Something in the water: aquatic microbial communities influence the larval amphibian gut microbiota, neurodevelopment and behaviour.

Microorganisms colonize the gastrointestinal tract of animals and establish symbiotic host-associated microbial communities that influence vertebrate physiology. More specifically, these gut microbial communities influence neurodevelopment through the microbiota-gut-brain (MGB) axis. We tested the hypothesis that larval amphibian neurodevelopment is affected by the aquatic microbial community present in their housing water. Newly hatched Northern Leopard Frog (Lithobates pipiens) tadpoles were raised in pond water that was unmanipulated (natural) or autoclaved. Tadpoles raised in autoclaved pond water had a gut microbiota with reduced bacterial diversity and altered community composition, had decreased behavioural responses to sensory stimuli, were larger in overall body mass, had relatively heavier brains and had altered brain shape when compared with tadpoles raised in natural pond water. Further, the diversity and composition of the gut microbiota were associated with tadpole behavioural responses and brain measurements. Our results suggest that aquatic microbial communities shape tadpole behaviour and brain development, providing strong support for the occurrence of the MGB axis in amphibians. Lastly, the dramatic role played by aquatic microbial communities on vertebrate neurodevelopment and behaviour should be considered in future wildlife conservation efforts.

Defining components of early thyroid hormone signalling through temperature-mediated activation of molecular memory in cultured Rana [lithobates] catesbeiana tadpole back skin.

Thyroid hormones (THs) are essential signalling molecules for the postembryonic development of all vertebrates. THs are necessary for the metamorphosis from tadpole to froglet and exogenous TH administration precociously induces metamorphosis. In American bullfrog (Rana [Lithobates] catesbeiana) tadpoles, the TH-induced metamorphosis observed at a warm temperature (24 °C) is arrested at a cold temperature (4 °C) even in the presence of exogenous THs. However, when TH-exposed tadpoles are shifted from cold to warm temperatures (4 â†’ 24 °C), they undergo TH-dependent metamorphosis at an accelerated rate even when the initial TH signal is no longer present. Thus, they possess a "molecular memory" of TH exposure that establishes the TH-induced response program at the cold temperature and prompts accelerated metamorphosis after a shift to a warmer temperature. The components of the molecular memory that allow the uncoupling of initiation from the execution of the metamorphic program are not understood. To investigate this, we used cultured tadpole back skin (C-Skin) in a repeated measures experiment under 24 °C only, 4 °C only, and 4 â†’ 24 °C temperature shifted regimes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA-sequencing (RNA-seq) analyses. RNA-seq identified 570, 44, and 890 transcripts, respectively, that were significantly changed by TH treatment. These included transcripts encoding transcription factors and proteins involved in mRNA structure and stability. Notably, transcripts associated with molecular memory do not overlap with those identified previously in cultured tail fin (C-fin) except for TH-induced basic leucine zipper-containing protein (thibz) suggesting that thibz may have a central role in molecular memory that works with tissue-specific factors to establish TH-induced gene expression programs.

How the Xenopus eleutheroembryonic thyroid assay compares to the amphibian metamorphosis assay for detecting thyroid active chemicals.

The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5'-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA.

Hobnail subtype of papillary carcinoma of the thyroid-a rare case with uncommon features.

The cytological features of the hobnail variant of papillary thyroid carcinoma may be subtle. It is important to recognize this variant because it may influence the corresponding surgical treatment and follow-up due to its aggressive nature. The hobnail subtype of papillary thyroid carcinoma is a rare entity with aggressive features. It presents extrathyroidal extension or lymph nodal metastasis in a high percentage of the cases.

Cadmium induces physiological and behavioral changes associated with 180 kDa NCAM lower expression and higher polysialic acid, in the African clawed Xenopus laevis tadpoles.

Heavy metals are released into the environment in increasing amounts from different natural and anthropogenic sources. Among them, cadmium contaminates aquatic habitats and represents a threat to Amphibians. To assess the risks of exposure to cadmium in the aquatic environment, we studied the survival rate of early tadpoles of Xenopus laevis under exposure to CdCl2 for 6 days in the concentration range between 0.15 and 150 µM of Cd2+. Tadpoles survived and reached stage 45 before feeding at all concentrations tested except 150 µM Cd2+, which significantly induced death. With an exposure of 15 µM Cd2+, tadpoles' mean body length decreased, heart rate increased, fastest swimming speed decreased, and distance traveled was greater compared to unexposed controls. Additionally, a witness of neuronal normal development, the neural cell adhesion molecules (NCAM) expression, was decreased. Moreover, this cell-surface glycoprotein exhibited higher polysialylation, a post-translational modification capable to reduce cell adhesion properties and to affect organ development. Our study highlights the effects of Cd2+ on a series of parameters including morphology, physiology, and behavior. They emphasize the deregulation of molecular NCAM suggesting this effector is an interesting biomarker to detect cadmic toxicity in early tadpoles.

Environmentally Relevant Levels of Antiepileptic Carbamazepine Altered Intestinal Microbial Composition and Metabolites in Amphibian Larvae.

There is growing concern about the potential ecological risks posed by pharmaceutical residues in the aquatic environment. However, our understanding of the toxic effects of antiepileptic pharmaceuticals, such as carbamazepine (CBZ), on aquatic animal larvae is still limited. In this study, the tadpoles of the black-spotted pond frog (Pelophylax nigromaculatus) were exposed to environmentally relevant concentrations of CBZ (0.3 and 3.0 µg/L) for 30 days, and their growth, intestinal microbial composition, and metabolites were investigated to assess the potential toxic effects of CBZ in non-targeted aquatic organisms. Some tadpoles died during exposure, but there was no significant among-group difference in the survival and growth rates. CBZ exposure significantly altered the composition of tadpole intestinal microbiota. Relative abundances of some bacterial genera (e.g., Blautia, Prevotella, Bacillus, Microbacterium, etc.) decreased, while others (e.g., Paucibacter, etc.) increased in CBZ-exposed tadpoles. Interestingly, CBZ-induced alterations in some bacteria might not necessarily lead to adverse outcomes for animals. Meanwhile, small molecular intestinal metabolites related to energy metabolism, and antioxidant and anti-inflammatory activities were also altered after exposure. Taken together, environmentally relevant levels of CBZ might alter the metabolic and immune performances of amphibian larvae by modifying the abundance of some specific bacteria and the level of metabolites in their intestines, thereby potentially causing a long-term effect on their fitness.

Quantitative macroinvertebrate bioassessment in seasonally astatic aquatic habitats, Part II. Applying the method.

Seasonally astatic aquatic habitats are important ecologically, municipally, and agriculturally. Regulatory agencies and conservation organizations have developed various plans for protecting or constructing temporary wetlands, resulting in habitat monitoring requirements, particularly as relates to restoration and constructed habitats. Unfortunately, there has been no effort to develop a unified, consistent method for wetland biological monitoring. In Part I, we presented a quantifiable, replicable method for assessing seasonally astatic wetlands, which would allow for direct comparison between individual wetlands, wetland sites, and wetland types. Here in Part II, we apply the method and present the results from more than a decade of a data on two disparate sites that support California vernal pool habitats. These habitats include natural, restored, and constructed vernal pools. Our results demonstrate that the method we present yields reliable, statistically useful, and actionable data and provides a better method for assessing astatic wetland ecological health and the persistence of federally listed vernal pool crustaceans than other methods so far employed.

Ranid Herpesvirus 3 Infection in Common Frog Rana temporaria Tadpoles.

Ranid herpesvirus 3 (RaHV3) is a recently discovered virus associated with skin disease in frogs. We detected RaHV3 DNA in free-ranging common frog (Rana temporaria) tadpoles, consistent with premetamorphic infection. Our finding reveals a critical aspect of RaHV3 pathogenesis, relevant for amphibian ecology and conservation and, potentially, for human health.

Evolutionary Tuning of Transient Receptor Potential Ankyrin 1 Underlies the Variation in Heat Avoidance Behaviors among Frog Species Inhabiting Diverse Thermal Niches.

Environmental temperature is a critical factor for all forms of life, and thermal tolerance defines the habitats utilized by a species. Moreover, the evolutionary tuning of thermal perception can also play a key role in habitat selection. Yet, the relative importance of thermal tolerance and perception in environmental adaptation remains poorly understood. Thermal conditions experienced by anuran tadpoles differ among species due to the variation in breeding seasons and water environments selected by parental frogs. In the present study, heat tolerance and avoidance temperatures were compared in tadpoles from five anuran species that spatially and temporally inhabit different thermal niches. These two parameters were positively correlated with each other and were consistent with the thermal conditions of habitats. The species difference in avoidance temperature was 2.6 times larger than that in heat tolerance, suggesting the importance of heat avoidance responses in habitat selection. In addition, the avoidance temperature increased after warm acclimation, especially in the species frequently exposed to heat in their habitats. Characterization of the heat-sensing transient receptor potential ankyrin 1 (TRPA1) ion channel revealed an amphibian-specific alternatively spliced variant containing a single valine insertion relative to the canonical alternative spliced variant of TRPA1, and this novel variant altered the response to thermal stimuli. The two alternatively spliced variants of TRPA1 exhibited different thermal responses in a species-specific manner, which are likely to be associated with a difference in avoidance temperatures among species. Together, our findings suggest that the functional change in TRPA1 plays a crucial role in thermal adaptation processes.

Organization of serotonergic system in Sphaerotheca breviceps (Dicroglossidae) tadpole brain.

The monoaminergic neurotransmitter 5-hydroxytryptamine (5-HT) is known to be involved in several physiological, behavioural and neuroendocrine functions in vertebrates. In this study, we investigated the distribution of 5-HT neuronal system in the central nervous system (CNS) of Sphaerotheca breviceps tadpoles at metamorphic climax stage. In the telencephalon, there was no 5-HT-immunoreactive (5-HT-ir) perikarya, but conspicuous fibres were observed in the olfactory bulb, pallium, subpallium and amygdala complexes. The preoptic area showed dense 5-HT-ir somata and cerebrospinal fluid contacting fibres, whereas a few varicose 5-HT-ir fibres were noticed in the suprachiasmatic nucleus. 5-HT-ir cells and fibres were found in the ventral, lateral dorsal subdivisions of the hypothalamus and in the nucleus tuberculi posterioris, but only 5-HT-ir fibres were localised in the periventricular area and pituitary gland. Numerous 5-HT-ir cells and/or fibres were detected in the thalamus, entopeduncular area and mesencephalic subdivisions. In the rhombencephalon, although 5-HT-ir cells and fibres were noticed in the subdivisions of the raphe nucleus and reticular formation, a moderate plexus of fibres was observed in the cerebellum, parabrachial nucleus and solitary tract. Distinct 5-HT-ir fibres, but no perikarya, were observed in the rostral spinal cord. Overall, extensively labelled 5-HT-ir cells and fibres in the CNS of the metamorphic tadpole suggest possible roles for the involvement of 5-HT in various somatosensory, behavioural and neuroendocrine functions during final stages of development.

Temperature and the microbial environment alter brain morphology in a larval amphibian.

Understanding how the global climate impacts the physiology of wildlife animals is of importance. Amphibians are particularly sensitive to climate change, and it is hypothesized that rising temperatures impair their neurodevelopment. Temperature influences the composition of the gut microbiota, which is critical to host neurodevelopment through the microbiota-gut-brain (MGB) axis. Most research investigating the link between the gut microbiota and neurodevelopment occurs in germ-free mammalian model systems, leaving the nature of the MGB axis in non-mammalian wildlife unclear. Here, we tested the hypothesis that the temperature and the microbial environment in which tadpoles were raised shapes neurodevelopment, possibly through the MGB axis. Newly hatched green frog tadpoles (Lithobates clamitans) were raised in natural pond water or autoclaved pond water, serving as an experimental manipulation of the microbiota by reducing colonizing microbes, at three different water temperatures: 14, 22 and 28°C. Neurodevelopment was analyzed through measures of relative brain mass and morphology of brain structures of interest. We found that tadpole development in warmer temperatures increased relative brain mass and optic tectum width and length. Further, tadpole development in autoclaved pond water increased relative optic tectum width and length. Additionally, the interaction of treatments altered relative diencephalon length. Lastly, we found that variation in brain morphology was associated with gut microbial diversity and the relative abundance of individual bacterial taxa. Our results indicate that both environmental temperature and microbial communities influence relative brain mass and shape. Furthermore, we provide some of the first evidence for the MGB axis in amphibians.

Complex Life Histories Alter Patterns of Mercury Exposure and Accumulation in a Pond-Breeding Amphibian.

Quantifying how contaminants change across life cycles of species that undergo metamorphosis is critical to assessing organismal risk, particularly for consumers. Pond-breeding amphibians can dominate aquatic animal biomass as larvae and are terrestrial prey as juveniles and adults. Thus, amphibians can be vectors of mercury exposure in both aquatic and terrestrial food webs. However, it is still unclear how mercury concentrations are affected by exogenous (e.g., habitat or diet) vs endogenous factors (e.g., catabolism during hibernation) as amphibians undergo large diet shifts and periods of fasting during ontogeny. We measured total mercury (THg), methylmercury (MeHg), and isotopic compositions (δ 13C, δ15N) in boreal chorus frogs (Pseudacris maculata) across five life stages in two Colorado (USA) metapopulations. We found large differences in concentrations and percent MeHg (of THg) among life stages. Frog MeHg concentrations peaked during metamorphosis and hibernation coinciding with the most energetically demanding life cycle stages. Indeed, life history transitions involving periods of fasting coupled with high metabolic demands led to large increases in mercury concentrations. The endogenous processes of metamorphosis and hibernation resulted in MeHg bioamplification, thus decoupling it from the light isotopic proxies of diet and trophic position. These step changes are not often considered in conventional expectations of how MeHg concentrations within organisms are assessed.

Maternal chemical defenses predict offspring defenses in a dendrobatid poison frog.

Within and among populations, alkaloid defenses of the strawberry poison frog (Oophaga pumilio) vary spatially, temporally, and with life history stage. Natural variation in defense has been implicated as a critical factor in determining the level of protection afforded against predators and pathogens. Oophaga pumilio tadpoles sequester alkaloids from nutritive eggs and are, thus, entirely dependent on their mothers for their defense. However, it remains unclear how tadpole alkaloid composition relates to that of its mother and how variation in maternally provisioned defenses might result in varying levels of protection against predators. Here, we demonstrate that natural variation in the alkaloid composition of a mother frog is reflected as variation in her tadpole's alkaloid composition. Tadpoles, like mother frogs, varied in their alkaloid composition but always contained the identical alkaloids found in their mother. Alkaloid quantity in tadpoles was highly correlated with alkaloid quantity in their mothers. Additionally, alkaloid quantity was the best predictor of tadpole palatability, wherein tadpoles with higher alkaloid quantities were less palatable. Mother frogs with greater quantities of alkaloids are, thus, providing better protection for their offspring by provisioning chemical defenses during one of the most vulnerable periods of life.

Metformin exposure altered intestinal microbiota composition and metabolites in amphibian larvae.

The antidiabetic pharmaceutical metformin (MET) is largely unmetabolized by the human body. Its residues are readily detectable in various aquatic environments and may have adverse impacts on the growth and survival of aquatic species. To date, its toxicological effects have scarcely been explored in non-fish species. Here, we exposed the tadpoles of black-spotted pond frog (Pelophylax nigromaculatus) to different concentrations (0, 1, 10 and 100 µg/L) of MET for 30 days and measured the body size, intestinal microbiota and metabolites to evaluate potential effects of MET exposure in amphibian larvae. MET exposure did not affect the growth and intestinal microbial diversity of tadpoles. However, intestinal microbial composition changed significantly, with some pathogenic bacteria (e.g., bacterial genera Salmonella, Comamonas, Stenotrophomonas, Trichococcus) increasing and some beneficial bacteria (e.g., Blautia, Prevotella) decreasing in MET-exposed tadpoles. The levels of some intestinal metabolites associated with growth and immune performance also changed significantly following MET exposure. Overall, our results indicated that exposure to MET, even at environmentally relevant concentrations, would cause intestinal microbiota dysbiosis and metabolite alteration, thereby influencing the health status of non-target aquatic organisms, such as amphibians.

Normal development in Xenopus laevis: A complementary staging table for the skull based on cartilage and bone.

BACKGROUND: Xenopus laevis is a widely used model organism in the fields of genetics and development, and more recently evolution. At present, the most widely used staging table for X. laevis is based primarily on external features and does not describe the corresponding skull development in detail. Here, we describe skull development in X. laevis, complete with labeled figures, for each relevant stage in the most widely used staging table. RESULTS: We find skull development in X. laevis is, for the most part, distinct at each of the previously established stages based on external anatomy. However, variation does exist in the timing of onset of ossification of certain bones in the skull, which results in a range of stages where a skull element first ossifies. The overall sequence of ossification is less variable than the timing of ossification onset. CONCLUSIONS: While events in skull development vary somewhat between specimens, and in comparison, to external events, this staging table is useful in showing both when bones first appear and for documenting the range of temporal variance in X. laevis skull development more accurately than previously done. Furthermore, when only skull data are available, the approximate stage of a specimen can now be determined.

The trunk-tail junctional region in Ciona larvae autonomously expresses tail-beating bursts at ∼20 second intervals.

Swimming locomotion in aquatic vertebrates, such as fish and tadpoles, is expressed through neuron networks in the spinal cord. These networks are arranged in parallel, ubiquitously distributed and mutually coupled along the spinal cord to express undulation patterns accommodated to various inputs into the networks. While these systems have been widely studied in vertebrate swimmers, their evolutionary origin along the chordate phylogeny remains unclear. Ascidians, representing a sister group of vertebrates, give rise to tadpole larvae that swim freely in seawater. In the present study, we examined the locomotor ability of the anterior and posterior body fragments of larvae of the ascidian Ciona that had been cut at an arbitrary position. Examination of more than 200 fragments revealed a necessary and sufficient body region that spanned only ∼10% of the body length and included the trunk-tail junction. 'Mid-piece' body fragments, which included the trunk-tail junctional region, but excluded most of the anterior trunk and posterior tail, autonomously expressed periodic tail-beating bursts at ∼20 s intervals. We compared the durations and intervals of tail-beating bursts expressed by mid-piece fragments, and also by whole larvae under different sensory conditions. The results suggest that body parts outside the mid-piece effect shortening of swimming intervals, particularly in the dark, and vary the burst duration. We propose that Ciona larvae express swimming behaviors by modifying autonomous and periodic locomotor drives that operate locally in the trunk-tail junctional region.

The role of environmental calcium in the extreme acid tolerance of northern banjo frog (Limnodynastes terraereginae) larvae.

Many aquatically respiring animals acutely exposed to low pH waters suffer inhibition of ion uptake and loss of branchial (gill) epithelial integrity, culminating in a fatal loss of body Na+. Environmental calcium levels ([Ca2+]e) are pivotal in maintaining branchial junction integrity, with supplemental Ca2+ reversing the negative effects of low pH in some animals. Tolerance of some naturally acidic environments by aquatic animals is further complicated by low [Ca2+]e, yet many of these environments are surprisingly biodiverse. How animals overcome the damaging actions of low pH and low environmental Ca2+ remains unknown. We examined the effects of [Ca2+]e on the response to low pH in larvae of the highly acid-tolerant frog Limnodynastes terraereginae. Acute exposure to low pH water in the presence of low (5 µmol l-1) [Ca2+]e increased net Na+ efflux. Provision of additional [Ca2+]e reduced net Na+ efflux, but the effect was saturable. Acclimation to both low and high (250 µmol l-1) [Ca2+]e improved the resistance of larvae to Na+ efflux at low pH. Exposure to the Ca2+ channel inhibitor ruthenium red resulted in an abrupt loss of tolerance in low pH-acclimated larvae. Acclimation to acidic water increased branchial gene expression of the intracellular Ca2+ transport protein calbindin, consistent with a role for increased transcellular Ca2+ trafficking in the tolerance of acidic water. This study supports a role for [Ca2+]e in promoting branchial integrity and highlights a potential mechanism via the maintenance of transcellular Ca2+ uptake in the acid tolerance of L. terraereginae larvae.

Phenotypic variation in Xenopus laevis tadpoles from contrasting climatic regimes is the result of adaptation and plasticity.

Phenotypic variations between populations often correlate with climatic variables. Determining the presence of phenotypic plasticity and local adaptation of a species to different environments over a large spatial scale can provide insight on the persistence of a species across its range. Amphibians, and in particular their larvae, are good models for studies of phenotypic variation as they are especially sensitive to their immediate environment. Few studies have attempted to determine the mechanisms that drive phenotypic variation between populations of a single amphibian species over a large spatial scale especially across contrasting climatic regimes. The African clawed frog, Xenopus laevis, occurs in two regions with contrasting rainfall regimes in southern Africa. We hypothesised that the phenotypic variation of life-history traits of X. laevis tadpoles emerges from a combination of plastic and genetic responses. We predicted that plasticity would allow the development of tadpoles from both regions in each environment. We also predicted that local adaptation of larval traits would drive the differentiation of reaction norms between populations and lower survival in tadpoles reared away from their home environment. We measured growth, time to metamorphosis, and survival in a reciprocal transplant experiment using outdoor mesocosms. Supporting our prediction, we found that the measured variation of all traits was explained by both adaptation and plasticity. However, the reaction norms differed between populations suggesting adaptive and asymmetric plasticity. All tadpoles experienced lower survival when translocated, but only translocated tadpoles from the winter rainfall region matched survival of local tadpoles. This has implications for the dynamics of translocated X. laevis into novel environments, especially from the winter rainfall region. Our discovery of their asymmetric capacity to overcome novel environmental conditions by phenotypic plasticity alone provides insight into their invasion success.

Maternal Provisioning of Alkaloid Defenses are Present in Obligate but not Facultative Egg Feeding Dendrobatids.

Poison frogs sequester alkaloid defenses from a diet of largely mites and ants. As a result, frogs are defended against certain predators and microbial infections. Frogs in the genus Oophaga exhibit complex maternal care, wherein mothers transport recently hatched tadpoles to nursery pools and return regularly to supply developing tadpoles with unfertilized (nutritive) eggs. Developing tadpoles are obligate egg feeders. Further, female O. pumilio and O. sylvatica maternally provision their nutritive eggs with alkaloid defenses, providing protection to their developing tadpoles at a vulnerable life-stage. In another genus of poison frog, Ranitomeya, tadpoles only receive and consume eggs facultatively, and it is currently unknown if mothers also provision these eggs (and thus their tadpoles) with alkaloid defenses. Here, we provide evidence that mother frogs of another species in the genus Oophaga (Oophaga granulifera) also provision alkaloid defenses to their tadpoles. We also provide evidence that Ranitomeya imitator and R. variabilis eggs and tadpoles do not contain alkaloids, suggesting that mother frogs in this genus do not provision alkaloid defenses to their offspring. Our findings suggest that among dendrobatid poison frogs, maternal provisioning of alkaloids may be restricted to the obligate egg-feeding members of Oophaga.

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.