systems. We propose to build a powerful and innovative comparative biomedical model based on modern genetic technologies that takes take advantage of X. laevis and S. tropicalis, as well as the whole Xenopus genus. Developmental Dynamics 238:1249-1270, 2009. (c) 2009 Wiley-Liss, Inc.

As paleopolyploid genomes evolve, the expression profiles of retained gene pairs are expected to diverge. To examine this divergence process on a large scale in a vertebrate system, we compare Xenopus laevis , which has retained ≈40% of loci in duplicate after a recent whole-genome duplication (WGD), with its unduplicated relative Silurana (Xenopus ) tropicalis . This comparison of ingroup pairs to an outgroup allows the direction of change in expression profiles to be inferred for a set of 1,300 X. laevis pairs, relative to their single orthologs in S. tropicalis , across 11 tissues. We identify 68 pairs in which X. laevis is inferred to have undergone a significant reduction of expression in at least two tissues since WGD. Of these pairs, one-third show evidence of subfunctionalization, with decreases in the expression levels of different gene copies in two different tissues. Surprisingly, we find that genes with slow rates of evolution are particularly prone to subfunctionalization, even when the tendency for highly expressed genes to evolve slowly is controlled for. We interpret this result to be an effect of allopolyploidization. We then compare the outcomes of this WGD with an independent one that happened in the teleost fish lineage. We find that if a gene pair was retained in duplicate in X. laevis , the orthologous pair is more likely to have been retained in duplicate in zebrafish, suggesting that similar factors, among them subfunctionalization, determined which gene pairs survived in duplicate after the two WGDs.

The estrogenic chemical bisphenol A (BPA) has multiple hormonal activities, but its effects on thyroid hormone (TH) action are not fully understood. TH is required for the metamorphosis of tadpoles into frogs, and thus tadpole tail regression, one of the most spectacular events in metamorphosis, can be used for studying the effect of BPA and related compounds (BPAs) on TH action. We found that 3,3 � ,5-triiodothyronine (T3)-induced tadpole tail regression in the wrinkled frog Rana rugosa (R. rugosa )i ss uppressed by BPA, tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA), and tetramethylbisphenol A (TMBPA). BPAs also inhibited spontaneous metamorphosis in the tropical clawed frog Silurana tropicalis (S. tropicalis )c ontrolled by endogenous circulating TH. These results indicate that BPAs are TH antagonists. For the African clawed frog Xenopus laevis (X. laevis), transgenic tadpoles carrying plasmid DNA containing TH response element (TRE) and 5 � -upstream promoter region of the TH receptor (TR) β A1 gene linked to an enhanced green fluorescent protein (EGFP) gene, T3 induced a strong EGFP expression in the hind limbs. This expression was suppressed by BPA, TBBPA, TCBPA and TMBPA, suggesting BPAs all act as antagonists to prevent the binding of T3 to TR, resulting in inhibition of TR-mediated gene expression. Much work has been done in examining the apoptotic and other detrimental effects of polychlorinated biphenyls (PCBs), but the role of mitochondrial damage in such toxic action remains obscure. There is evidence however that mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptosis. We investigated the effect of 4-hydroxy-3,3 � ,4 � ,5-tetrachlorobiphenyl (4-OH-TCB) on isolated rat liver mitochondria. In order to help clarify this, biochemical analysis revealed that 4-OH-TCB induced calcium release, reactive oxygen species (ROS) generation, depolarization, swelling and cytochromec (Cyt. c )r elease in a time- and concentration-dependent manner. These 4-OH-TCB-induced changes in mitochondrial function were found to be inhibited by the presence of cyclosporin A (CsA), a specific inhibitor of MPT, suggesting a classic type of MPT. It is concluded that 4-OH-TCB induces MPT together with ROS generation, causing apoptosis in certain cells.

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H+/K+-ATPase β subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.

Mycobacterium liflandii causes a fatal frog disease in captive anurans. Here we report, to our knowledge, the first epizootic of mycobacteriosis in a European colony of clawed frogs (Silurana tropicalis), previously imported from a United States biologic supply company. Our findings suggest the emerging potential of this infection through international trade.

Selected aromatic azo and benzidine based dyes are priority compounds under the Government of Canada's Chemical Management Plan (CMP) for environmental risk assessments. Organic compounds undergo chemical and biological transformations when they interact with environmental matrices and biotic species; identifying the transformation products is thus a critical component of the risk assessment process. Here, we used zero valent iron (ZVI) to initiate the reduction of the diazo compound dye Disperse Yellow 7 (DY 7). Using state-of-the-art accurate mass Liquid Chromatography-Quadrupole Time of Flight-Mass Spectroscopy (LC-QToF-MS), four transformation products were conclusively identified, while a fifth product was tentatively ascertained. The conclusively established transformation products included p-phenylenediamine (p-PDA, a known genotoxin), 4-aminoazobenzene (4-AAB, a category 2 carcinogen) and 4-aminobiphenyl (4-ABP, a category 1 human carcinogen). 4-ABP is thought to form via a benzidine rearrangement; this is the first report of DY 7 undergoing a benzidine rearrangement. Given the importance of reduction processes in the metabolism of organic contaminants by aquatic species, we used LC-MS/MS to analyze sediment samples that had been generated previously upon exposure of Western clawed frogs (Silurana tropicalis) to DY 7 (at exposure levels where cellular stress was observed in S. tropicalis). We found p-PDA, 4-AAB, and 4-ABP were present in all exposures, but not in any of the sediment controls, demonstrating that upon release of DY 7 to the aquatic environment, sediment dwelling organisms will metabolize DY 7 to generate known (and suspected) human carcinogens, including through a previously unreported in vivo benzidine rearrangement to produce 4-ABP.

Phylogenetic studies can reveal patterns of evolutionary change, including the gain or loss of elaborate courtship traits in males. Male African clawed frogs generally produce complex and rapid courtship vocalizations, whereas female calls are simple and slow. In a few species, however, male vocalizations are also simple and slow, suggesting loss of male-typical traits. Here, we explore features of the male vocal organ that could contribute to loss in two species with simple, slow male calls. In Xenopus boumbaensis, laryngeal morphology is more robust in males than in females. Larynges are larger, have a more complex cartilaginous morphology and contain more muscle fibers. Laryngeal muscle fibers are exclusively fast-twitch in males but are both fast- and slow-twitch in females. The laryngeal electromyogram, a measure of neuromuscular synaptic strength, shows greater potentiation in males than in females. Male-specific physiological features are shared with X. laevis, as well as with a species of the sister clade, Silurana tropicalis, and thus are likely ancestral. In X. borealis, certain aspects of laryngeal morphology and physiology are sexually monomorphic rather than dimorphic. In both sexes, laryngeal muscle fibers are of mixed-twitch type, which limits the production of muscle contractions at rapid intervals. Muscle activity potentiation and discrete tension transients resemble female rather than male X. boumbaensis. The de-masculinization of these laryngeal features suggests an alteration in sensitivity to the gonadal hormones that are known to control the sexual differentiation of the larynx in other Xenopus and Silurana species.

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).

It is known that estrogen-like environmental pollutants can feminise gonadal differentiation in frogs resulting in female-biased sex-ratios at metamorphosis. The long-term effects on reproductive function in frogs following larval exposure to pollutants are less known. Amphibian test systems which allow life-cycle studies are therefore needed. The aim of the present study was to characterise long-term estrogenic effects on the reproductive system of the emerging model species Xenopus (Silurana) tropicalis following larval exposure to ethynylestradiol (EE(2)). EE(2) is a synthetic estrogen that has been detected in sewage effluents and in surface waters. Newly hatched tadpoles (Niewkoop Faber (NF) stage 48) were exposed to the nominal EE(2) concentrations 0 (control), 1, 10, and 100 nM (with analytical chemistry support) until complete metamorphosis (NF stage 66). Effects on the reproductive organs were determined in juveniles (1 month after metamorphosis) and in 9-month-old frogs. Larval exposure to EE(2) caused female-biased phenotypic sex-ratios in both juvenile and adult frogs, which is in agreement with previous work on other frog species. Nearly all (97%) of the 63 EE(2)-exposed 9-month-old frogs had ovaries. Histological evaluation of the gonads of the 9-month-old frogs showed that they were sexually mature. Among the adult frogs with ovaries there was a dose-dependent increase in the frequency of individuals lacking oviducts. Adult frogs exposed to 100 nM EE(2) that had ovaries but no oviducts had lower levels of estrogen receptor alpha (ERalpha) mRNA in the brain than control animals and those exposed to 100 nM EE(2) that had ovaries as well as oviducts. EE(2) exposure did not cause any significant changes in ERalpha mRNA levels in the ovaries of the adult frogs. The reduced level of ERalpha mRNA in the brain of individuals with ovaries lacking oviducts suggests an organizing effect of EE(2) on the central nervous system. The results show that transient early life-stage exposure to an environmental pollutant can induce effects on the reproductive organs and the central nervous system that persist into adulthood. Overall, our data suggest that X. tropicalis, which has a shorter generation time than the well-established model species Xenopus laevis, is a suitable model organism for research on developmental reproductive toxicity in anuran species.

This investigation attempts to clarify the effects of the plastic monomer bisphenol A (BPA) and related chemicals on 3,3′,5-triiodothyronine (T3)-induced and spontaneous anuran tadpole tail regression. T3-induced tail regression was found to be suppressed by BPA and tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA), and tetramethylbisphenol A (TMBPA). T3-treated Rana rugosa tadpole tails displayed marked apoptotic features, including DNA fragmentation and ladder-like profiles, as opposed to essentially little or no fragmentation and ladder formation for BPA, TBBPA, TCBPA and TMBPA-treated tails. BPA and related compounds also inhibited Silurana tropicalis spontaneous metamorphosis controlled by endogenous circulating thyroid hormone (TH). These results indicate that BPA and related compounds are TH antagonists. In transgenic Xenopus laevis tadpoles carrying plasmid DNA containing TH response element (TRE) and 5′-upstream promoter region of the TH receptor (TR) bA1 gene linked to a green fluorescent protein (EGFP) gene, T3 induced a strong EGFP expression in the hind limbs, while T3 plus BPA, TBBPA, TCBPA or TMBPA suppressed the expression, suggesting BPA and related chemicals all act in preventing the binding of T3 to TR, resulting in inhibition of TR-mediated gene expression.

The ABCA4 (ABCR) gene encodes a retinal-specific ATP-binding cassette transporter. Mutations in ABCA4 are responsible for several recessive macular dystrophies and susceptibility to age related macular degeneration (AMD). The protein appears to function as a flippase of all-trans-retinaldehyde and/or its derivatives across the membrane of outer segment disks and is a potentially important element in recycling visual cycle metabolites. However, the understanding of ABCA4's role in the visual cycle is limited due to the lack of a direct functional assay. An evolutionary analysis of ABCA4 may aid in the identification of conserved elements, the preservation of which implies functional importance. To date, only human, murine, and bovine ABCA4 genes are described. We have identified ABCA4 genes from African (Xenopus laevis) and Western (Silurana tropicalis) clawed frogs. A comparative analysis describing the evolutionary relationships between the frog ABCA4s, annotated T. rubripes ABCA4, and mammalian ABCA4 proteins was carried out. Several segments are conserved in both intradiscal loop (IL) domains, in addition to the transmembrane and ATP-binding domains. Nonconserved segments were found in the IL and cytoplasmic linker domains. Maximum likelihood analyses of the aligned sequences strongly suggest that ABCA4 was subject to purifying selection. Collectively, these data corroborate the current evolutionary model where two distinct ABCA half-transporter progenitors were combined to form a full ABCA4 progenitor in ancestral chordates. We speculate that evolutionary alterations may increase the retinoid metabolite recycling capacity of ABCA4 and may improve dark adaptation.

Hemoglobin, myoglobin, neuroglobin, and cytoglobin are four types of vertebrate globins with distinct tissue distributions and functions. Here, we report the identification of a fifth and novel globin gene from fish and amphibians, which has apparently been lost in the evolution of higher vertebrates (Amniota). Because its function is presently unknown, we tentatively call it globin X (GbX). Globin X sequences were obtained from three fish species, the zebrafish Danio rerio, the goldfish Carassius auratus, and the pufferfish Tetraodon nigroviridis, and the clawed frog Silurana tropicalis. Globin X sequences are distinct from vertebrate hemoglobins, myoglobins, neuroglobins, and cytoglobins. Globin X displays the highest identity scores with neuroglobin (approximately 26% to 35%), although it is not a neuronal protein, as revealed by RT-PCR experiments on goldfish RNA from various tissues. The distal ligand-binding and the proximal heme-binding histidines (E7 and F8), as well as the conserved phenylalanine CD1 are present in the globin X sequences, but because of extensions at the N-terminal and C-terminal, the globin X proteins are longer than the typical eight alpha-helical globins and comprise about 200 amino acids. In addition to the conserved globin introns at helix positions B12.2 and G7.0, the globin X genes contain two introns in E10.2 and H10.0. The intron in E10.2 is shifted by 1 bp in respect to the vertebrate neuroglobin gene (E11.0), providing possible evidence for an intron sliding event. Phylogenetic analyses confirm an ancient evolutionary relationship of globin X with neuroglobin and suggest the existence of two distinct globin types in the last common ancestor of Protostomia and Deuterostomia.