African Clawed Frog Xenopus Laevis Research Articles

The future of amphibian immunology: Opportunities and challenges

Historically, amphibians have been essential to our understanding of vertebrate biology and animal development. Because development from egg to tadpole to adult frog can be directly observed, amphibians contributed greatly to our understanding of not only vertebrate animal development but also the development of the immune system. The South African clawed frog (Xenopus laevis) has been key to many of these findings. For example, using Xenopus as a model, the comparative immunology community learned about the contribution of hematopoietic stem cells to development of the immune system and about the diversity of antibodies, B cells, T cells and antigen presenting cells. Amphibians offer many advantages as unique potential model systems to address questions about immune skin interactions, host responses to mycobacteria, the diverse functions of interferons, and immune and mucosal interactions. However, there are also many challenges to advance the research including the lack of specific reagents and well annotated genomes of diverse species. While much is known, many important questions remain. The aim of this short commentary is to look to the future of comparative immunology of amphibians as a group. By identifying some important questions or “information-deficit” areas of research, I hope to pique the interest of younger developing scientists and persuade funding agencies to continue to support comparative immunology studies including those of amphibians.

Comparative effects of polyvinyl chloride microplastics on the brittle star Ophiactis virens and the amphibian Xenopus laevis

In this study, we investigated the effects of PVC microplastics (PVC-MPs) using two different animal models: the brittle star Ophiactis virens, and the African clawed frog Xenopus laevis. This is the first study using an environmental relevant sample of PVC-MPs obtained through mechanical fragmentation of a common PVC plumbing pipe. Exposure experiments on brittle star were performed on the adult stage for a duration of 14 days, while those on African clawed frog were performed on the embryogenic developmental stage according to the standardized FETAX protocol (Frog Embryo Teratogenesis Assay-Xenopus). For both models, different endpoints were analysed: mortality, developmental parameters, behavioural assays and histological analyses on target organs by optical and electronic microscopy. Results showed that the concentration of 0.1 μg mL−1 PVC do not cause any adverse effects in both models (common NOEC concentration), while exposure to 1 μg mL−1 PVC adversely affected at least one species (common LOEC concentration). In particular arm regeneration efficiency was the most affected parameters in O. virens leading to a significantly lower differentiation pattern at 1 μg mL−1 PVC. On the contrary, in X. laevis larvae histopathological analyses and behavioural tests were the most susceptible endpoints, exhibiting several abnormal figures and different swimming speed at 10 μg mL−1 PVC. Histopathological analyses revealed a higher abundance of degenerating cells, pyknotic nuclei and cellular debris in the gut of exposed larvae in respect to control. The comparative analyses performed in this work allowed to characterize the specificity of action of the PVC-MPs on the two species, underlining the importance of exploring a large spectrum of endpoints to offer adequate protection in the emerging fields of microplastic research.

Feral frogs, native newts, and chemical cues: identifying threats from and management opportunities for invasive African Clawed Frogs in Washington state.

Invasive species threaten biodiversity globally. Amphibians are one of the most threatened vertebrate taxa and are particularly sensitive to invasive species, including other amphibians. African clawed frogs (Xenopus laevis) are native to Southern Africa but have subsequently become invasive on multiple continents-including multiple parts of North America-due to releases from the pet and biomedical trades. Despite their prevalence as a global invader, the impact of X. laevis remains understudied. This includes the Pacific Northwest of the USA, which now hosts multiple expanding X. laevis populations. For many amphibians, chemical cues communicate important information, including the presence of predators. Here, we tested the role chemical cues may play in mediating interactions between feral X. laevis and native amphibians in the Pacific Northwest. We tested whether native red-legged frog (Rana aurora) tadpoles display an antipredator response to non-native frog (X. laevis) or native newt (rough-skinned newts, Taricha granulosa) predator chemical stimuli. We found that R. aurora tadpoles exhibited pronounced anti-predator responses when exposed to chemical cues from T. granulosa but did not display anti-predator response to invasive X. laevis chemical cues. We also began experimentally testing whether T. granulosa-which produce a powerful neurotoxin tetrodotoxin (TTX)-may elicit an anti-predator response in X. laevis, that could serve to deter co-occupation. However, our short-duration experiments found that X. laevis were attracted to newt chemical stimuli rather than deterred. Our findings show that X. laevis likely poses a threat to native amphibians, and that these native species may also be particularly vulnerable to this invasive predator, compared to native predators, because toxic native newts may not limit X. laevis invasions. Our research provides some of the first indications that native Pacific Northwest species may be threatened by feral X. laevis and provides a foundation for future experiments testing potential management techniques for X. laevis.

Large-landscape connectivity models for pond-dwelling species: methods and application to two invasive amphibians of global concern

ContextFunctional connectivity models are essential in identifying major dispersal pathways and developing effective management strategies for expanding populations of invasive alien species. However, the extrapolation of models parameterized within current invasive ranges may not be applicable even to neighbouring areas, if the models are not based on the expected responses of individuals to landscape structure.ObjectivesWe have developed a high-resolution connectivity model for both terrestrial and aquatic habitats using solely potential sources. The model is used here for the invasive, principally-aquatic, African clawed frog Xenopus laevis, which is a species of global concern.MethodsAll ponds were considered as suitable habitats for the African clawed frog. Resistance costs of lotic aquatic and terrestrial landscape features were determined through a combination of remote sensing and laboratory trials. Maximum cumulative resistance values were obtained via capture-mark-recapture surveys, and validation was performed using independently collected presence data. We applied this approach to an invasive population of the American bullfrog, Lithobates catesbeianus, in France to assess its transferability to other pond-dwelling species.ResultsThe model revealed areas of high and low functional connectivity. It primarily identified river networks as major dispersal pathways and pinpointed areas where local connectivity could be disrupted for management purposes.ConclusionOur model predicts how the dispersal of individuals connect suitable lentic habitats, through river networks and different land use types. The approach can be applied to species of conservation concern or interest in pond ecosystems and other wetlands, including aquatic insects, birds and mammals, for which distribution data are limited or challenging to collect. It serves as a valuable tool for forecasting colonization pathways in expanding populations of both native and invasive alien species and for identifying regions suitable for preventive or adaptive control measures.

Prdm15 acts upstream of Wnt4 signaling in anterior neural development of Xenopus laevis.

Mutations in PRDM15 lead to a syndromic form of holoprosencephaly (HPE) known as the Galloway-Mowat syndrome (GAMOS). While a connection between PRDM15, a zinc finger transcription factor, and WNT/PCP signaling has been established, there is a critical need to delve deeper into their contributions to early development and GAMOS pathogenesis. We used the South African clawed frog Xenopus laevis as the vertebrate model organism and observed that prdm15 was enriched in the tissues and organs affected in GAMOS. Furthermore, we generated a morpholino oligonucleotide-mediated prdm15 knockdown model showing that the depletion of Prdm15 leads to abnormal eye, head, and brain development, effectively recapitulating the anterior neural features in GAMOS. An analysis of the underlying molecular basis revealed a reduced expression of key genes associated with eye, head, and brain development. Notably, this reduction could be rescued by the introduction of wnt4 RNA, particularly during the induction of the respective tissues. Mechanistically, our data demonstrate that Prdm15 acts upstream of both canonical and non-canonical Wnt4 signaling during anterior neural development. Our findings describe severe ocular and anterior neural abnormalities upon Prdm15 depletion and elucidate the role of Prdm15 in canonical and non-canonical Wnt4 signaling.

Intraspecific morphological and genetic variation in South African populations of a polystomatid flatworm parasite

The African Clawed Frog Xenopus laevis, a global invader, exhibits a marked phylogeographic divergence among native populations in southern Africa, which seems to enhance its invasive potential. The polystomatid flatworm, Protopolystoma xenopodis, is the frog’s most frequently co-introduced metazoan parasite. In an integrative approach, we utilised morphometrics and molecular markers to assess variation in P. xenopodis in its native range. We measured twelve key morphological characters from 23 flatworms and compared these statistically between flatworms collected from the northern- and southernmost distribution in South Africa. Phylogenetic analyses were based on three concatenated markers, namely 28S and 12S rDNA and COX1, from six flatworms. The combination of five morphological characters, which involve egg size, gut morphology and size of the attachment hooks, differentiated northern and southern populations of P. xenopodis. The multilocus phylogenetic analyses showed a cluster of northern P. xenopodis and two southern lineages with more basal positioning. These findings demonstrate a relatively high level of intraspecific variation in P. xenopodis in its native range. The presented intraspecific variation of P. xenopodis could be potentially informative to trace geographic origin in its non-native range.

Comparing the effects of three neonicotinoids on embryogenesis of the South African clawed frog Xenopus laevis

Neonicotinoids (NEOs) are widely used insecticides that are ubiquitous in agricultural use. Since NEOs are found in natural waters as well as in tap water and human urine in regions where NEOs are widely used, NEOs pose a potential hazard to non-target organisms such as animals and humans. Some of the commonly detected NEOs are imidacloprid (IMD), thiamethoxam (TMX), and its metabolite clothianidin (CLO). Although previously published scientific information, including an assessment of the environmental risks, particularly for bees, had resulted in a ban on the outdoor use of these three NEOs in the EU – their use is now only permitted in closed greenhouses – these NEOs continue to be used in agriculture in many other parts of the world. Therefore, a detailed study and comparison of the effects of NEOs on the embryonic development of non-target organisms is needed to further define the risk profiles.Embryos of the South African clawed frog Xenopus laevis, a well-established aquatic model, were exposed to different concentrations of IMD, TMX, or CLO (0.1–100 mg/L) to study and compare the possible effects of a single contaminant in natural water bodies on early embryogenesis. The results included a reduced body length, a smaller orbital space, impaired cranial cartilage and nerves, and an altered heart structure and function. At the molecular level, NEO exposure partially resulted in an altered expression of tissue-specific factors, which are involved in eye, cranial placode, and heart development.Our results suggest that the NEOs studied negatively affect the embryonic development of the non-target organism X. laevis. Since pesticides, especially NEOs, pollute the environment worldwide, it is suggested that they are strictly controlled and monitored in the areas where they are used. In addition, the question arises as to whether pesticide metabolites also pose a risk to the environment and need to be investigated further so that they can be taken into account when registering ingredients.

Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts for Comparative Cell Biology and Evolutionary Studies.

Cytoplasmic extracts prepared from eggs of the African clawed frog Xenopus laevis are extensively used to study various cellular events including the cell cycle, cytoskeleton dynamics, and cytoplasm organization, as well as the biology of membranous organelles and phase-separated non-membrane-bound structures. Recent development of extracts from eggs of other Xenopus allows interspecies comparisons that provide new insights into morphological and biological size variations and underlying mechanisms across evolution. Here, we describe methods to prepare cytoplasmic extracts from eggs of the allotetraploid Marsabit clawed frog, Xenopus borealis, and the diploid Western clawed frog, Xenopus tropicalis. We detail mixing and "hybrid" experiments that take advantage of the physiological but highly accessible nature of extracts to reveal the evolutionary relationships across species. These new developments create a robust and versatile toolbox to elucidate molecular, cell biological, and evolutionary questions in essential cellular processes.

EPIGENETIC AGE DYNAMICS AND EMBRYONIC MORTALITY POINT TO GASTRULATION AS AGING GROUND ZERO IN XENOPUS LAEVIS

Abstract Recent studies on embryonic development revealed a natural rejuvenation event followed by epigenetic aging. Here, we developed a frog aging clock based on the DNA methylation profile in the African clawed frog Xenopus laevis. Application of this clock to Xenopus laevis early embryonic development identified a decrease in epigenetic age during early embryogenesis, with the minimal epigenetic age reached around gastrulation. We further examined the individual developmental trajectories of 6,457 embryos and found that this minimum age is also accompanied by a higher incidence of disrupted development. Taken together, our data point to gastrulation as a critical stage for aging and natural rejuvenation, characterized by the lowest epigenetic age and increased mortality, defining aging ground zero as the basepoint where rejuvenation ends and the aging process begins.

Morphological, acoustic and genetic identification of a reproducing population of the invasive African clawed frog Xenopuslaevis (Anura, Pipidae) recently discovered in Belgium.

Using external morphology of adults and tadpoles, osteology from high-resolution microcomputed tomography, vocalization analysis, and DNA sequence data, the identity of a reproducing Belgian population of invasive Xenopus at the current northernmost edge of the distribution of the genus in Europe was assessed. All data concur to an identification as Xenopus (Xenopus) laevis (Daudin, 1802). Genetically it is most closely related to populations of the Cape region in South Africa. No studies on the natural history of the Belgian Xenopus population and its impact on the local environment have been made to date.

The Distribution of the Germ Plasm Gene <i>germes</i> among Anurans

The germes gene is a marker of germ plasm and primordial germ cells (PGC) described in the African clawed frog Xenopus laevis. It is known that overexpression of its mutant form negatively affects the formation and migration of PGC. However, until now it was not known how widely this gene is represented in animals of different phylogenetic groups. In this work, we performed bioinformatic analysis of genomic and transcriptome sequences of animals with germ plasm. It turned out that germes homologs are present only in representatives of the genera Xenopus and Hymenochirus of the family Pipidae (order Anura). The obtained results were confirmed by RT-PCR analysis of the expression of germes orthologs in the ovaries of six representatives of different Anura families. Phylogenetic analysis of cloned sequences of germes homologs suggests the appearance of this gene in the ancestors of Pipidae and its secondary loss in the genus Pseudohymenochirus. It is also identified that the amino acid sequences of the functional domains of the Germes protein are rather conservative.

Functional dissection and assembly of a small, newly evolved, W chromosome-specific genomic region of the African clawed frog Xenopus laevis.

Genetic triggers for sex determination are frequently co-inherited with other linked genes that may also influence one or more sex-specific phenotypes. To better understand how sex-limited regions evolve and function, we studied a small W chromosome-specific region of the frog Xenopus laevis that contains only three genes (dm-w, scan-w, ccdc69-w) and that drives female differentiation. Using gene editing, we found that the sex-determining function of this region requires dm-w but that scan-w and ccdc69-w are not essential for viability, female development, or fertility. Analysis of mesonephros+gonad transcriptomes during sexual differentiation illustrates masculinization of the dm-w knockout transcriptome, and identifies mostly non-overlapping sets of differentially expressed genes in separate knockout lines for each of these three W-specific gene compared to wildtype sisters. Capture sequencing of almost all Xenopus species and PCR surveys indicate that the female-determining function of dm-w is present in only a subset of species that carry this gene. These findings map out a dynamic evolutionary history of a newly evolved W chromosome-specific genomic region, whose components have distinctive functions that frequently degraded during Xenopus diversification, and evidence the evolutionary consequences of recombination suppression.

The Functioning of the Frog’s Single Ventricle

African clawed frog (Xenopus laevis) has double-inlet and single-outlet single ventricle supporting both systemic and pulmonary circulations, with normal lifespan of 25-30 years. The relationship between pulmonary and systemic blood flows was investigated using echocardiography. Twelve female African clawed frogs, age 18-24 months, mean weight 173±19 grams were studied. Heart rate, percutaneous oxygen saturation, echocardiography derived 2D and blood flow in all cardiovascular structures were measured in two temperature setting, 17◦C (normal environment for frogs) and 22 C. In eight frogs cerebral Near-Infra-Red Spectroscopy (NIRS) was measured. Mean oxygen saturation was 66±9% with temperature of 22◦C and 85±6% with 17◦C (P<0.001), while there was no statistical difference between mean heart rates, respectively 48 13 and 42±7 beats/min. Doppler echocardiography showed a mean total pulmonary blood flow (QP) =173.0 ml/min and mean total systemic blood flow (QS) = 99.3ml/min, with mean QP/QS of 1.74:1.00. The only statistical correlation between size of the cardiovascular structures and body weight was found for the pulmonary veins, with correlation coefficient = 0.71 (P=0.02). NIRS measurements showed a reverse correlation (coefficient = -0.21) with oxygen saturation. The frog’s heart has peculiar morphologic and functional characteristics allowing normal quality of life for many years despite the presence of single ventricle. Baseline QP/QS under general anesthesia was significant for increased total pulmonary blood flow. Exposure to environmental temperature 5◦C higher than normal caused peripheral vasodilatation, with subsequent decrease of QP/QS and reduced oxygenation.

The impact of the insecticide acetamiprid on the embryogenesis of the aquatic model organism Xenopus laevis

Acetamiprid (ACT) is used extensively in agriculture worldwide, although data on ACT concentrations in natural water bodies and its impact on aquatic organisms are limited. To study whether ACT influences the embryogenesis of the South African clawed frog Xenopus laevis, embryos were incubated in ACT solutions from 0.01 to 100 mg/L. The low concentrations were chosen on the basis of concentrations already found in nature. ACT treatment leads to shorter embryo lengths, intestine malformation and reduced eye areas. It also affects the cranial cartilage and cardiac development as well as the embryo’s mobility. The expression of tissue-specific marker genes is affected as well. Thus, our study suggests that pesticides may lead to an increased mortality of non-target organisms and emphasizes the importance of regular testing for ACT concentrations in nature. Our study provides an overview of ACT effects and can therefore be used as a basis for an ACT risk assessment.

Effects of mifepristone, a model compound with anti-progestogenic activity, on the development of African clawed frog (Xenopus laevis)

The objective of this study was to assess the effects of a model substance with anti-progestogenic activity on development of African clawed frog (Xenopus laevis) from tadpole to juvenile stage. Mifepristone, a synthetic progesterone receptor-blocking steroid hormone used in medicine as an abortifacient, was chosen as a model compound with anti-progestogenic activity. In the experiment, African clawed frog tadpoles were exposed to mifepristone at three concentrations (2, 21, and 215 ng L−1). A control group was exposed to dimethyl sulfoxide (DMSO; 0.001 %). The experiment started when tadpoles reached stages 47–48 according to Nieuwkoop and Faber (NF; 1994) and continued until stage NF 66, when metamorphosis was complete. Exposure to mifepristone had no significant effect on the rate of tadpole development, occurrence of morphological anomalies, weight, body length, or sex ratio. Mortality was within an acceptable range of 0–3.6 % throughout the test and did not differ among the groups. Histopathological examination of the gonads and thyroid gland revealed no significant changes. Therefore, we can conclude that mifepristone had no negative effect on development of the African clawed frog up to juvenile stage. Nevertheless, at the highest tested mifepristone concentration (215 ng L−1), gene expression analysis revealed up-regulation of mRNA expression of nuclear progesterone receptor (npr), membrane progesterone receptor (mpr), estrogen receptor beta (esrβ), and luteinizing hormone (lh) in the brain–pituitary complex of exposed frogs at stage NF 66. Higher mRNA expression of npr was also found in frogs exposed to 22 ng L−1 mifepristone compared to the solvent control. These findings confirmed the anti-progestogenic activity of mifepristone in frogs because the up-regulation of progesterone receptors occurs if progesterone availability in the body is reduced. All the observed changes in combination may have negative consequences for reproduction and reproductive behavior later in life.

Investigation of Potassium Chloride for Euthanasia of Anesthetized African Clawed Frogs (Xenopus laevis).

Euthanasia is frequently performed in amphibians, but techniques are currently limited in number and variable in effectiveness. The current study examined the use of potassium chloride (KCl) for euthanasia of anesthetized African clawed frogs (Xenopus laevis). Twenty adult, female African clawed frogs were anesthetized by immersion in buffered tricaine methanesulfonate (MS-222) for 5 min beyond loss of righting reflex. Frogs were then randomly assigned to receive one of 4 treatments: KCl via intracardiac injection (10 mEq/kg; IC, n = 5), intracoelomic injection (100 mEq/kg; ICe, n = 5), immersion (4,500 mEq/L; IMS, n = 5), or no treatment (C, n = 5). After treatment, serial heart rate was measured via Doppler device until either the loss of Doppler sounds, a 60-min endpoint (IC, ICe, IMS), or recovery (C). Times to loss of righting reflex, loss of Doppler sounds, and/or recovery were recorded. Plasma potassium concentrations were measured immediately after Doppler sound cessation in frogs in IC (n = 1), ICe (n = 2), and IMS (n = 5). Injection failure occurred in 1 IC frog, and 1 Ice frog regained spontaneous movement 4 min after treatment administration. Data from these 2 frogs were not included in statistical analysis. Doppler sound cessation occurred in 4 of 4, 4 of 4, 0 of 5, and 0 of 5 frogs in IC, ICe, IMS, and C, respectively. Median (range) times to Doppler sound cessation in IC and ICe were 6 (0 to 16) s and 18 (10 to 25) min, respectively. Plasma potassium concentration was greater than 9.0 mmol/L in sampled frogs. Intracardiac KCl at 10 mEq/kg and intracoelomic KCl at 100 mEq/kg were effective for euthanasia of anesthetized African clawed frogs. Return to MS-222 solution after KCl administration may be warranted to prevent unintended, premature anesthetic recovery prior to death.

Development and initial testing of EcoToxChip, a novel toxicogenomics tool for environmental management and chemical risk assessment.

New approach methods (NAMs) are increasingly important to help accelerate the pace of ecological risk assessment and offer more ethical, affordable, and efficient alternatives to traditional toxicity tests. In the present study, we describe the development, technical characterization and initial testing of a toxicogenomics tool, EcoToxChip (384-well qPCR array), to support chemical management and environmental monitoring for three laboratory model species - fathead minnow (Pimephales promelas), African clawed frog (Xenopus laevis), and Japanese quail (Coturnix japonica). Chip design, including gene selection, was informed by a diverse end-user group and quality control metrics (e.g. primer assay, reverse transcription and PCR efficiency) performed well based on a priori established criteria. Correlation with RNAseq data provided additional confidence in this novel toxicogenomics tool. While the present study represents an initial testing of only 24 EcoToxChips for each of the model species, the results provide increased confidence in the robustness/reproducibility of EcoToxChips for evaluating perturbations in gene expression associated with chemical exposure and thus, this NAM, combined with early-life stage toxicity testing, could augment current efforts for chemical prioritization and environmental management.

DNA methylation clocks for clawed frogs reveal evolutionary conservation of epigenetic aging.

To address how conserved DNA methylation-based epigenetic aging is in diverse branches of the tree of life, we generated DNA methylation data from African clawed frogs (Xenopus laevis) and Western clawed frogs (Xenopus tropicalis) and built multiple epigenetic clocks. Dual species clocks were developed that apply to both humans and frogs (human-clawed frog clocks), supporting that epigenetic aging processes are evolutionary conserved outside mammals. Highly conserved positively age-related CpGs are located in neural-developmental genes such as uncx, tfap2d as well as nr4a2 implicated in age-associated disease. We conclude that signatures of epigenetic aging are evolutionary conserved between frogs and mammals and that the associated genes relate to neural processes, altogether opening opportunities to employ Xenopus as a model organism to study aging.

Glyphosate without Co-formulants affects embryonic development of the south african clawed frog Xenopus laevis

BackgroundGlyphosate (GLY) is the most widely used herbicide in the world. Due to its mode of action as an inhibitor of the 5-enolpyruvylshikimate-3-phosphate synthase, an important step in the shikimate pathway, specifically in plants, GLY is considered to be of low toxicity to non-target organisms. However, various studies have shown the negative effects of GLY on the mortality and development of different non-target organisms, including insects, rodents, fish and amphibians. To better understand the various effects of GLY in more detail, we studied the effects of GLY without co-formulants during the embryogenesis of the aquatic model organism Xenopus laevis. ResultsA treatment with GLY affected various morphological endpoints in X. laevis tadpoles (body length, head width and area, eye area). Additionally, GLY interfered with the mobility as well as the neural and cardiac development of the embryos at stage 44/45. We were able to detect detailed structural changes in the cranial nerves and the heart and gained insights into the negative effects of GLY on cardiomyocyte differentiation. ConclusionThe application of GLY without co-formulants resulted in negative effects on several endpoints in the early embryonic development of X. laevis at concentrations that are environmentally relevant and concentrations that reflect the worst-case scenarios. This indicates that GLY could have a strong negative impact on the survival and lives of amphibians in natural waters. As a result, future GLY approvals should consider its impact on the environment.

Forgotten Clones: The Birth of Cloning and the Biological Revolution. By NathanCrowe. Pittsburgh, PA: University of Pittsburgh Press. 2021. pp. 336. $60 (Hardcover).

Forgotten Clones: The Birth of Cloning and the Biological Revolution. By NathanCrowe. Pittsburgh, PA: University of Pittsburgh Press. 2021. pp. 336. $60 (Hardcover).