Premetamorphic Tadpoles Research Articles

Tadpoles rely on mechanosensory stimuli for communication when visual capabilities are poor

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species.

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

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

Changes in Tryptophan Hydroxylase 2 and 5HT1A Receptor Density May Contribute to Developmental Variation in Response to Serotonin in the in vitro Bullfrog Brainstem

Bullfrog, Lithobates catesbeiana, is a vertebrate animal model that can be used to study toxicology and developmental physiology. Our research is focused on better understanding vulnerability to brainstem respiratory centers in the pathology of sudden infant death syndrome (SIDS). We propose that a link between nicotine exposure during development leads to changes in the serotonergic signaling cascades of respiratory coordinating neurons. Caudal raphe neurons send serotonergic fibers that arborize and terminate directly on or near respiratory neurons in the brainstem. In bullfrogs, serotonin elicits a developmentally dependent effect on respiratory motor output. Late metamorphic tadpoles demonstrate an increase in lung burst activity with exposure to low concentrations of serotonin (≤ 0.5μM 5-hydroxytryptamine; 5-HT) mediated in part by 5-HT1a receptor (5HT1aR) activation. This developmental change in activity may be due to changes in the number of serotonergic fibers and/or 5-HT1a receptor density. Using the NIH Basic Local Alignment Search Tool (BLAST) we compared human and bullfrog 5HT1aR and tryptophan hydroxylase 2 (TH2) protein sequences and found a 34 and 65 percent identity match respectively. Quantitative western blot analysis of both proteins indicates developmental shifts in the serotonergic network during metamorphosis. These results were supported by immunohistochemical staining of 5HT1aR and TH2 in caudal raphe containing brainstem sections from pre-metamorphic tadpoles and post-metamorphic frogs. Impairments in the maturation of this serotonergic network may underlie the toxidrome associated with developmental nicotine exposure and further support the increased risk of SIDS following that exposure. This research was supported by the University of Wisconsin- La Crosse (UWL) through support from the UWL College of Science and Health, two UWL Undergraduate Research Creative Grants and a UWL FY23/24 Faculty Research Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

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

Functional organization of vestibulospinal inputs responsible for tail postural control in larval Xenopus.

In all vertebrates, maintaining trunk posture primarily depends on descending commands originating from brainstem vestibulospinal nuclei. Despite being broadly outlined across species, the detailed anatomical and operational structure of these vestibulospinal networks remains poorly understood. Xenopus frogs have previously served as an excellent model for exploring such anatomical and functional aspects in relation to the animal's behavioral requirements. In this study, we examined the reflex motor reactions induced by vestibular stimulation in pre-metamorphic tadpoles. Our findings indicate that natural vestibular stimulation in the horizontal plane yields greater efficacy compared to stimulation in other planes, a phenomenon replicated in a frequency-dependent manner through specific galvanic stimulation (GVS) of the horizontal semicircular canals. With the exception of a very rostral cluster of neurons that receive vestibular inputs and project to the spinal cord, the overall anatomical segregation of vestibulospinal nuclei in the brainstem mirrors that observed in juvenile frogs. However, our results suggest closer similarities to mammalian organization than previously acknowledged. Moreover, we demonstrated that vestibulospinal cells project not only to spinal motoneurons in rostral segments but also to more distal segments that undergo regression during metamorphosis. Lastly, we illustrated how vestibular-induced spinal reflexes change during larval development, transitioning from tail swim-based activity to rostral trunk bursting responses, likely anticipating postural control in post-metamorphic frogs.

Metamorphic gene regulation programs in Xenopus tropicalis tadpole brain.

Amphibian metamorphosis is controlled by thyroid hormone (TH), which binds TH receptors (TRs) to regulate gene expression programs that underlie morphogenesis. Gene expression screens using tissues from premetamorphic tadpoles treated with TH identified some TH target genes, but few studies have analyzed genome-wide changes in gene regulation during spontaneous metamorphosis. We analyzed RNA sequencing data at four developmental stages from the beginning to the end of spontaneous metamorphosis, conducted on the neuroendocrine centers of Xenopus tropicalis tadpole brain. We also conducted chromatin immunoprecipitation sequencing (ChIP-seq) for TRs, and we compared gene expression changes during metamorphosis with those induced by exogenous TH. The mRNA levels of 26% of protein coding genes changed during metamorphosis; about half were upregulated and half downregulated. Twenty four percent of genes whose mRNA levels changed during metamorphosis had TR ChIP-seq peaks. Genes involved with neural cell differentiation, cell physiology, synaptogenesis and cell-cell signaling were upregulated, while genes involved with cell cycle, protein synthesis, and neural stem/progenitor cell homeostasis were downregulated. There is a shift from building neural structures early in the metamorphic process, to the differentiation and maturation of neural cells and neural signaling pathways characteristic of the adult frog brain. Only half of the genes modulated by treatment of premetamorphic tadpoles with TH for 16 h changed expression during metamorphosis; these represented 33% of the genes whose mRNA levels changed during metamorphosis. Taken together, our results provide a foundation for understanding the molecular basis for metamorphosis of tadpole brain, and they highlight potential caveats for interpreting gene regulation changes in premetamorphic tadpoles induced by exogenous TH.

Persistent augmentation of fictive air breathing by hypoxia: An in vitro study of the role of GABAB signaling in pre-metamorphic tadpoles

Tadpole development is influenced by environmental cues and hypoxia can favor the emergence of the neural networks driving air breathing. Exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (∼0% O2; 15 min) leads to a progressive increase in fictive air breaths (∼3 fold) in the hours that follow stimulation. Here, we first determined whether this effect persists over longer periods (<18 h); we then evaluated maturity of the motor output by comparing the breathing pattern of hypoxia-exposed brainstems to that of preparations from adult bullfrogs under basal conditions. Because progressive withdrawal of GABAB-mediated inhibition contributes to the developmental increase in fictive lung ventilation, we then hypothesised that hypoxia reduces respiratory sensitivity to baclofen (selective GABAB-agonist). Experiments were performed on isolated brainstem preparations from pre-metamorphic tadpoles (TK stages IV to XIV); respiratory-related neural activity was recorded from cranial nerves V/VII and X before and 18 h after exposure to hypoxia (0% O2 + 2% CO2; 25 min). Time-control experiments (no hypoxia) were performed. Exposing pre-metamorphic tadpoles to hypoxia did not affect gill burst frequency, but augmented the frequency of fictive lung bursts and the incidence of episodic breathing levels intermediate between pre-metamorphic and adult preparations.Addition of baclofen to the aCSF (0,2 μM – 20 min) reduced lung burst frequency, but the response of hypoxia-exposed brainstems was greater than controls. We conclude that acute hypoxia facilitates development and maturation of the motor command driving air breathing. We propose that a greater number of active rhythmogenic neurons expressing GABAb receptors contributes to this effect.

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

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

Comparison of transcriptome responses of the liver, tail fin, and olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles disrupted by thyroid hormones and estrogen

Thyroid hormones (THs) are important developmental regulators in vertebrates, including during the metamorphosis of a tadpole into a frog. Metamorphosis is a post-embryonic developmental period initiated by TH production in the tadpole thyroid gland. The two main bioactive forms of TH are L-thyroxine (T4) and 3,5,3’-triiodothyronine (T3); these hormones have overlapping but distinct mechanisms of action. Premetamorphic tadpoles are highly responsive to TH and can be induced to metamorphose through exogenous TH exposure, making them an important model for both the study of vertebrate TH signaling and endocrine disrupting chemicals (EDCs). It is important to differentiate TH-mediated responses from estrogenic responses in premetamorphic tadpoles when assessing dysregulation by EDCs as crosstalk between the two endocrine systems is well-documented. Herein, we compare the RNA-sequencing-derived transcriptomic profiles of three TH-responsive tissues (liver, olfactory epithelium, and tail fin) in premetamorphic bullfrog (Rana [Lithobates] catesbeiana) tadpoles exposed to T3, T4, and estradiol (E2). These profiles were generated using the latest available genome assembly for the species. The data indicate that there is a clear distinction, and little overlap, between the transcriptomic responses elicited by E2 and the THs. In contrast, within the THs, the T3- and T4-induced transcriptomic profiles generally show considerable overlap; however, the degree of overlap is highly tissue-dependent, illustrating the importance of distinguishing the two THs and the affected signaling pathways within the target tissue type when evaluating hormone active agents. The data herein also show that E2 and TH treatment can uniquely induce significant changes in expression of their respective “classic” bioindicator transcripts vtg (E2) and thra, thrb, and thibz (THs). However, care must be taken in the interpretation of increased vep or esr1 transcripts as a change in transcript levels can be induced by THs rather than solely E2.

Upregulation of proto-oncogene ski by thyroid hormone in the intestine and tail during Xenopus metamorphosis

Thyroid hormone (T3) is important for adult organ function and vertebrate development, particularly during the postembryonic period when many organs develop/mature into their adult forms. Amphibian metamorphosis is totally dependent on T3 and can be easily manipulated, thus offering a unique opportunity for studying how T3 controls postembryonic development in vertebrates. Numerous early studies have demonstrated that T3 affects frog metamorphosis through T3 receptor (TR)-mediated regulation of T3 response genes, where TR forms a heterodimer with RXR (9-cis retinoic acid receptor) and binds to T3 response elements (TREs) in T3 response genes to regulate their expression. We have previously identified many candidate direct T3 response genes in Xenopus tropicalis tadpole intestine. Among them is the proto-oncogene Ski, which encodes a nuclear protein with complex function in regulating cell fate. We show here that Ski is upregulated in the intestine and tail of premetamorphic tadpoles upon T3 treatment and its expression peaks at stage 62, the climax of metamorphosis. We have further discovered a putative TRE in the first exon that can bind to TR/RXR in vitro and mediate T3 regulation of the promoter in vivo. These data demonstrate that Ski is activated by T3 through TR binding to a TRE in the first exon during Xenopus tropicalis metamorphosis, implicating a role of Ski in regulating cell fate during metamorphosis.

The Diencephalon Contributes to Hypoxia‐Induced Facilitation of Fictive Air Breathing in Pre‐Metamorphic Tadpoles

Introduction and hypothesis The onset of air breathing is an important milestone in the physiological development of the American bullfrog (Lithobates catesbeianus). The expression of the motor command driving lung ventilation is inhibited during the early tadpole stages and is progressively disinhibited as the animal enters metamorphosis. Environmental and endocrine signals regulate the onset of metamorphosis and our previous work showed that exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (98% N2 + 2% CO2; 15 min) facilitates the expression of air breathing, as indicated by an increase in fictive air breaths that persists for at least 2h after the end of hypoxic stimulation. Based on the growing evidence indicating that structures located rostral to the brainstem contribute to respiratory control, we tested the hypothesis that the presence of rostral structures augments hypoxia-induced facilitation of fictive air breathing in pre-metamorphic tadpoles. Methods Experiments were performed on pre-metamorphic tadpoles (TK stages V to XI). The central nervous system as isolated and placed in a recording chamber where it was superfused with an oxygenated artificial cerebrospinal fluid (98% O2, 2% CO2; pH = 7.9). The preparation was randomly assigned to one of three groups corresponding to a distinct level of brain transection: 1) Medullary preparation, 2) Diencephalon and 3) Telencephalon. The neural correlates of gill and lung ventilation was assessed by placing suction electrodes onto cranial nerves V and X. Following 1h of recovery, fictive breathing was recorded under baseline condition (15 min) before being exposed to hypoxia (98% N2 + 2% CO2; 15 min). The preparation was returned to baseline condition and time-ependent changes were recorded 30 min, 1h and 2h post-hypoxia. Fictive gill and lung breathing frequency and amplitude was measured in each time point. In a distinct series of experiments, preparations from each groupwere maintained under basal conditions to assess stability of the preparations over the course of the protocol. Result Hypoxia induced time-dependent changes in lung burst frequency that were greater than those observed in “time control” experiments. The largest increase was observed in preparations with diencephale whereas medullary preparations did not respond. Gill burst frequency remained constant; changes in burst amplitude (gill or lung) were not significant. Conclusion and perspectives The lack of response of medullary preparations is consistent with data showing that the locus coeruleus is an important central O2 sensing structure. However, results obtained from diencephalon preparations indicate the presence of structures that potentiate the long-term stimulatory effects of hypoxia on fictive air breathing. The fact that the time-dependent rise in lung burst frequency was not as important in more intact preparations suggest that more rostral structures attenuate those effects.

Uncovering early thyroid hormone signalling events through temperature-mediated activation of molecular memory in the cultured bullfrog tadpole tail fin

Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing a crucial role in postembryonic development. The best-studied mechanism of TH response is through modulating gene expression, however TH’s involvement in coordinating the early steps in the TH signal transduction pathway is still poorly understood. The American bullfrog, Rana [Lithobates] catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development in the form of metamorphosis of the tadpole into a frog can be experimentally induced by TH exposure. The rate of TH-induced metamorphosis can be modulated by temperature where sufficiently cold temperatures (5 °C) completely halt precocious metamorphosis. Interestingly, when premetamorphic tadpoles exposed to exogenous THs at 5 °C are shifted to permissive temperatures (24 °C), their metamorphic rate exceeds that of TH-exposed tadpoles at the permissive temperature. This suggests that a molecular memory of TH exposure is retained at 5 °C even after THs are cleared at this low temperature. However, the molecular memory machinery is poorly understood. Herein we use RNA-seq analysis to identify potential components of the molecular memory in cultured tail fin that allows for the recapitulation of the molecular memory phenomenon. Eighty-one gene transcripts were TH-responsive at 5 °C compared to matched controls indicating that the molecular memory is more complex than previously thought. Many of these transcripts encode transcription factors including thyroid hormone-induced B/Zip, thibz, and a novel krüppel-like factor family member, klfX. Actinomycin D and cycloheximide treatment had no effect on their TH induction suggesting that a change in transcription or translation is not required. Rather a change in RNA stability may be a possible mechanism contributing to the molecular memory. The ability to manipulate temperature and TH response in cultured organs provide an exciting opportunity to further elucidate the early TH signalling mechanisms during postembryonic development.

Retinoid-X receptor agonists increase thyroid hormone competence in lower jaw remodeling of pre-metamorphic Xenopus laevis tadpoles.

Thyroid hormone (TH) signaling plays critical roles during vertebrate development, including regulation of skeletal and cartilage growth. TH acts through its receptors (TRs), nuclear hormone receptors (NRs) that heterodimerize with Retinoid-X receptors (RXRs), to regulate gene expression. A defining difference between NR signaling during development compared to in adult tissues, is competence, the ability of the organism to respond to an endocrine signal. Amphibian metamorphosis, especially in Xenopus laevis, the African clawed frog, is a well-established in vivo model for studying the mechanisms of TH action during development. Previously, we've used one-week post-fertilization X. laevis tadpoles, which are only partially competent to TH, to show that in the tail, which is naturally refractive to exogenous T3 at this stage, RXR agonists increase TH competence, and that RXR antagonism inhibits the TH response. Here, we focused on the jaw that undergoes dramatic TH-mediated remodeling during metamorphosis in order to support new feeding and breathing styles. We used a battery of approaches in one-week-old tadpoles, including quantitative morphology, differential gene expression and whole mount cell proliferation assays, to show that both pharmacologic (bexarotene) and environmental (tributyltin) RXR agonists potentiated TH-induced responses but were inactive in the absence of TH; and the RXR antagonist UVI 3003 inhibited TH action. Bex and TBT significantly potentiated cellular proliferation and the TH induction of runx2, a transcription factor critical for developing cartilage and bone. Prominent targets of RXR-mediated TH potentiation were members of the matrix metalloprotease family, suggesting that RXR potentiation may emphasize pathways responsible for rapid changes during development.

Sperm associated antigen 7 is activated by T3 during Xenopus tropicalis metamorphosis via a thyroid hormone response element within the first intron.

Thyroid hormone (T3) affects many diverse physiological processes such as metabolism, organogenesis, and growth. The two highly related frog species, diploid Xenopus tropicalis and pseudo tetraploid Xenopus laevis, have been used as models for analyzing the effects of T3 during vertebrate development. T3 regulates T3-inducible gene transcription through T3 receptor (TR)-binding to T3-response elements (TREs). We have previously identified sperm associated antigen 7 (spag7) as a candidate T3 target gene that is potentially involved in adult stem cell development and/or proliferation during intestinal metamorphosis. To investigate whether T3 regulates spag7 directly at the transcriptional level via TR, we first conducted qRT-PCR to analyze its expression during natural and T3-induced metamorphosis and found that spag7 was up-regulated during natural metamorphosis in the intestine, tail, brain and hindlimb, peaking at the climax of metamorphosis in all those organs, and upon T3 treatment of premetamorphic tadpoles. Next, we demonstrated that an intronic TRE in spag7, first identified through bioinformatic analysis, could bind to TR in vitro and in vivo during metamorphosis. A dual luciferase assay utilizing a reconstituted frog oocyte transcription system showed that the TRE could mediate promoter activation by liganded TR. These results indicate that spag7 expression is directly regulated by T3 through the TRE in the first intron during metamorphosis, implicating a role for spag7 early during T3-regulated tissue remodeling and resorption.

Gut microbial ecology of Xenopus tadpoles across life stages

Background: The microorganism world living in amphibians is still largely under-represented and under-studied in the literature. Among anuran amphibians, African clawed frogs of the Xenopus genus stand as well-characterized models with an in-depth knowledge of their developmental biological processes including their metamorphosis. In this study, we analyzed the succession of microbial communities and their activities across diverse body habitats of Xenopus tropicalis using different approaches including flow cytometry and 16s rDNA gene metabarcoding. We also evaluated the metabolic capacity of the premetamorphic tadpoles gut microbiome using metagenomic and metatranscriptomic sequencing. Results: We analyzed the bacterial components of the Xenopus gut microbiota, the adult gut biogeography, the succession of communities during ontogeny, the impact of the alimentation in shaping the tadpoles gut bacterial communities and the transmission of skin and fecal bacteria to the eggs. We also identified the most active gut bacteria and their metabolic contribution to tadpole physiology including carbohydrate breakdown, nitrogen recycling, essential amino-acids and vitamin biosynthesis. Conclusions: We present a comprehensive new microbiome dataset of a laboratory amphibian model. Our data provide evidences that studies on the Xenopus tadpole model can shed light on the interactions between a vertebrate host and its microbiome. We interpret our findings in light of bile acids being key molecular components regulating the gut microbiome composition during amphibian development and metamorphosis. Further studies into the metabolic interactions between amphibian tadpoles and their microbiota during early development and metamorphosis should provide useful information on the evolution of host-microbiota interactions in vertebrates.

Orexin-A inhibits fictive air breathing responses to respiratory stimuli in the bullfrog tadpole (Lithobates catesbeianus).

In pre-metamorphic tadpoles, the neural network generating lung ventilation is present but actively inhibited; the mechanisms leading to the onset of air breathing are not well understood. Orexin (ORX) is a hypothalamic neuropeptide that regulates several homeostatic functions, including breathing. While ORX has limited effects on breathing at rest, it potentiates reflexive responses to respiratory stimuli mainly via ORX receptor 1 (OX1R). Here, we tested the hypothesis that OX1Rs facilitate the expression of the motor command associated with air breathing in pre-metamorphic bullfrog tadpoles (Lithobates catesbeianus). To do so, we used an isolated diencephalic brainstem preparation to determine the contributions of OX1Rs to respiratory motor output during baseline breathing, hypercapnia and hypoxia. A selective OX1R antagonist (SB-334867; 5-25 µmoll-1) or agonist (ORX-A; 200 nmol l-1 to 1 µmoll-1) was added to the superfusion media. Experiments were performed under basal conditions (media equilibrated with 98.2% O2 and 1.8% CO2), hypercapnia (5% CO2) or hypoxia (5-7% O2). Under resting conditions gill, but not lung, motor output was enhanced by the OX1R antagonist and ORX-A. Hypercapnia alone did not stimulate respiratory motor output, but its combination with SB-334867 increased lung burst frequency and amplitude, lung burst episodes, and the number of bursts per episode. Hypoxia alone increased lung burst frequency and its combination with SB-334867 enhanced this effect. Inactivation of OX1Rs during hypoxia also increased gill burst amplitude, but not frequency. In contrast with our initial hypothesis, we conclude that ORX neurons provide inhibitory modulation of the CO2 and O2 chemoreflexes in pre-metamorphic tadpoles.

A comprehensive look at Xenopus gut microbiota: effects of feed, developmental stages and parental transmission

Background The microorganism world living in amphibians is still largely under-represented and under-studied in the literature. Among anuran amphibians, African clawed frogs of the Xenopus genus stand as well-characterized models with an in-depth knowledge of their developmental biological processes including their metamorphosis. In this study, we analyzed the succession of microbial communities and their activities across diverse body habitats of Xenopus tropicalis using different approaches including flow cytometry and 16s rDNA gene metabarcoding. We also evaluated the metabolic capacity of the premetamorphic tadpole’s gut microbiome using metagenomic and metatranscriptomic sequencing.

Acute and chronic toxicity tests of systemic insecticides, four neonicotinoids and fipronil, using the tadpoles of the western clawed frog Silurana tropicalis

Extensive use of neonicotinoids and fipronil, which are popular systemic insecticides used in Japanese rice paddies, has raised concerns about their impacts on nontarget aquatic organisms such as amphibians. This study employed premetamorphic tadpoles of Silurana tropicalis and addressed the toxicity of four neonicotinoids (acetamiprid, clothianidin, dinotefuran, and imidacloprid) and fipronil. Acute toxicity tests were conducted under a 96-h semistatic exposure regime and median lethal concentration (LC50) values were calculated at 24-h intervals. All LC50 values of the four neonicotinoids exceeded 100 mg/L, suggesting their low acute toxicity to amphibians. Fipronil yielded much lower LC50 values (3.00–1.34 mg/L) and was highly toxic compared to the four neonicotinoids. Additionally, exposure to fipronil at >1 mg/L induced axial malformations, suggesting its teratogenicity. However, the LC50 values of fipronil were three orders of magnitude higher than the realistic concentrations in paddy water. Chronic toxicity tests were conducted with morphometric, gravimetric, and thyroid-histological endpoints. Premetamorphic tadpoles were exposed to each insecticide at two test concentrations: 0.1 and 1.0 mg/L for the four neonicotinoids; and 1/100 and 1/10 of the 96-h LC50 value for fipronil. Exposure to each insecticide continued until all tadpoles in the control reached late prometamorphic stages or the initial stage of metamorphic climax. At test termination, all insecticides showed no significant differences in any of the endpoints between the respective controls and chemical exposure groups. Overall, our results suggest that these insecticides alone do not directly affect amphibians through their larval stages at concentrations that likely occur in paddy water.

Dynamics of perineuronal nets over amphibian metamorphosis.

Extracellular matrix materials known as perineuronal nets (PNNs) have been shown to have remarkable consequences for the maturation of neural circuits and stabilization of behavior. It has been proposed that, due to the possibly long-lived biochemical nature of their components, PNNs may be an important substrate by which long-term memories are stored in the central nervous system. However, little empirical evidence exists that shows that PNNs are themselves stable once established. Thus, the question of their temporal dynamics remains unresolved. We leverage the dramatic morphological and behavioral transformations that occur during amphibian metamorphosis to show that PNNs can be highly dynamic in nature. We used established lectin histochemistry to show that PNNs undergo drastic reconstruction during the metamorphic transition. Pre-metamorphic tadpoles have abundant lectin-labeled pericellular material, which we interpret to be PNNs, surrounding neurons throughout the central nervous system. During the metamorphic transition, these structures degrade, and begin to reform in the months following metamorphosis. We show that PNN sizes and staining intensity further change over metamorphosis, suggesting compositional rearrangement. We found PNNs in brain regions with putative homology to regions in mammals with known PNN function, and in other shared regions where PNN function is unknown. Our results suggest that PNNs are susceptible to remodeling by endogenous mechanisms during development. Interpreting the roles of PNNs in circuit maturation and stability requires understanding their temporal relationship with the neurons and synapses they surround. Our work provides further impetus to investigate this relationship in tandem with developmental and behavioral studies.

Toxicity assessment at different experimental scenarios with glyphosate, chlorpyrifos and antibiotics in Rhinella arenarum (Anura: Bufonidae) tadpoles

The presence of pesticides as well as that of several antibiotics provided at a great scale to poultry, cattle, and swine in aquatic environments within agroecosystems is a matter of growing concern. The objective of the present study was to characterize the sublethal effects of four environmental toxic compounds at two experimental pollution scenarios on the morphology, development and thyroid (T4), acetylcholinesterase (AChE) and glutathione S-transferase (GST) levels in Rhinella arenarum tadpoles. The first experimental pollution scenario aimed to evaluate the individual and mixed toxicity (50:50% v/v) of a glyphosate-based herbicide (GBH) and the antibiotic ciprofloxacin (CIP) on earlier developmental stages. The second experimental pollution scenario aimed to evaluate the effects of other toxic compounds (the insecticide chlorpyrifos (CP) and the antibiotic amoxicillin (AMX)) added to the ones from the first scenario on previously exposed premetamorphic tadpoles. In all the treatments of the first pollution scenario, the most conspicuous effect observed in early-stage tadpoles was a high prevalence of morphological abnormalities. Exposure to GBH and to its mixture with CIP also led to a significant decrease in T4 levels and lower development. Both pollutant combinations from the second experimental scenario significantly increased T4 levels, inhibited AChE activities, and led to lower development, whereas the quaternary mixture led to a significant decrease in GST levels. The alterations here revealed by our approaches in several morphological and biochemical endpoints allow characterizing the ecotoxicological risk for anurans exposed to complex mixtures of pollutants that frequently occur in aquatic systems.