Metamorphic Climax Research Articles

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.

Crystalline basement from Laguna Amarga metamorphic complex in the high Andes of western Catamarca, Argentina (27° 15’ - 27° 40’ south): Petrology, structure and geodynamic implications

The Laguna Amarga Metamorphic Complex is a major stratigraphic component of the southern Central Andes. The occurrence of crystalline basement pieces extensively exposed in the western Andes of Catamarca (Argentina), has been known for decades, nevertheless, a lack of detailed field-based and geochronological survey has precluded an accurate correlation of the Laguna Amarga Complex with other basement exposures in the Central Andes. The Laguna Amarga Metamorphic Complex outcrops in several fault-bounded blocks spread over about 360 km2, always underlying Neopaleozoic strata and Neogene volcanic strata from the Central Volcanic Zone. The northern part of the complex consists of amphibolite-facies metamorphic rocks derived from sedimentary and igneous precursors grouped in an association named Cazadero Grande. The original stratigraphic succession was interbedded siliciclastic sediments, carbonaceous, and mafic rocks. The southern segment of the complex is dominated by granulite-facies migmatites and ortho-amphibolites grouped in an association called Los Aparejos. In the Cazadero Grande Association, four deformational events are recognized. The first three phases gave rise to an imbricate S-verging over-thrusting system and prevailing tectonic transport to the south in response to progressive non-coaxial strains. The mineral assemblages linked to the metamorphic climax equilibrated coeval with the early stages of fabric-forming deformation. The posthumous fourth phase of deformation produces gentle folds with kilometer-scale wavelengths whose axial planes (non-penetrative) have an NNE strike, and are as related to Andean deformation. Three incremental deformation events coeval with the metamorphic climax are registered in the Los Aparejos Association that indicate a kinematics transport to the east. First U–Pb zircon ages are reported for the Laguna Amarga Metamorphic Complex. Age distributions are polymodal, but the dominant populations make well-defined clusters. The most prominent concord age peak is at 389 Ma, revealing the age of the metamorphic climax. Another noteworthy aspect is the presence of dominant Mesoproterozoic concord age peaks (1119-1082 Ma), interpreted as inheritance ages of their protolith. A point to highlight is the absence of ages reflecting the source with Brasiliano-Pan-African orogenic ages that dominates detrital provenance from the West Gondwana. The Mesoproterozoic protolith ages allow the correlation of this metamorphic complex with nearby basement detached from Laurentian realms. The analysis of the ages shows that the metamorphic peak and the generalized deformation that affected the entire basement of the complex developed in the Middle Devonian (Eifelian), this being a very little known event for the Paleozoic evolution of western Gondwana. The work discusses how a better understanding of the crystalline basement of the high Andes impacts the geodynamic history of the proto-Andean Gondwana margin. It also provides new information on the geodynamic configuration of the Lower to Middle Paleozoic and its link with the processes of accretion-collision of terranes.

An Environmentally Relevant Mixture of Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Does Not Conform to Additivity in Northern Leopard Frogs Exposed Through Metamorphosis.

Per- and polyfluoroalkyl substances (PFAS) are chemicals associated with adverse health effects. At aqueous film-forming foam sites, they occur as mixtures, with perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) commonly co-occurring in the highest concentrations. Although PFOS and PFHxS toxicities have been studied, few studies have tested their potential interaction. Using Rana pipiens, the present study compared toxicities of a 1:1 PFOS:PFHxS mixture to PFOS and PFHxS individually with the prediction that responses would be additive. Gosner stage 25 (GS 25) tadpoles were exposed through metamorphosis (GS 46) to 0.5 and 1 ppb PFOS or PFHxS alone or to a mixture of 0.5 ppb PFOS and 0.5 ppb PFHxS. Tadpoles were weighed and measured (snout-vent length [SVL]) at day 31, metamorphic climax (GS 42), and GS 46. These values were used to calculate the scaled mass index (SMI), a measure of body condition. Body burdens were quantified on day 31 and at GS 46. The PFOS and PFHxS body burdens were elevated relative to controls at GS 46. No effects were observed on survival, SVL, or mass. Single PFAS effects included a 17% reduction in SMI at day 31 (0.5 ppb PFHxS) and a 1.1-day longer metamorphic period (1 ppb PFHxS) relative to controls. Mixture results deviated from additivity-SMIs were higher than expected on day 31 and lower than expected at GS 42. In addition, time to GS 42 in the PFAS mixture exceeded expected additivity by 12 days. Results from a chronic exposure to a 1:1 PFOS:PFHxS mixture resulted in changes in body condition and length of metamorphosis that deviated from additivity. More PFAS mixture toxicity studies conducted at relevant ratios and concentrations are needed. Environ Toxicol Chem 2022;41:3007-3016. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Naphthenic acid fraction compounds reduce the reproductive success of wood frogs (Rana sylvatica) by affecting offspring viability

Understanding the toxicity of organic compounds in oil sands process-affected water (OSPW) is necessary to inform the development of environmental guidelines related to wastewater management in Canada's oil sands region. In the present study, we investigated the effects of naphthenic acid fraction compounds (NAFCs), one of the most toxic components of OSPW, on mating behaviour, fertility, and offspring viability in the wood frog (Rana sylvatica). Wild adult wood frogs were exposed separately from the opposite sex to 0, 5, or 10 mg/L of OSPW-derived NAFCs for 24 h and then combined in outdoor lake water mesocosms containing the same NAFC concentrations (n = 2 males and 1 female per mesocosm, n = 3 mesocosms per treatment). Mating events were recorded for 48 h and egg masses were measured to determine adult fertility. NAFC exposure had no significant effect on mating behaviour (probability of amplexus and oviposition, amplexus and oviposition latency, total duration of amplexus and number of amplectic events) or fertility (fertilization success and clutch size). Tadpoles (50 individuals per mesocosm at hatching, and 15 individuals per mesocosm from 42 d post-hatch) were reared in the same mesocosms under chronic NAFC exposure until metamorphic climax (61–85 d after hatching). Offspring exposed to 10 mg/L NAFCs during development were less likely to survive and complete metamorphosis, grew at a reduced rate, and displayed more frequent morphological abnormalities. These abnormalities included limb anomalies at metamorphosis, described for the first time after NAFC exposure. The results of this study suggest that NAFCs reduce wood frog reproductive success through declines in offspring viability and therefore raise the concern that exposure to NAFCs during reproduction and development may affect the recruitment of native amphibian populations in the oil sands region.

Skeletal description of Pelophylax caralitanus (Anura: Ranidae): Comparison with other ranids.

A description of the skeletal development of ranids is scarce despite being well known in the family Ranidae. Herein, the description of several species of two genera representing the family Ranidae from Turkey is studied wherein the larval and adult cranial skeletons of Pelophylax caralitanus are described and compared with that of the water frog (Pelophylax bedriagae) and mountain frog (Rana macrocnemis). The data are based on cleared and double-stained specimens of 20 Gosner developmental Stages (26-46). The first element to ossify in P. caralitanus is the parasphenoid (Stage 30), followed by the exoccipitals (Stage 34) and prootics (Stage 35). The metamorphic climax commences at Stage 42 during which several modifications to the chondrocranium will unfold. In addition, ranids demonstrate remarkable differences between the water (Pelophylax sp.) and mountain (Rana sp.) frog species: (a) the ventromedial bridge between the corpora of the suprarostral cartilage in the larval upper jaw, (b) the number of processes of the central corpus in the hyobranchial skeleton, and (c) the differences in the ossification time and sequence between Pelophylax sp. and Rana sp. A detailed description of the larval osteological features of P. caralitanus conforms to the current phylogenetic position and provides a model for comparison with other ranids.

Effects of a Neonicotinoid Insecticide and Population Density on Behavior and Development of Wood Frogs (Rana sylvatica).

Amphibians have been facing global declines over the last decades from direct and indirect effects of anthropogenic activities. A contributor to declines is waterway contamination from agricultural runoffs of pesticides such as neonicotinoids. Beyond direct and indirect effects of the pesticide, few studies have investigated the possible interactions between neonicotinoids and natural environmental stressors across larval development, which could alter the strength and direction of observed neonicotinoid effects. The present study used a fully crossed design to investigate how a concentration of imidacloprid (a neonicotinoid; 10 µg/L) measured in surface waters interacted with low and high population densities (0.33 and 1 tadpole/L, respectively), an important environmental stressor, to influence behavior and development across metamorphosis in wood frogs (Rana sylvatica), known to breed in agricultural landscapes. Behaviors were measured in the absence and presence of predation cues using open-field tests at three distinct developmental stages, up to the metamorph stage. We found that imidacloprid did not interact with population density or independently affect behaviors in the absence of predation cues. However, individuals raised at high density compared with low density were more active at an early developmental stage but less active at metamorphic climax. Furthermore, both density and imidacloprid independently decreased the natural freezing response of tadpoles to predation cues. Finally, we found that distance traveled in the open-field test was weakly repeatable between aquatic stages but not repeatable across metamorphosis, a pattern that was not affected by treatments. The present study provides novel insights on the ecotoxicology of imidacloprid in the presence of a natural stressor, highlighting the importance of including behavioral assays and natural stressors in studies of amphibian ecotoxicology. Environ Toxicol Chem 2022;41:2968-2980. © 2022 SETAC.

Thyroid hormone-dependent and independent processes of red blood cell transition from larval to adult type during metamorphosis in Xenopus laevis.

Amphibian metamorphosis results in drastic whole-body remodeling. Thyroid hormone (TH) drives most of these metamorphic changes. A prominent event during this remodeling is the red blood cell (RBC) transition from larval to adult forms, which exclusively contain larval and adult hemoglobin, respectively. However, the role of TH in RBC transition remains unclear. Here we reconfirmed that RBC transition of Xenopus laevis is completed much later than morphological metamorphosis. Further, larval and adult RBCs/erythroblasts proliferated both in the erythropoietic liver and in circulation during metamorphic climax. RBC transition was also confirmed in Rana ornativentris, but in contrast to X. laevis, adult RBC-specific proliferation was observed from the early climax stages. We also revealed in either species that RBC transition occurs in the liver prior to circulating RBCs. Moreover, anemia induction using phenylhydrazine during the prometamorphosis of X. laevis caused precocious RBC transition even when TH synthesis was blocked, resulting in metamorphosis-arrested larvae in which most of RBCs were of adult type. These results indicate that a decline in larval RBCs facilitates RBC transition during metamorphosis in a TH-independent manner. Further, combined administration of phenylhydrazine and TH induced precocious appearance of adult RBCs in early prometamorphic X. laevis tadpoles, whereas individual treatment with phenylhydrazine or TH did not cause precocious RBC transition; this suggests that TH is required to initiate RBC transition by promoting the differentiation of adult erythroblasts during early prometamorphosis in X. laevis. These results show that TH-dependent and independent processes are present in RBC transition in X. laevis.

Predatory influence of dragonfly larvae and water scorpions on eggs and tadpoles of Indosylvirana temporalis (Anura: Ranidae)

We assessed in the laboratory the vulnerability of Bronzed frog (Indosylvirana temporalis) eggs and tadpoles to two potential sit-and-wait insect predators, larvae of a dragonfly (Pantala flavescens; Odonata: Libellulidae) and adult water scorpions (Laccotrephes sp.; Hemiptera: Nepidae). We exposed a series of different developmental stages of I. temporalis (from eggs to metamorphic climax stage) to these two predators. The results of this study showed that larvae of P. flavescens preyed on eggs and tadpoles of I. temporalis but only to stage 36. Laccotrephes sp. did not prey on eggs of I. temporalis but on tadpoles of all stages (22 to 42). This difference in predation rate was likely due to the gape size of the predators. The larvae of P. flavescens are gape-limited and cannot prey on larger tadpoles (above stage 36). Adults Laccotrephes sp. are non-gape-limited predators, using a segmented beak to pierce I. temporalis and suck the body fluids. They captured small to large tadpoles by quickly grabbing and immobilizing them using the front pair of raptorial legs. The present study shows that both predatory insects are a threat to I. temporalis at early and later stages of larval development.

Resilience of larval wood frogs (Rana sylvatica) to hydrocarbons and other compounds released from naturally weathered diluted bitumen in a boreal lake.

The risks to aquatic wildlife from spills of diluted bitumen (dilbit) into inland waters are poorly understood. In this paper, we describe the response of larval wood frogs (Rana sylvatica) to hydrocarbons and other compounds released from experimental spills of dilbit in a temperate boreal lake. To simulate a wide range of environmentally relevant oil spill scenarios, different volumes of Cold Lake Winter Blend dilbit (0, 1.5, 2.9, 5.5, 18, 42, 82, and 180L) were added to 10m diameter in-lake limnocorrals. Larvae (n=360) were reared (from Gosner Stage (GS) 25 to ∼42) in land-based aquatic microcosms, where they were first exposed to clean water during a 2-week baseline phase, and then (at GS ∼30), to contaminated water withdrawn from the limnocorrals for 3 weeks. We observed no statistically significant trends in survival, growth, or development of larvae as a consequence of exposure to the chemical compounds released from naturally weathered dilbit. Likewise, neither cytochrome P450 1A biomarkers nor levels of thyroid hormones in wood frogs near metamorphic climax were significantly related to volume of the oil spills. However, there was a modest statistically significant decrease in larval activity (up to 8.7% relative to the control), but no change in other behavioral metrics (i.e., sociality or space use). Our work adds to the limited body of literature on the effects of unconventional oils on aquatic wildlife and helps to inform risk assessments regarding pipeline projects.

From Water to Land: The Structural Construction and Molecular Switches in Lungs during Metamorphosis of Microhyla fissipes.

Simple SummaryThe functionalization of lungs is a necessity for most anurans to breathe on land. Previous studies have focused on the morphological and physiological functions of amphibian lungs, while the microstructural changes and molecular mechanisms that underpin the functional maturation of lungs remain under-researched. We used integrated histology and transcriptomics to study the critical cytological and molecular events associated with lung maturation in Microhyla fissipes. The results illuminated the molecular processes and their coordination in lung development, providing an insight into the transition of amphibians from aquatic to terrestrial life stages.Most anurans must undergo metamorphosis to adapt to terrestrial life. This process enhances the air-breathing ability of the lungs to cope with the change in oxygen medium from water to air. Revealing the structural construction and molecular switches of lung organogenesis is essential to understanding the realization of the air-breathing function. In this study, histology and transcriptomics were conducted in combination to explore these issues in Microhyla fissipes’ lungs during metamorphosis. During the pro-metamorphic phase, histological structural improvement of the alveolar wall is accompanied by robust substrate metabolism and protein turnover. The lungs, at the metamorphic climax phase, are characterized by an increased number of cilia in the alveolar epithelial cells and collagenous fibers in the connective tissues, corresponding to the transcriptional upregulation of cilia and extracellular matrix-related genes. Post-metamorphic lungs strengthen their contracting function, as suggested by the thickened muscle layer and the upregulated expression of genes involved in muscle contraction. The blood–gas barrier is fully developed in adult lungs, the transcriptional features of which are tissue growth and regulation of differentiation and immunity. Importantly, significant transcriptional switches of pulmonary surfactant protein and hemoglobin facilitate air breathing. Our results illuminated four key steps of lung development for amphibians to transition from water to land.

Effects of sodium perchlorate and 6-propylthiouracil on metamorphosis and thyroid gland histopathology in the European common frog (Rana temporaria).

Several chemicals have been identified as thyroid hormone axis disrupting chemicals (THADCs) able to interfere with the thyroid hormone system during fetal life and early life stages, thereby impairing neurodevelopment in mammals and inducing development and growth disorders in fish and amphibians. However, identification of THADCs is particularly challenging, and thyroid modalities are currently only assessed in vivo by mammalian and amphibian tests. The aquatic African clawed frog (Xenopus laevis/tropicalis) is the model species of the amphibian test guidelines developed by the OECD and the United States Environmental Protection Agency, but as most European amphibians are semi-aquatic, concern has been raised whether the sensitivity of native European species is comparable to Xenopus. A shortened version of the OEDC test guideline 241 (Larval Amphibian Growth and Development Assay, LAGDA) was used to investigate the effects of two model THADCs on the metamorphosis and thyroid histopathology in the European common frog (Rana temporaria). R. temporaria eggs were collected on the field and exposed till metamorphic climax to sodium perchlorate (11.9-426.5 μg/L perchlorate concentrations) and 6-propylthiouracil (PTU: 1.23-47.7mg/L). PTU severely delayed metamorphosis and affected several thyroid gland histopathological endpoints at slightly lower concentrations compared to Xenopus. As opposed to what was described in similar Xenopus studies, we observed no effect of perchlorate on the investigated endpoints. Interspecies differences may be linked to mechanisms of action.

Anatomy of a deformed upper crust fragment from western Alpine Corsica (France): insights into continental subduction processes

ABSTRACT In Corsica Island (France), a belt of metamorphic continental units derived from the European margin is exposed at the western rim of the Alpine Corsica. This study provides a complete anatomy of one of these units (Tour de Valletto Unit) consisting of a Palaeozoic basement covered by an Early Jurassic – late Eocene sedimentary succession. This unit records a polyphase tectono-metamorphic history acquired during the late Eocene – lower early Miocene convergence processes involving the Adria and Europe continental plates. Using a multidisciplinary approach, we reconstructed in detail the P-T path of the Tour de Valletto Unit. The path shows a metamorphic climax at blueschist facies conditions suggesting that the unit was accreted at c. 40 km of depth. After its accretion, the Tour de Valletto Unit was exhumed up to the Earth surface following a path characterized by an increase of temperature and a decrease of pressure. The reconstructed tectono-metamorphic history of the Tour de Valletto Unit is compared with those of the other continental units located in western Alpine Corsica to discuss the exhumation processes active during continental subduction.

Tetrabromobisphenol A Disturbs Brain Development in Both Thyroid Hormone-Dependent and -Independent Manners in Xenopus laevis.

Although tetrabromobisphenol A (TBBPA) has been well proven to disturb TH signaling in both in vitro and in vivo assays, it is still unclear whether TBBPA can affect brain development due to TH signaling disruption. Here, we employed the T3-induced Xenopus metamorphosis assay (TIXMA) and the spontaneous metamorphosis assay to address this issue. In the TIXMA, 5–500 nmol/L TBBPA affected T3-induced TH-response gene expression and T3-induced brain development (brain morphological changes, cell proliferation, and neurodifferentiation) at premetamorphic stages in a complicated biphasic concentration-response manner. Notably, 500 nmol/L TBBPA treatment alone exerted a stimulatory effect on tadpole growth and brain development at these stages, in parallel with a lack of TH signaling activation, suggesting the involvement of other signaling pathways. As expected, at the metamorphic climax, we observed inhibitory effects of 50–500 nmol/L TBBPA on metamorphic development and brain development, which was in agreement with the antagonistic effects of higher concentrations on T3-induced brain development at premetamorphic stages. Taken together, all results demonstrate that TBBPA can disturb TH signaling and subsequently interfere with TH-dependent brain development in Xenopus; meanwhile, other signaling pathways besides TH signaling could be involved in this process. Our study improves the understanding of the effects of TBBPA on vertebrate brain development.

Larval Exposure to Polychlorinated Biphenyl-126 Led to a Long-Lasting Decrease in Immune Function in Postmetamorphic Juvenile Northern Leopard Frogs, Lithobates pipiens.

Amphibian populations are decreasing worldwide, and pollution is a contributing factor. Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants known to exert immunotoxicity. To assess impacts of PCBs on frogs, we exposed Lithobates pipiens tadpoles to a diet of PCB-126 (0-5 ng PCB-126/g wet food) through metamorphic climax. Postmetamorphic frogs were immunized with keyhole limpet hemocyanin (KLH); then production of KLH-specific IgY, as well as total IgY and IgM, was measured (Trial I). A second larval study (0 and 7.3 ng PCB-126/g wet food) was performed to investigate whether PCB altered antigenic responses in prometamorphic tadpoles (Gosner Stage 36-39), and to measure the innate immune response of postmetamorphic frogs (Trial II). After larval PCB-126 exposure, both KLH-specific IgY levels and complement activity were reduced. Because postmetamorphic frogs carried a body burden of PCB-126 (2.4 ng/g or less), we wanted to determine whether the effect on immune response was due to larval exposure or to the resulting body burden as frogs. To test this, we reared tadpoles under control conditions (no PCB), and limited PCB exposure to postmetamorphosis only by injecting 2-week-old frogs with 10 ng PCB-126/g (Trial III). The resulting body burden (3.4 ng/g) was similar to that of frogs in Trial I, but we no longer detected suppression of KLH-specific IgY or hemolytic activity. These results suggest life-stage-specific immune responses; however, because we administered PCB-126 differently between trials, it is premature to conclude that these differences are intrinsically life stage dependent, and further study is warranted. Regardless, our study demonstrated a long-lasting effect of larval PCB-126 exposure that persisted through metamorphosis and suppressed frog immunity. Environ Toxicol Chem 2022;41:81-94. © 2021 SETAC.

Morphology and molecular mechanisms of tail resorption during metamorphosis in Rana chensinensis tadpole (Anura: Ranidae)

The tail resorption process was an inevitable and pivotal transformation during amphibian metamorphosis. The present study investigated the mechanisms of tail resorption through histological and transcriptome analysis in Rana chensinensis. The results showed that tail resorption was initiated before the onset of metamorphic climax, and dramatically regressed after metamorphic climax by external-morphology measurement. The drastic disintegration of tail muscle and notochord occurred at Gs42-44, which were consistent with the trend of thyroid follicular cell height. Besides, expression level analysis and functional annotation of DEGs (differentially expressed genes) were conducted through RNA-seq analysis of the tail. Our study also analyzed the expression of genes related to oxidative stress, autophagy, apoptosis and degradation of cellular components in the tail of R. chensinensis. This study enriched the R. chensinensis transcriptome database and laid the foundation of further analysis of tail resorption.

Temporal and spatial transcriptomic dynamics across brain development in Xenopus laevis tadpoles.

Amphibian metamorphosis is a transitional period that involves significant changes in the cell-type populations and biological processes occurring in the brain. Analysis of gene expression dynamics during this process may provide insight into the molecular events underlying these changes. We conducted differential gene expression analyses of the developing Xenopus laevis tadpole brain during this period in two ways: first, over stages of the development in the midbrain and, second, across regions of the brain at a single developmental stage. We found that genes pertaining to positive regulation of neural progenitor cell proliferation as well as known progenitor cell markers were upregulated in the midbrain prior to metamorphic climax; concurrently, expression of cell cycle timing regulators decreased across this period, supporting the notion that cell cycle lengthening contributes to a decrease in proliferation by the end of metamorphosis. We also found that at the start of metamorphosis, neural progenitor populations appeared to be similar across the fore-, mid-, and hindbrain regions. Genes pertaining to negative regulation of differentiation were upregulated in the spinal cord compared to the rest of the brain, however, suggesting that different programs may regulate neurogenesis there. Finally, we found that regulation of biological processes like cell fate commitment and synaptic signaling follow similar trajectories in the brain across early tadpole metamorphosis and mid- to late-embryonic mouse development. By comparing expression across both temporal and spatial conditions, we have been able to illuminate cell-type and biological pathway dynamics in the brain during metamorphosis.

Accumulation of microplastics in tadpoles from different functional zones in Hangzhou Great Bay Area, China: Relation to growth stage and feeding habits

Microplastics (MPs) are ubiquitous in freshwater ecosystems, including inland small waterbodies (e.g., ponds and ditches), which are unique habitats for tadpoles. The uptake of MPs by tadpoles is influenced by their habitat, life stage, and feeding strategy. In this study, we investigated MP levels in small waterbodies in three different functional zones from the Hangzhou Great Bay Area, China, and resident tadpoles at different stages of metamorphosis with different feeding habits. Our results indicated that MPs in all three sampling areas were predominantly fibers; and the highest abundances of MPs were observed in water (4.70 ± 2.30 items/L) and sediment (728 ± 324 items/kg) from a textile industrial area, likely the result of nearby human activities. There was no significant difference in MP number in tadpoles between areas; however, omnivorous tadpoles with labial teeth and horny beaks ingested more MPs than did filter feeders. Based on their developmental characteristics, the collected tadpoles were categorized as: pre-metamorphosis, pro-metamorphosis, and metamorphic climax. The MP levels exhibited an upward trend, and MP size gradually increased as tadpole development progressed. This suggests that MPs may accumulate in tadpoles as they grow and potentially affect their metamorphosis from larvae to frogs.

Anuran development: A reinvestigation of the conus arteriosus and gill formation in Bufo bufo throughout metamorphosis using micro-CT.

Using high‐resolution X‐ray micro‐CT imaging of whole Bufo bufo specimens, we acquired detailed 3D descriptions of the changing morphology of the cardiac outflow structures, in particular the conus arteriosus through larval development and the transition. Previous findings regarding anuran conal structures were contradictory, depending on the specifics of the 2D imaging methods used by different authors. Our descriptions of conal morphology at different developmental stages show that early tadpoles initially only have one opening at the ventricular‐conal junction and only one cavum within their conus; however, the forming septum coni soon divides the conus into two chambers, the cavum pulmocutaneum and the cavum aorticum. This is accompanied by the development of a second small opening at the ventricular‐conal junction. The separated chambers continue into the aortic arches. Following the aortic arches into the area where gills will form, we describe how blood vessels associated with the external gills develop from vessels arising from the truncus arteriosus. The external gills soon undergo partial absorption. During the transition from external to internal gills, the gill filaments retreat asymmetrically into a gill chamber formed by a hyoidal cover contacting the animal's ventral side, leaving only a single opening on the animal's left side, the opercular spout. B. bufo retains its internal gills up to metamorphic climax, with the aortic arches arising from the conus arteriosus still leading into the gills. Our 3D image data are publicly available and will provide a sound morphological basis for future studies.

Frogs Respond to Commercial Formulations of the Biopesticide Bacillus thuringiensis var. israelensis, Especially Their Intestine Microbiota.

It is generally believed that Bacillus thuringiensis var. israelensis (Bti) biopesticides are harmless to non-target organisms; however, new research shows controversial results. We exposed acutely and chronicallyLithobates sylvaticusandAnaxyrus americanus tadpoles until metamorphic climax to VectoBac 200G (granules) and VectoBac 1200L (aqueous suspension) at 300-20,000 ITU/L covering field-relevant concentrations and higher. The data show that the exposure parameters tested did not affect significantly the survival, total length, total weight, hepatosomatic index, gonadosomatic index, the expression of genes of interest (i.e., related to xenobiotic exposure, oxidative stress, and metamorphosis), and the intestine tissue layer detachment ofL. sylvaticusandA. americanus in a concentration-response pattern. In contrast, VectoBac 200G significantly increased the median time to metamorphosis ofL. sylvaticus tadpoles by up to 3.5 days and decreased the median by up to 1 day inA. americanus. VectoBac 1200L significantly increased the median time to metamorphosis ofL. sylvaticusandA. americanustadpoles by up to 4.5 days. Also, the exposure to VectoBac 200G and 1200L altered the intestine bacterial community composition inA. americanus at application rates recommended by the manufacturer, which led to an increase in the relative abundance of Verrucomicrobia, Firmicutes, Bacteroidetes, and Actinobacteria. Changes in the intestine microbiota might impact the fitness of individuals, including the susceptibility to parasitic infections. Our results indicate that the effect of Bti commercial products is limited; however, we recommend that Bti-spraying activities in amphibian-rich ecosystems should be kept minimal until there is more conclusive research to assess if the changes in the time to metamorphosis and microbiota can lead to negative outcomes in amphibian populations and, eventually, the functioning of ecosystems.

Water-borne corticosterone assay is a valid method in some but not all life-history stages in Northern Leopard Frogs

There is a particular need to develop conservation tools for use in amphibian populations, which are declining rapidly. Glucocorticoid hormones like corticosterone (CORT) are often used as biomarkers of amphibian stress. A relatively new method of assessing CORT in amphibians is to measure CORT concentrations in water that has held amphibians (water-borne (WB) CORT). Here, we tested whether WB CORT is a valid measure of CORT in larval and metamorphic Northern Leopard Frogs (Lithobates pipiens). We assessed whether levels of WB CORT are different among groups of animals that should have different levels of CORT due to a handling challenge, a pharmacological challenge (ACTH), or developmental stage. We also assessed whether WB CORT was correlated with plasma CORT within individuals. Results indicated that measurement of WB CORT is valid in prometamorphic tadpoles because injection with ACTH increased WB CORT, and WB CORT and plasma CORT levels were correlated within an animal in most cases. However, were unable to fully validate the use of WB CORT in metamorphic frogs (metamorphs) because although injection with ACTH elevated levels of WB CORT, WB CORT was not correlated with plasma CORT within individual metamorphs. Also, there was no correlation between WB CORT and plasma CORT in early stage (premetamorphic) tadpoles or tadpoles undergoing metamorphic climax, indicating that WB CORT is not sensitive enough to detect natural variation of organismal CORT in these groups. Together, results indicated that WB CORT is a valid method of assessing plasma CORT in Northern Leopard Frogs, but only for some life-history stages. Our results illustrate the importance of carefully validating the use of WB CORT for appropriate interpretation of results.