Available data, including results from six invivo studies conducted by the United States (US) Environmental Protection Agency (EPA) were reviewed to assess the potential of 2-ethylhexyl 4-hydroxybenzoate (2-EHHB) to affect endocrine pathways. 2-EHHB is a paraben, and parabens are used in the cosmetics, food, and pharmaceutical industries. It was selected for testing based on invitro bioactivity scores from androgen receptor (AR) and estrogen receptor (ER) pathway models provided in the US EPA Computational Toxicology Chemicals Dashboard. The following assays were performed using US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 and Tier 2 test guidelines: Hershberger, male pubertal, amphibian metamorphosis assay (AMA), fish short-term reproduction assay (FSTRA; medaka, fathead minnow, and zebrafish), larval amphibian growth and development assay (LAGDA), and medaka extended one-generation reproduction test (MEOGRT). Published data from uterotrophic assays performed in Japan were also reviewed. Based on this evaluation, estrogen agonism was the best supported mechanism of action and there was no clear support for thyroid hormone perturbation. Among the invivo assays reviewed, the Japanese medaka and African clawed frog were the most sensitive test species. Results from the MEOGRT indicated decreased fecundity (all generations) at concentrations ≥ 5.32 μg/L. Decreased fecundity and fertilization at ≥ 10.6 μg/L in the medaka FSTRA support those results. LAGDA results indicated delayed development at ≥ 5.33 μg/L, but the mechanism was unclear based on the available data.

Regulation of the agonist sensitivity of neurotransmitter receptors is critical for proper functioning of neuronal circuits and is, therefore, conserved across evolutionary time. Mutations that alter agonist sensitivity are often pathological in humans. A brain-expressing nicotinic acetylcholine receptor (nAChR) from the frog Xenopus tropicalis shows ∼20× greater sensitivity to ACh as orthologs from human, chickens, and other frogs prompt us to examine the molecular basis for this extreme sensitivity. We identified a single amino acid substitution in the third transmembrane domain (M3) of the X. tropicalis α4 nAChR subunit, F294 (S in other vertebrate orthologs), that confers the high sensitivity. Surprisingly, we noted variation at this site in sequences deposited in NCBI, suggesting either allelic variation or RNA editing. By sequencing genomic DNA and mRNA (cDNA) from the same individuals from two different colonies of X. tropicalis, we determined that a possible source of this variation is RNA editing. The unedited receptor from X. tropicalis (S294) has a similar ACh sensitivity as those from other vertebrates. Further work must be done to examine possible adaptations of edited receptors and if the frog's brain compensates for an increase in sensitivity since increases in agonist sensitivity lead to pathology in humans.

Predicting acute developmental toxicity of chemicals in embryos of the African clawed frog (Xenopus laevis): Calibration and validation of regression-based quantitative structure activity relationship models for hazard assessment of chemicals in anuran amphibians.

Repeated survival surgical oocyte collection in African clawed frogs (Xenopus laevis) is commonly performed to access high-quality, viable oocytes for research procedures and to reduce the number of animals used in research. Despite this common practice, there is no evidence-based limit for the total number of survival celiotomies that can be performed. To provide an improved reference, a retired colony of experimental frogs (n = 31) was euthanized for gross and histopathologic evaluation and then compared with 4 experimentally naive age-matched controls (n = 4). Experimental animals underwent 4-11 celiotomies (average 6 ± 2) and were between 180 and 1,646 days (average 448 ± 245.2 days) from their last surgery. Body weight, residual skin, and coelomic wall sutures were counted. Surgically incised skin and coelomic wall musculature were collected for histopathology. A cumulative skin score and a coelomic wall score were developed to reflect all histopathologic evidence of acute and chronic inflammation. The cumulative skin score was significantly predicted by the number of surgeries (P = 0.003), but not predicted by the number of skin sutures remaining (P = 0.14). The coelomic wall score was significantly predicted by the number of coelomic sutures placed (P < 0.001) and the number of surgeries (P < 0.001). Neither one of the histopathology scores correlated to the number of days since the last surgery (P > 0.2). As one would expect, increasing the number of surgeries and coelomic sutures correlated with the histopathologic finding of a chronic inflammatory process in the skin. Although a specific recommendation for a maximum number of surgeries could not be discerned, this study provides an improved reference for Xenopus survival surgical oocyte collection underscoring the recommendation to use absorbable sutures within the body cavity and to remove all external nonabsorbable sutures 10-14 days postoperatively.

In this study, the biocatalytic activity of four steroid-transforming strains isolated from the African frog Xenopus laevis and identified as Streptomyces rochei towards pregnane steroids has been investigated. All the isolated strains facilitated the reduction of the C20-carbonyl group and the structures of the metabolites were confirmed by mass spectrometric (MS) and 1H NMR spectroscopic analyses. Hydrocortisone and progesterone were poorly transformed by the streptomycete strains, whereas cortexolone (Reichstein's substance S) was effectively biotransformed, yielding more than 90% of 17α,20β,21α-trihydroxy-4-pregnen-3-one (20β-S). Primarily, 20α-reduction was detected when the microbial isolates were incubated with 17α-hydroxyprogesterone with the yield of 17α,20α-dihydroxy-4-pregnen-3-one (17,20α-P) reaching 70%. The biological activity of 20β-S was evaluated in Danio rerio. The results demonstrated that 20β-S modulated stress- and anxiety-related behavioral responses and activated Pgr-dependent transcriptional pathways in the brain and ovarian tissues. These observations support the potential relevance of the synthesized progestin as a functional regulator in teleost physiology. The findings enhance our understanding of the biodiversity of steroid-transforming actinomycetes inhabiting amphibians and can be successfully employed for the effective microbiological synthesis of biologically active 20-hydroxylated progestins that serve as bioregulators in teleosts.

Major Histocompatibility Complex (MHC) molecules are central to vertebrate adaptive immunity, and MHC genes serve as key models in evolutionary genomics, offering insight into birth-and-death evolution, gene duplication, and the maintenance of genetic diversity. However, the organization and evolution of the MHC in species with giant genomes, such as salamanders, remain poorly understood. Here, we use comparative genomics, expression across multiple ontogenetic stages and tissues, as well as polymorphism data to investigate MHC evolution in newts. Contrary to earlier suggestions of a massively expanded MHC in salamanders, we find that the core MHC region remains relatively compact, demonstrating that genome gigantism does not scale proportionally in this region. Our finding also challenges the model of coevolution between a single classical MHC-Ia gene and antigen processing genes (APGs), revealing instead several polymorphic and highly expressed putative MHC-Ia located at varying distances from the APGs. MHC-I genes exhibit lineage-specific duplications and signs of concerted evolution, resulting in poorly resolved phylogenies. In contrast, MHC-II genes are more conserved and exhibit extensive trans-species polymorphism. Expression and polymorphism patterns identify putative nonclassical MHC-Ib genes, likely repeatedly derived from MHC-Ia genes, paralleling patterns seen in mammals but contrasting with the situation in fish and Xenopus frogs. In all seven studied species, some MHC-Ib genes show high relative expression during the larval stage but not at adulthood, suggesting a role in larval immunity. Our results underscore the importance of salamanders for understanding the evolution of complex regions in giant genomes and the architecture of the tetrapod MHC.

ABSTRACTTranscription factors are typically thought to play a limited role in developmental evolution due to their high pleiotropic nature. However, such constraints may be relaxed following gene duplication or when proteins are organized into structural and functional modules, opening avenues for evolutionary innovation. Here, we integrate expression and genomic data to investigate the evolutionary dynamics of Hox gene duplicates in the allotetraploid frog Xenopus laevis. Despite overall conservation across the Hox clusters, we find that HoxB4L has acquired expression during maternally regulated stages and is evolving under positive selection. Protein‐level changes include the number, length, and sequence of functionally important protein regions. Our results indicate that HoxB4L has escaped ancestral constraints and is undergoing maternal neofunctionalization as a result of cis‐regulatory divergence and structural protein modifications. These findings illustrate how transcription factors can overcome developmental constraints and contribute to novel functions during early development.

Biotic resistance theory posits a negative relationship between diversity and invasibility. The mechanisms behind this can be variable, but studies are often focused on competition between many resident and introduced species. However, individual species may interact more strongly in communities, some of which may be effective predators and, thus, could offer stronger biotic resistance, broadening biotic resistance theory beyond diversity. In species with complex life histories, biotic resistance may be stage-specific, with resisting species affecting select life stages. The relationship between native eastern mosquitofish ( Gambusia holbrooki ) and the non-native tropical clawed frog ( Xenopus tropicalis ; TCF), provides a model system to investigate stage-specific biotic resistance. Preliminary observations suggest TCFs in their introduced range in Florida, USA are more likely to occupy ponds without fish and we hypothesise that mosquitofish provide predatory biotic resistance to early TCF life stages. We tested the hypothesis that the strength of predatory biotic resistance to species with complex life histories would be based on life stage. We also examined how the strength of predatory biotic resistance can be mitigated by predator density, cannibalism by adult life stages and habitat complexity. Hypotheses were tested using wild-caught and lab-reared individuals in controlled laboratory experiments. Early TCF life stages, lacking defensive traits, suffer near-total mortality due to predation and harassment, with vulnerability decreasing during and after metamorphosis. Mosquitofish density and habitat complexity had minimal effects, while the combination of adult frogs and mosquitofish resulted in facilitation between the two predators, increasing tadpole predation. This study underscores the need for thorough testing of the biotic resistance hypothesis across life stages and shows promise for the control of TCFs in Florida. Mosquitofish could be used as a biological control to reduce the recruitment of TCFs by eliminating the larval stage.

African clawed frogs (Xenopus) have a high rate of genome duplication, which may catalyze evolution-including of sex chromosomes. To explore this, for each of four species in the subgenus Silurana, we analyzed sex-associated genetic variation, and in the diploid species X. tropicalis, we explored population structure. We found that the sex-linked regions in all four species are homologous, and we infer that X. calcaratus has an unusual sex determination system with three sex chromosomes, which was previously known only in X. tropicalis. Our results evidence two independent allotetraploidization in Silurana, admixture across ploidy levels, and demonstrate that the most recent allotetraploidization that generated the X. calcaratus lineage occurred after population subdivision arose in X. tropicalis. Thus, this unusual triple sex chromosome system has been maintained independently in two different species for a protracted period and through an allotetraploidization event. Simulations indicate that genetic drift should eliminate one of the sex chromosomes, suggesting that there may be unidentified benefits to maintaining this complex system.

The doublesex and mab-3 related transcription factor 1 (dmrt1) plays a crucial role in metazoan sexual differentiation. This gene, or its paralogs, independently became triggers for sex determination several times, including in the tetraploid African clawed frog Xenopus laevis. To explore functional evolution of this gene, we generated knockout lines of each of two dmrt1 homeologs in X. laevis and an ortholog in the closely related diploid Western clawed frog X. tropicalis. Our findings evidence sex-specific functional evolution following duplication by allotetraploidization in an ancestor of X. laevis. In females, dmrt1 was essential for fertility and oogenesis in the Xenopus ancestor, but this important function was lost (subfunctionalized) in one X. laevis homeolog (dmrt1.S) after allotetraploidization. In males – in sharp contrast – dmrt1 was not essential for fertility and spermatogenesis in the Xenopus ancestor, but this essentiality was acquired (neofunctionalized) in the other X. laevis homeolog (dmrt1.L) after allotetraploidization. Transcriptomic analysis of the mesonephros/gonad complex during sexual differentiation identifies distinctive patterns of dysregulation in male and female knockouts of dmrt1.L and dmrt1.S relative to same-sex wildtype siblings, including possible autocatalysis of dmrt1.L and activation of the female-determining gene dm-w. Previous work demonstrates that dm-w was recently derived from partial gene duplication of dmrt1.S – a gene that our analysis demonstrates is non-essential in both sexes. Thus, in X. laevis, a developmental system was pushed past a “tipping point” to a novel state where sexual differentiation is now orchestrated by a sex-specific duplicate of a dispensable gene.

The insecticide acetamiprid is a neonicotinoid still approved for outdoor use worldwide. For active use, there are formulations that contain other often unpublished co-formulants in addition to acetamiprid. This study investigated effects of four different acetamiprid formulations (Careo Rosenspray, Careo, Mospilan, Danjiri) in a concentration range of 0.01-100 mg a.i./L on embryonic development of Xenopus laevis. The formulations led to an increase in embryo mortality with LC50-values between 0.5 and 53.2 mg a.i./L depending on the formulation. At sublethal acetamiprid concentrations in Mospilan and Danjiri, the external appearance (body length, oedemas), the early neural development (eyes, heads, brains, cranial nerves) as well as the heart morphology and function were disturbed. Our results show that strict adherence to recommended handling and application conditions of acetamiprid formulations is essential to protect X. laevis. Therefore, pesticide risk assessment and approval renewal procedures must include commercially available combined formulations rather than solely evaluating the active ingredient.

The rise of antibiotic resistance has created an urgent need for the discovery of new antimicrobial peptides (AMPs), prompting various screening strategies. However, the mechanisms of action of AMPs are often overlooked during screening and optimization. Here, a mechanism‐driven screening approach is introduced using machine learning‐based computational models to identify peptide sequences that target bacterial membranes and form pores. From the metaproteomes of poison frogs, African clawed frogs, and human skin, seven peptides are identified and validated, each exhibiting antimicrobial activity with minimal hemolysis and cytotoxicity. These peptides demonstrated membrane disruption in liposome leakage assays, with three showing broad‐spectrum activity against Gram‐positive and Gram‐negative bacteria. Single‐molecule experiments confirmed peptide oligomerization on membranes, while electrophysiological measurements verified pore formation by the three broad‐spectrum AMPs, suggesting a correlation between pore‐forming ability and broad‐spectrum antimicrobial activity. This approach offers a promising mechanism‐driven strategy for discovering new antimicrobial agents to combat antibiotic resistance.

Simple four-neuron computational models comprising bilateral pairs of excitatory dIN and inhibitory cIN neurons were used to test several hypotheses concerning the role of electrogenic sodium pumps in shaping swimming CPG output in Xenopus tadpoles. The initial model had no sodium pumps and generated continuous swim-like rhythmic activity. In real tadpoles, activity-dependent “dynamic” sodium pumps are proposed to mediate the post-swim ultraslow after-hyperpolarization (usAHP) apparent in most cINs, that reduces subsequent swim episode durations, producing a form of short-term motor memory (STMM). Dynamic pumps were therefore incorporated into model cINs, which then generated a usAHP causing swimming episodes to self-terminate, and when inter-swim intervals were varied the model also replicated STMM. In real tadpoles no usAHP is normally apparent in dINs, but one can be revealed by pharmacologically blocking the hyperpolarization-activated current, Ih, which is exclusively expressed in dINs. Dynamic pumps and HCN channels mediating Ih were therefore added to the model dINs. If HCN conductance was locked at its resting level, the dINs now showed a substantial pump-generated usAHP, but this was almost completely cancelled when HCN conductance was allowed to respond normally. Complete cancellation could be achieved by including a speculative cAMP-mediated modulation of the HCN activation kinetics. The models thus confirm the plausibility of published hypotheses regarding the generation of the usAHP in cINs, its apparent absence in dINs due to masking by Ih, and its role in mediating STMM. They also suggest the involvement of the usAHP in swim termination, and possible regulation by cyclic nucleotides.

Transposable elements (TEs) occupy a significant fraction of a wide variety of eukaryotic genomes and can be domesticated into functional sequences harbouring a coding or regulatory potential. While studies in mammals have revealed that retrotransposons can frequently give rise to tissue-specific transcriptional enhancers our understanding of this phenomenon in other vertebrate groups is scarcer. Here, we examined TE occupancy at tissue-specific nucleosome free regions (NFRs) which are not annotated as promoters in the amphibian model organism Xenopus tropicalis. We report three distinct miniature inverted-repeat TEs (MITEs) enriched at distal liver-specific NFRs and belonging to the hAT, Harbinger and Kolobok superfamilies of DNA transposons. These MITEs show a marked depletion at NFRs specific to the bone tissue, probably reflecting a process of negative selection. In addition, we show that they are enriched for transcription factor binding sites known to be bound by key regulators of liver biology, hematopoiesis, and the immune system, and that they are more likely to be located in the vicinity of genes specifically expressed in the liver than other MITE copies that are not associated to a NFR. We also find that these MITEs are not present at orthologous positions in the genome of the related allotetraploid frog Xenopus laevis, while they globally are abundant in this species. We discuss how independent bursts of MITE amplification followed by subsequent domestication episodes might independently have given rise to liver-specific transcriptional enhancers in the Xenopus tropicalis lineage.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00384-7.

Improving extracellular vesicle isolation from non-mammalian blood plasma: Enhancing efficiency through adjusted osmolarity.

Antibiotics are among the most frequently detected pharmaceuticals in aquatic environments, raising concern about their effects on nontarget organisms. Roxithromycin (ROX), a semisynthetic macrolide widely used in human medicine, occurs in wastewater and surface waters, yet its developmental and biochemical impacts on aquatic vertebrates remain poorly understood. Here, we comparatively assessed the sublethal toxicity of ROX in zebrafish (Danio rerio) and frog (Xenopus laevis) embryos using standardized assays. Embryos were exposed for 96 h to nominal concentrations of 88-1000 mg/L. No lethality occurred in either species. However, zebrafish exhibited high malformation rates (46%-92%), dominated by pericardial edema and spinal curvature, and a marked reduction in heart rate within 24 h. In contrast, X. laevis displayed lower and non-dose-dependent malformations (≤ 19%), mainly gut and abdominal edema. Biochemical markers revealed distinct species-specific responses: In zebrafish, only glutathione (GSH) increased at the highest concentration, whereas in X. laevis, glutathione S-transferase (GST) and GSH increased at all concentrations, glutathione reductase (GR) was inhibited, and catalase (CAT) decreased at the highest concentration. UV-Vis analyses confirmed that ROX remained mostly undissolved, with acid-conditional values representing analytical upper bound estimates rather than true dissolved concentrations. Accordingly, exposures are interpreted as suspensions with limited dissolved fractions. These findings show that zebrafish are more prone to cardiac and morphological disruption, while X. laevis exhibits stronger biochemical stress responses. Overall, the study highlights the ecological relevance of macrolide antibiotics and supports the use of cross-species testing in environmental risk assessment of pharmaceuticals in aquatic ecosystems.

This study presents the draft genome sequence of a steroid-transforming actinomycete isolated from the Xenopus laevis frog. Based on the 16S rRNA gene sequence and the DNA-DNA hybridization in silico, the isolated strain was identified as Streptomyces rochei MTOC-St3. The data obtained expand knowledge of the diversity of steroid-transforming actinomycetes inhabiting amphibians.

The African clawed frog Xenopus laevis is invasive on four continents, and is recognized as one of the invasive amphibians that generates the greatest impacts in the ecosystems it invades. Although its diet has been studied in its native habitat and invaded areas, its trophic role is still unclear, especially in the communities it invades. We studied the diet of X. laevis , and looked at its stable isotope signatures and its bioaccumulation of heavy metals, to gain a better understanding of its trophic role. The diet was found to consist mainly of aquatic invertebrates, with some consumption of the native fish Cheirodon pisciculus . The isotope analysis revealed that the assimilation of prey by X. laevis is unrelated to the most-consumed item. Xenopus laevis occupied a high trophic position in its own stream and was segregated from fish in by its use of trophic resources. Despite its high trophic position, only biomagnification of copper and zinc was found in relation to some prey, but not manganese or arsenic.

Developing eye optics, determined by the lens and cornea, must coordinate with the axial length of growing eyes to focus light onto the retina to form an image. It was found that zebrafish (Danio rerio) lens nuclei are initially anteriorly localized in optical axes in larvae, then centralize at older stages. An anteriorly placed lens nucleus would increase lens power, thereby likely enabling a functional optical system in larvae, where eye axial length is short. To assess if alike mechanisms occur in other aquatic animals, we studied the clawed frog, Xenopus laevis, a fully aquatic species similarly relying on vision for survival at stages where eyes are small. We found the Xenopus tadpole lens nucleus also shifted from an anterior to a central location in the optical axis during the prometamorphosis period. Similarly, in eyes regenerated after embryonic ablation, tadpole lens nuclei are anteriorly localized then centralize before metamorphosis, recapitulating the same pattern as control developing eyes. Moreover, lens nuclei localization in optical axes in developing and regenerated Xenopus eyes show close correlations to axial eye length. Close correlation of these two parameters suggests lens nuclei centralization is required for a functional optical system by coordinating the focal length. Our findings suggest a conserved evolutionary mechanism for eye optical development in at least two aquatic species. Understanding key mechanisms regulating crosstalk between eye optics and eye axial length will aid in discovering mechanisms of optical development and future therapies to prevent or delay formation of refractive error when these two properties mismatch.

Abstract Description Disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), are compounding global amphibian declines. Bd infections are confined to amphibian skin, and there are many published studies describing the roles of amphibian skin commensals and skin-produced antimicrobial components in anti-Bd defenses. In contrast, relatively little is known about the roles of skin-resident immune cells, such as mast cells, in amphibian antifungal immunity. Indeed, mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Thus, we investigated the roles of frog (Xenopus laevis) mast cells during Bd infections. Our findings indicate that frog mast cells confer antifungal protection by reducing neutrophil infiltration into Bd-infected skins, ablating inflammation-associated damage therein and promoting mucus production by cutaneous mucus glands. Akin to mammalian mast cells, which produce copious amounts of the pleiotropic interleukin-4 (IL4) cytokine, frog mast cells confer their anti-Bd protection at least in part through IL4 production. Together, our findings underscore the importance of skin-resident immune cells in amphibian anti-Bd defenses and suggest a new direction to explore host-chytrid pathogen interactions. Funding Sources Support: NSF 2147466, NSF 2131061 (LG) and NSF 2147467 (LR-S). Topic Categories Veterinary and Comparative Immunology (VET)