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Gestagens are active ingredients in human and veterinary drugs with progestogenic activity. Two gestagens—progesterone (P4), and the synthetic P4 analogue, melengestrol acetate (MGA)—are approved for use in beef cattle agriculture in North America. Both P4 and MGA have been measured in surface water receiving runoff from animal agricultural operations. This project aimed to assess the morphometric and molecular consequences of chronic exposures to P4, MGA, and their mixture during Western clawed frog metamorphosis. Chronic exposure (from embryo to metamorphosis) to MGA (1.7 µg/L) or P4 + MGA (0.22 µg/L P4 + 1.5 µg/L MGA) caused a considerable dysregulation of metamorphic timing, as evidenced by an inhibition of growth, narrower head, and lack of forelimb emergence in all animals. Molecular analysis revealed that chronic exposure to the mixture induced an additive upregulation of neurosteroid-related (GABAA receptor subunit α6 (gabra6) and steroid 5-alpha reductase 1 (srd5α1) gene expression in brain tissue. Chronic P4 exposure (0.26 µg/L P4) induced a significant upregulation of the expression hypothalamic–pituitary–gonadal (HPG)-related genes (ipgr, erα) in the gonadal mesonephros complex (GMC). Our data suggest that exposure to P4, MGA, and their mixture induces multiple endocrine responses and adverse effects in larval Western clawed frogs. This study helps to better our understanding of the consequences of chronic gestagen exposure and suggests that the implications and risk of high gestagen use in beef cattle feeding operations may extend to the aquatic environment.

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.

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.

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.

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.

A 150-day post-metamorphosis (dpm) partial lifecycle study exposing Silurana tropicalis to

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.

The African clawed frogs of the subgenus Silurana comprise both diploid and tetraploid species. The root of the polyploidization event leading to the extant Xenopus calcaratus, X. mellotropicalis, and X. epitropicalis is not fully understood so far. In X. mellotropicalis, we previously proposed 2 evolutionary scenarios encompassing complete (scenario A) or incomplete (scenario B) translocation of a heterochromatic block from chromosome 9 to 2 in a diploid ancestor. To resolve this puzzle, we performed FISH coupled with tyramide signal amplification (FISH-TSA) using 5 X. tropicalis and X. mellotropicalis single copy gene probes (gyg2, cept1, fn1, ndufs1, and sf3b1) reflecting borders of the heterochromatic blocks in X. tropicalis chromosome 9 (XTR 9) and X. mellotropicalis chromosome 9b (XME 9b) and XME 2a. cDNA sequencing recognized both homoeologous genes in X. mellotropicalis. Comparison of gene physical mapping between X. tropicalis and X. mellotropicalis clearly confirmed complete rather than incomplete translocation t(9;2) of the heterochromatic block in the diploid predecessor and thus favored scenario A regarding the formation of an ancestral allotetraploid karyotype.

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.

Indoor dust contains a multitude of industrial chemicals, and ingestion of dust is considered an important exposure route to organic contaminants. Some of these contaminants have been shown to interfere with the thyroid system, which may result in significant consequences on public health. The amphibian metamorphosis is a thyroid hormone-dependent process, which can be used as an in vivo model for studies on thyroid hormone-disrupting potency. Three contaminants of indoor dust were tested on metamorphosing Silurana (Xenopus) tropicalis tadpoles. The tested chemicals were Tris (1,3-dichloroisopropyl) phosphate (TDCiPP), tetrabromobisphenol-A (TBBPA) and propylparaben (PrP). Measurements reflecting general growth, development progress and thyroid epithelial cell height were performed on the exposed tadpoles as well as chemical analyses of the exposure water. It was shown that TDCiPP acts as a thyroid hormone-disrupting chemical in metamorphosing tadpoles by causing increased epithelial cell height in thyroid glands after exposure to a nominal concentration of 0.010 mg/L and in higher concentrations. TBBPA caused reductions in general growth of tadpoles at the nominal concentration 0.125 mg/L, and PrP caused acute toxicity at the nominal concentration 12.5 mg/L. However, no evident indications of specific thyroid-disrupting effects caused by TBBPA or PrP were observed.

Naphthenic acids (NAs) are carboxylic acids naturally occurring in crude oils and bitumen and are suspected to be the primary toxic substances in wastewaters associated with oil refineries and mining of oil sands. Oil sands process-affected water (OSPW) generated by the extraction of bitumen from oil sands are a major source of NAs and are currently stored in tailings ponds. We report on the acute lethality and teratogenic effects of aquatic exposure of Silurana (Xenopus) tropicalis embryos to commercial NA extracts and from the acid extractable organics (AEOs) fraction of a Canadian OSPW. Using electrospray ionization-high resolution mass spectrometry, we determined that the O2 species proportion were 98.8, 98.9 and 58.6% for commercial mixtures Sigma 1 (S1M) and Sigma 2 (S2M), and AEOs, respectively. The 96h LC50 estimates were 10.4, 11.7, and 52.3 mg/L for S1M, S2M, and the AEOs, respectively. The 96h EC50 estimates based on frequencies of developmental abnormalities were 2.1, 2.6, and 14.2 mg/L for S1...

The impact of the perfluoro-chemical, perfluorooctanesulfonate (PFOS), on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined because of its ubiquity in the environment, bioaccumulative nature and potential to disturb endocrine activity. A partial life cycle study exposing S. tropicalis to varying concentrations of PFOS 0.06, 0.13, 0.25, 0.50 and 1.0 mg PFOS/L [nominal]) was conducted. Gonad and plasma samples were collected from juvenile control specimens and organisms exposed to PFOS from early embryo through 150 days post-metamorphosis. Gonad CYP17, aromatase and 5α-reductase activities were measured. Plasma estradiol, testosterone, dihydrotestosterone (DHT) and gonadal testosterone were measured in both males and females. Increased plasma DHT and gonadal testosterone were found in PFOS-treated juvenile male S. tropicalis compared to controls. Decreased plasma estradiol, but not testosterone, was detected in PFOS-treated female S. tropicalis compared to controls. Plasma DHT was not detected and an increase in gonadal testosterone was detected in PFOS-treated female frogs. Female S. tropicalis exposed to PFOS exhibited a concentration-related decrease in the mean aromatase activity, but not 5α-reductase. PFOS exposure in male frogs induced a concentration-related increase in 5α-reductase activity, but did not alter aromatase activity compared to control frogs. A concentration-related increase in CYP 17,20-lyase activity, but not 17-hydroxylase activity, was found in both female and male S. tropicalis exposed to PFOS.

Phthalates are compounds used in polymers to increase their flexibility and are now ubiquitous in the environment as a result of widespread use. Because few studies have focused on the adverse effects of these chemicals in aquatic species, the present study aimed to determine the effects of phthalate diesters in amphibians. Western clawed frog (Silurana tropicalis) tadpoles were acutely exposed to water spiked with monomethyl phthalate (MMP; 1.3-1595.5 mg/L), dimethyl phthalate (DMP; 0.03-924.0 mg/L), or dicyclohexyl phthalate (DCHP; 0.3-99.3 mg/L). Because few studies have addressed the toxicity of these specific phthalates in most organisms, the present study used higher concentrations of these chemicals to determine their toxicity pathways in amphibians and at the same time investigate a suite of genes known to be altered by the well-studied phthalates. Both DMP and DCHP increased larval mortality (9.1-924.0 mg/L DMP and 4.1-99.3 mg/L DCHP), increased frequency of malformations in tadpoles (0.1-34.1 mg/L DMP and 4.1-19.0 mg/L DCHP), and up-regulated cellular stress-related messenger-RNA (mRNA) levels (4.1 mg/L DCHP). To characterize the molecular toxicity pathway of these phthalates in tadpoles, transcriptome analysis was conducted using a custom microarray. Parametric analysis of gene set enrichment revealed important changes in the expression of genes related to drug metabolism and transport, liver metabolism, xenobiotic clearance, and xenobiotic metabolism after DMP and DCHP treatments, although these responses were less pronounced with MMP (the metabolite of DMP). The present study is one of the few studies that demonstrated complementarity between gene expression analysis and organismal effects. Environ Toxicol Chem 2016;35:2511-2522. © 2016 SETAC.

While the identification of conserved processes across multiple taxa leads to an understanding of fundamental developmental mechanisms, the ways in which different animals fail to conform to common developmental processes can elucidate how evolution modifies development to result in the vast array of morphologies seen today-the developmental mechanisms that lead to anatomical variation. Odontogenesis-how teeth are initiated and formed-is well suited to the examination of both developmental conservation and phenotypic diversity. We suggest here that the study of early tooth development, the period of odontogenic band development, reveals departures from conserved mechanisms that question the role of players in the developmental process. In the earliest stages of odontogenesis, Sonic hedgehog (Shh) gene expression is interpreted as critical evidence of tooth initiation prior to any histological indication. However, a detailed examination of studies of tooth development across a wide range of taxa reveals that several vertebrate species fail to conform to the expectations of the Shh Consensus Model, calling for a reconsideration of the assumed causality of epithelial Shh in tooth initiation. We present new Shh gene expression data for an amphibian, the frog Silurana (Xenopus) tropicalis. In these animals, craniofacial and odontogenic developmental processes are more disjunct, and thereby provide a natural test of the hypothesis that Shh is immediately required for subsequent tooth development. Our results suggest that Shh expression may actually be related to the formation of the mouth rather than a required precursor to subsequent tooth formation. Anat Rec, 299:1099-1109, 2016. © 2016 Wiley Periodicals, Inc.

Amphibian gonadal differentiation involves the action of sex steroids. Recent research indicates that the anti-Müllerian hormone (AMH) is involved in testicular development in some lower vertebrate species. For amphibians there is a lack of data on ontogenetic expression of the AMH receptor AMHR2/amhr2 and of progesterone receptors (PGRS/pgrs). Here we expand the knowledge on amphibian sex differentiation by characterizing ontogenetic mRNA levels of amh, amhr2, intracellular and membrane pgrs (ipgr and mpgr beta) and cytochrome P450 19a1 (cyp19a1) (ovarian marker) in the urogenital complex of the model species Xenopus (Silurana) tropicalis. Furthermore, we characterized the ontogenetic development of the Müllerian ducts (precursors of the female reproductive tract) histologically. The developmental period investigated spanned from beginning of gonadal differentiation, Nieuwkoop and Faber (NF) stage 51, to 4weeks post-metamorphosis. The Müllerian ducts were first observed at NF 64 in both sexes. Male-enhanced amh mRNA levels from NF 53/54 to 6days post-metamorphosis and female-enhanced cyp19a1 levels from NF 53 to 4weeks post-metamorphosis were noted. The sexually dimorphic mRNA level profile was more distinct for amh than for cyp19a1. The pgrs mRNA levels increased over the studied period and showed no sex differences. At later developmental stages, the amhr2 mRNA level was increased in putative females compared with males. Our findings suggest that AMH has a role in gonadal differentiation in X. tropicalis. We propose relative gonadal amh mRNA level as a testicular marker during early gonadal development in amphibians.

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.

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.

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.

Western clawed frog (Silurana tropicalis) embryos were exposed to control, low (nominally 0.5 mg L(-1)) and high (nominally 1 mg L(-1)) arsenate (As(V)) culture water concentrations to investigate the effects of arsenic (As) on different life stages, namely tadpole (Nieuwkoop and Faber stage 56, NF56) and frog stages (NF66). The effects were assessed by measuring arsenic(+3) and DNA methyltransferases (AS3MT and DNMT1), as well as As speciation in the tissues. The As content in frog tissues increased with water As concentration. The As species observed by high performance liquid chromatography - inductively coupled plasma mass spectrometry (HPLC-ICPMS) were mostly inorganic, dimethylarsinic acid (DMA) and trimethylarsine oxide (TMAO). With solid state X-ray absorption near edge structure (XANES) analysis, arsenobetaine/tetramethylarsonium ion were also seen. AS3MT levels decreased upon low As exposure in NF56, rising again to control levels at the high As exposure. In NF66 tissues, on the other hand, AS3MT decreased only with NF66 high As exposure. DNMT1 increased with exposure, and this was statistically significant only for the high As exposure at both life stages. Thus these enzymes seem to be affected by the As exposure. Methylation of As to form monomethylarsonate (MMA), DMA and TMAO in the frogs appeared to be inversely related to AS3MT levels. A possible interpretation of this finding is that when AS3MT is higher, excretion of MMA + DMA + TMAO is more efficient, leaving lower concentrations in the tissues, with the opposite effect (less excretion) when AS3MT is lower; alternatively, other enzymes or linked genes may affect the methylation of As.

Sex-biased gene expression can evolve from sex-specific selection and is often associated with sex-linked genes. Gene duplication is a particularly effective mechanism for the generation of sex-biased genes, in which a new copy can help resolve intralocus sexual conflicts. This study assesses sex-biased gene expression in an amphibian with homomorphic ZW sex chromosomes, the Western clawed frog Silurana (Xenopus)tropicalis. Previous work has shown that the sex chromosomes in this species are mainly undifferentiated and pseudoautosomal. Consistent with ongoing recombination between the sex chromosomes, this study detected little evidence for the general sexualization of sex-linked regions. A subset of genes closely linked to the sex determining locus displays a tendency for male-biased expression and elevated rates of evolution relative to genes in other genomic locations. This may be a symptom of an early stage of sex chromosome differentiation driven by, for example, chromosomal degeneration or natural selection on genes in this portion of the Z chromosome. Alternatively, it could reflect variation between the sexes in allelic copy number coupled with a lack of dosage compensation. Irrespective of the genomic location, lineage-specific genes and recently duplicated genes had significantly high levels of sex-biased expression, offering insights into the early transcriptional differentiation of young genes.

The T2 family of miniature inverted-repeat transposable elements (T2-MITE) is a prevalent MITE family found in both Xenopus(Silurana) tropicalis and X. laevis. Some subfamilies, particularly T2-A1 and T2-C, may have originated prior to the diversification of the 2 Xenopus lineages and currently include active members in X. tropicalis, whereas another subfamily, T2-E, may have lost its transposition activity even earlier. The distribution of each T2-MITE subfamily in X. tropicalis was investigated and compared to evaluate the evolutionary dynamics of the T2-MITE subfamilies. The subfamilies showed differences in chromosomal distribution, uniformity of insertion density on scaffolds, ratios of upstream to downstream insertions with respect to genes, and their distance from genes. Among these, the T2-C subfamily was interesting because it was frequently inserted upstream and close to genes and because genes with close insertions of this subfamily showed high correlations in spatial expression patterns. This unique distribution and long-lived transposition activity may reflect a mutual relationship evolved between this subfamily and the host.

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.

B chromosomes are additional elements to standard karyotypes observed in different species of fishes, especially in Curimatidae. However, despite studies demonstrating the occurrence of Bs, little is known about their origin and evolution. To better understand the genomic composition and evolutionary processes involving B chromosomes, microdissection of B microchromosomes in Steindachnerina insculpta was conducted. Chromosome painting revealed the totally hybridized B and markings on A chromosomes both in S. in sculpta and in Cyphocharax spilotus, demonstrating a strong homology between these different species. In specimens of C. modestus, which do not have Bs, the signals were observed on A chromosomes. Cloning and sequencing of some B fragments revealed that the B microchromosome in S. insculpta is composed of repetitive elements, homologous to the DIRS-4 LTR retrotransposon of Xenopus (Silurana) tropicalis. FISH with clone pSi48 with the DIRS-4 retroelement revealed signals on all A chromosomes in the 2 species and also on the B, suggesting the insertion of repetitive elements in these species.

Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawed frog (Xenopus laevis), the Western clawed frog (Silurana/X. tropicalis), the Japanese bell-ring frog (Buergeria buergeri), and the Japanese wrinkled frog (Rana rugosa). Comparative mapping data revealed that the sex chromosomes of X. laevis, X. tropicalis and R. rugosa are different chromosome pairs; however, the sex chromosomes of X. tropicalis and B. buergeri are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in B. buergeri, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences.

International audience; Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus fraseri Boulenger, 1905 (Pipidae) led to identification of 13 host-defense peptides. The primary structures of the peptides demonstrate that they belong to the magainin (3 peptides), peptide glycine-leucine-amide, PGLa (4 peptides), and xenopsin-precursor fragment, XPF (2 peptides) families, first identified in Xenopus laevis, together with caerulein precursor fragment-related peptides, CPF-RP (4 peptides), first identified in Silurana tropicalis. In addition, the secretions contain a molecular variant of xenopsin displaying the substitution Arg(4)→Lys compared with X. laevis xenopsin and peptide glycine-tyrosine-amide (PGYa) (GRIIPIYPEFERVFA KKVYPLY.NH2) whose function is unknown. The most potent antimicrobial peptide identified is CPF-RP-F1 (GFGSVLGKALKFGANLL.NH2) with MIC=12.5μM against Staphylococcus aureus and 50μM against Escherichia coli. On the basis of similarities in morphology and advertisement calls, X. fraseri has been placed in a species group that includes the octoploids Xenopus amieti and Xenopus andrei, and the tetraploid Xenopus pygmaeus. Cladistic analyses based upon the primary structures of magainin, PGLa, and CPF-RP peptides support a close evolutionary relationship between X. fraseri, X. amieti and X. andrei but suggest a more distant relationship with X. pygmaeus.

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.

Estrogens and their receptors are present at very early stages of vertebrate embryogenesis before gonadal tissues are formed. However, the cellular source and the function of estrogens in embryogenesis remain major questions in developmental endocrinology. We demonstrate the presence of estrogen-synthesizing enzyme aromatase and G protein-coupled estrogen receptor (GPER) proteins throughout early embryogenesis in the model organism, Silurana tropicalis. We provide the first evidence of aromatase in the vertebrate lateral line. High levels of aromatase were detected in the mantle cells of neuromasts, the mechanosensory units of the lateral line, which persisted throughout the course of development (Nieuwkoop and Faber stages 34-47). We show that GPER is expressed in both the accessory and hair cells. Pharmacological activation of GPER with the agonist G-1 disrupted neuromast development and migration. Future study of this novel estrogen system in the amphibian lateral line may shed light on similar systems such as the mammalian inner ear.

Exploration behaviour is a complex trait that may have strong implications for the fitness of individuals and the persistence of populations. Understanding the different exploration strategies is necessary to understand how animals may adapt to changes in their environment including human-induced habitat fragmentation. Behavioural syndromes are often thought to characterize exploration behaviour, and within a population, individual strategies may vary from ‘bold’ to ‘shy’. Although our understanding of behavioural syndromes has increased enormously over the past decade, little is known about the presence of such syndromes in frogs. Yet, frogs are particularly sensitive to changes in their environment because of their ectothermic physiology and low mobility. Here, we investigate the exploration behaviour of wild-caught male frogs under laboratory conditions to test whether distinct behavioural strategies exist. We demonstrate the presence of different behavioural syndromes with two of the syndromes that can be categorized as ‘bold’ and ‘shy’, and a third one that is clearly intermediate. These behavioural strategies are, however, independent of variation in morphology and locomotor performance indicating that these two components of mobility (i.e. behaviour and locomotor capacity) are decoupled and could thus respond differentially to selection on mobility.

The objectives of this study were to examine the effects of potassium perchlorate (KClO 4 ) treatment on androgen- and thyroid hormone (TH)-related transcript levels during gonadogenesis in the frog Silurana tropicalis . Androgen- and TH-related gene expression was examined in gonad–mesonephros complex (GMC) and liver tissues at stage NF 56 and stage NF 60. These stages of development coincide with the period of sexual differentiation. Real-time RT-PCR analyses revealed that androgen- and TH-related transcript levels in the GMC and liver of stage NF 56 and NF 60 frogs are responsive to KClO 4 exposure during prometamorphosis. An increase in srd5α2 mRNA levels in hepatic tissues of KClO 4 -treated NF 56 tadpoles suggests an important role for hepatic tissues in androgen metabolism. Gene transcript differences highlight possible stage- and tissue-specific sensitivities to KClO 4 . A greater number of TH- and androgen-related transcriptional changes were discerned in the hepatic tissues compared to the gonads, and overall fewer transcriptional changes were observed in stage NF 60 tadpoles compared to stage NF 56 larvae. Perchlorate suppressed somatic and hind-limb development during the 96-d exposure period. Treatment with KClO 4 had no significant effect on sex ratios, however a notable reduction in the percentage of males (33.3% M: 66.7% F) in the highest KClO 4 concentration (107 μg/L) was observed. Overall, these findings suggest that KClO 4 has secondary androgenic disrupting properties in addition to its known primary thyroid hormone-disrupting role.

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.

Azo compounds are used in a variety of industrial applications, such as textile colorant. Azo dyes have been found to contaminate aquatic environments and it has been shown that these compounds could potentially be toxic or induce endocrine disruption in aquatic organisms. However, there are few data available on the toxicity of these dyes, specifically Acid Red 97 (AR97) and Bismarck Brown Y (BBY). The aim of this study was to determine the toxicity and the endocrine-disrupting properties of AR97 and BBY in frogs. As fugacity modeling predicted that both compounds would sorb to sediment, sediment exposures were performed using a geometric range of concentrations (0, 1, 10, 100 and 1,000 ppm). Both AR97 and BBY dyes were not lethal to Silurana tropicalis embryos; however, BBY significantly induced malformations. Gene expression analysis of oxidative stress and mutagen-related genes was performed in BBY-treated larvae. There were significant two-fold increases of the tumor-suppressing protein p53 and heat shock protein 70 mRNA at 1,000 ppm suggesting that BBY induces cellular stress in early S. tropicalis development. Transcripts of the heat shock protein 90 did not change. Furthermore, reproductive-related genes were assessed and a 2.1-fold change was observed in the mRNA of the steroidogenic acute regulatory protein while steroid 5 alpha-reductase type 2 and androgen receptor transcript levels did not vary among treatments. In conclusion, high concentrations of BBY lead to increased developmental defects in frog embryogenesis and early larval development.

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.