Metamorphic Climax Research Articles

Endocrine Disruption Alters Developmental Energy Allocation and Performance in Rana temporaria.

Environmental change exposes wildlife to a wide array of environmental stressors that arise from both anthropogenic and natural sources. Many environmental stressors with the ability to alter endocrine function are known as endocrine disruptors, which may impair the hypothalamus-pituitary-thyroid axis resulting in physiological consequences to wildlife. In this study, we investigated how the alteration of thyroid hormone (TH) levels due to exposure to the environmentally relevant endocrine disruptor sodium perchlorate (SP; inhibitory) and exogenous L-thyroxin (T4; stimulatory) affects metabolic costs and energy allocation during and after metamorphosis in a common amphibian (Rana temporaria). We further tested for possible carry-over effects of endocrine disruption during larval stage on juvenile performance. Energy allocated to development was negatively related to metabolic rate and thus, tadpoles exposed to T4 could allocate 24% less energy to development during metamorphic climax than control animals. Therefore, the energy available for metamorphosis was reduced in tadpoles with increased TH level by exposure to T4. We suggest that differences in metabolic rate caused by altered TH levels during metamorphic climax and energy allocation to maintenance costs might have contributed to a reduced energetic efficiency in tadpoles with high TH levels. Differences in size and energetics persisted beyond the metamorphic boundary and impacted on juvenile performance. Performance differences are mainly related to strong size-effects, as altered TH levels by exposure to T4 and SP significantly affected growth and developmental rate. Nevertheless, we assume that juvenile performance is influenced by a size-independent effect of achieved TH. Energetic efficiency varied between treatments due to differences in size allocation of internal macronutrient stores. Altered TH levels as caused by several environmental stressors lead to persisting effects on metamorphic traits and energetics and, thus, caused carry-over effects on performance of froglets. We demonstrate the mechanisms through which alterations in abiotic and biotic environmental factors can alter phenotypes at metamorphosis and reduce lifetime fitness in these and likely other amphibians.

Oxidative stress in Pelophylax esculentus complex frogs in the wild during transition from aquatic to terrestrial life

During life, anuran individuals undergo drastic changes in the course of transition from aquatic to terrestrial habitat, when they are faced with metabolically demanding processes (growth, responses to developmental pressures), which result in increased production of reactive oxygen species (ROS), signaling molecules involved in development that can induce oxidative damage and stress. This situation can be further complicated by environmental influences. The aim of this study was to investigate oxidative stress parameters in naturally developing Pelophylax esculentus complex frogs during four developmental periods: premetamorphosis, prometamorphosis, metamorphic climax and juvenile stage, in order to examine changes in the response of the antioxidative system (AOS) and oxidative damage during the transition from aquatic to terrestrial life. Results show that ontogenetic shifts in anurans are accompanied by different levels of damage and AOS responses, which vary from the increased first-line enzymatic activities during the early period of development (premetamorphosis), through increased changes in the non-enzymatic complement during the metamorphic climax, to changes in both the enzymatic and non-enzymatic components observed in juvenile individuals. Premetamorphic individuals and individuals in metamorphosis displayed higher levels of lipid peroxidation, indicating that direct exposure to the environment for the first time and the modulation of organs are the most susceptible stages for oxidative damage. On the other hand, lower oxidative damage in juveniles points to the ability of their AOS to efficiently respond to challenges of the terrestrial environment. This study highlights the importance of ROS and the AOS of anurans in response to different developmental and/or environmental pressures that individuals face.

Las Cañas plutonic complex: Geodynamic implications during the Famatinian magmatism in northeast of Sierra de San Luis, Argentina

Las Cañas Plutonic Complex (LCPC) is a small representative of the Famatinian Ordovician plutonism (470.4 ± 8.1 Ma) located in the northeastern sector of Sierra Grande de San Luis, Argentina. The LCPC was emplaced into a metasedimentary sequence, which reached their metamorphic climax under high-temperature/low-pressure conditions (∼680 ± 37 °C and 550 ± 120 MPa). The bodies of the LCPC are zoned by rocks of different compositions that represent ultrabasic, basic, intermediate and acidic trends (hornblendites, gabbros, tonalites, and granodiorites). Their petrological, mineralogical and geochemical characteristics indicate that multiple processes formed them, mainly crystal fractionation of magnesian, calc-alkaline and metaluminous magmas (typical of arc), but also different assimilation degrees of crustal melts from the fusion of metawackes involving AFC processes. Fieldwork (structural and petrological data), the geochemical characteristics of rocks [eg. Low Sr/Y and (Gd/Yb)C-N], and thermobarometric results, indicate a relatively shallow fractionalization for the LCPC, which is in line with a pre-collisional arc stage linked to an extensive retro-arc setting for the Famatinian orogeny in Sierra de San Luis.

Ontogenetic development of the oral apparatus and oropharyngeal cavity in bullfrog tadpoles (Lithobates catesbeianus, Shaw 1802)

ObjectiveThe present study aimed to describe the morphology of oral apparatus and oral cavity of bullfrog tadpoles during their development and metamorphosis. DesignThe oral apparatus and oropharyngeal cavity of tadpoles from hatching up to metamorphosis stage was dissected for further analysis. These structures were fixed in Karnovsky solution, afterwards in osmium tetroxide and metalized in palladium gold and electron-micrographed using the scanning electron microscope. ResultsThe development of oral apparatus started with the formation and keratinization of the jaw sheaths and labial teeth followed by the formation of marginal and sub-marginal papillae. Degeneration of oral apparatus and formation of mouth was observed during metamorphosis. From stage-42 (metamorphic climax) to stage-43, the jaw sheath and labial tooth rows were disappeared progressively while the size and number of labial papillae were decreased. At stage-44, mouth formation started with the development of anterior and posterior labium though the labial papillae were still present. At stage-45 and 46, mouth was already formed, being very similar to the adult and characterized by the progressive increase in size. ConclusionThe sequence of events that happen during the development of oral apparatus of Lithobates catesbeianus Shaw, 1802 tadpoles follows the same pattern as occur in other anuran species but metamorphic atrophy of the oral apparatus follows the sequence of morphogenesis.

A novel type of prolactin expressed in the bullfrog pituitary specifically during the larval period

Prolactin (PRL) is one of the major hormones that control amphibian metamorphosis. Recently, a PRL (PRL1B) gene that is different from the known PRL (PRL1A) gene has been found in the genomes of several amphibian species. In order to ascertain whether the PRL1B gene is expressed in the bullfrog (Rana catesbeiana) pituitary, cloning of cDNA encoding PRL1B in the pituitary of the premetamorphic bullfrog tadpole was attempted. The bullfrog PRL1B amino acid sequence predicted from the obtained cDNA showed 62% identity with those of Xenopus PRL1Bs that have been presumed from the genome sequences, whereas the sequence identity between bullfrog PRL1A and PRL1B was 48%. A molecular phylogenetic tree showed that bullfrog PRL1B is most appropriately grouped with amphibian PRL1Bs. Quantitative PCR analysis revealed that the mRNA expression levels of bullfrog PRL1B in the pituitary were high during pre- and prometamorphosis, sharply declined at metamorphic climax and became undetectable after metamorphosis. In contrast, PRL1A mRNA levels were relatively low during pre- and prometamorphosis, rose at climax and remained high after metamorphosis. Immunohistochemical study using antibodies against partial peptides of PRL1A and PRL1B revealed that most of the PRL1A- and PRL1B-immunoreactive cells in the larval pituitary were distributed separately, but that some of the cells immunoreactive with both antibodies were also present. Western blot analysis with the larval pituitary extract indicated that PRL1B-immunoreactive band appeared at the position of molecular weight ca. 22.1 kDa and PRL1A-immunoreactive band at the position of ca. 22.8 kDa. The results obtained in this experiment suggest the possibility that PRL1B plays as-yet-unknown role(s) during the pre-climactic period of metamorphosis. This is the first report on the existence of PRL1B as a protein in the amphibian larval pituitary.

Expression of hyaluronan synthases upregulated by thyroid hormone is involved in intestinal stem cell development during Xenopus laevis metamorphosis.

During amphibian intestinal remodeling, thyroid hormone (TH) induces adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian one. We have previously shown that hyaluronan (HA) is newly synthesized and plays an essential role in the development of the stem cells via its major receptor CD44 in the Xenopus laevis intestine. We here focused on HA synthase (HAS) and examined how the expression of HAS family genes is regulated during natural and TH-induced metamorphosis. Our quantitative RT-PCR analysis indicated that the mRNA expression of HAS2 and HAS3, but not that of HAS1 and HAS-rs, a unique Xenopus HAS-related sequence, is upregulated concomitantly with the development of adult epithelial primordia consisting of the stem/progenitor cells during the metamorphic climax. In addition, our in situ hybridization analysis indicated that the HAS3 mRNA is specifically expressed in the adult epithelial primordia, whereas HAS2 mRNA is expressed in both the adult epithelial primordia and nearby connective tissue cells during this period. Furthermore, by treating X. laevis tadpoles with 4-methylumbelliferone, a HA synthesis inhibitor, we have experimentally shown that inhibition of HA synthesis leads to suppression of TH-upregulated expression of leucine-rich repeat-containing G protein-coupled 5 (LGR5), an intestinal stem cell marker, CD44, HAS2, HAS3, and gelatinase A in vivo. These findings suggest that HA newly synthesized by HAS2 and/or HAS3 is required for intestinal stem cell development through a positive feedback loop and is involved in the formation of the stem cell niche during metamorphosis.

Chronic effects of mercury on Bufo gargarizans larvae: Thyroid disruption, liver damage, oxidative stress and lipid metabolism disorder

Mercury is severely detrimental to organisms and is ubiquitous in both terrestrial and aquatic ecosystems. In the present study, we examined the effects of chronic mercury (Hg) exposure on metamorphosis, body size, thyroid microstructures, liver microstructural and ultrastructural features, and transcript levels of genes associated with lipid metabolism, oxidative stress and thyroid hormones signaling pathways of Chinese toad (Bufo gargarizans) tadpoles. Tadpoles were exposed to mercury concentrations at 0, 6, 12, 18, 24 and 30 µg/L from Gosner stage 26–42 of metamorphic climax. The present results showed that high dose mercury (24 and 30 µg/L) decelerated metamorphosis rate and inhibited body size of B. gargarizans larvae. Histological examinations have clearly exhibited that high mercury concentrations caused thyroid gland and liver damages. Moreover, degeneration and disintegration of hepatocytes, mitochondrial vacuolation, and endoplasmic reticulum breakdown were visible in the ultrastructure of liver after high dose mercury treatment. Furthermore, the larvae exposed to high dose mercury demonstrated a significant decrease in type II iodothyronine deiodinase (Dio2) and thyroid hormone receptor α and β (TRα and TRβ) mRNA levels. Transcript level of superoxide dismutase (SOD) and heat shock protein (HSP) were significantly up regulated in larvae exposed to high dose mercury, while transcript level of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was significantly down regulated. Moreover, exposure to high dose mercury significantly down regulated mRNA expression of carnitine palmitoyltransferase (CPT), sterol carrier protein (SCP), acyl-CoA oxidase (ACOX) and peroxisome proliferator-activated receptor α (PPAPα), but significantly up regulated mRNA expression of fatty acid elongase (FAE), fatty acid synthetase (FAS) and Acetyl CoA Carboxylase (ACC). Therefore, we conclude that high dose mercury induced thyroid function disruption, liver oxidative stress and lipid metabolism disorder by damaging thyroid and liver cell structures and altering the expression levels of relevant genes.

Evaluating Iodide Recycling Inhibition as a Novel Molecular Initiating Event for Thyroid Axis Disruption in Amphibians.

The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces available iodide for thyroid hormone synthesis, which leads to hormone insufficiency in tissues and subsequent negative developmental consequences. IYD activity is especially critical under conditions of lower dietary iodine and in low iodine environments. Our objective was to evaluate the toxicological relevance of IYD inhibition in a model amphibian (Xenopus laevis) used extensively for thyroid disruption research. First, we characterized IYD ontogeny through quantification of IYD mRNA expression. Under normal development, IYD was expressed in thyroid glands, kidneys, liver, and intestines, but minimally in the tail. Then, we evaluated how IYD inhibition affected developing larval X. laevis with an in vivo exposure to a known IYD inhibitor (3-nitro-l-tyrosine, MNT) under iodine-controlled conditions; MNT concentrations were 7.4-200 mg/L, with an additional 'rescue' treatment of 200 mg/L MNT supplemented with iodide. Chemical inhibition of IYD resulted in markedly delayed development, with larvae in the highest MNT concentrations arrested prior to metamorphic climax. This effect was linked to reduced glandular and circulating thyroid hormones, increased thyroidal sodium-iodide symporter gene expression, and follicular cell hypertrophy and hyperplasia. Iodide supplementation negated these effects, effectively rescuing exposed larvae. These results establish toxicological relevance of IYD inhibition in amphibians. Given the highly conserved nature of the IYD protein sequence and scarcity of environmental iodine, IYD should be further investigated as a target for thyroid axis disruption in freshwater organisms.

To eat or not to eat: ontogeny of hypothalamic feeding controls and a role for leptin in modulating life-history transition in amphibian tadpoles.

Many animal life histories entail changing feeding ecology, but the molecular bases for these transitions are poorly understood. The amphibian tadpole is typically a growth and dispersal life-history stage. Tadpoles are primarily herbivorous, and they capitalize on growth opportunities to reach a minimum body size to initiate metamorphosis. During metamorphic climax, feeding declines, at which time the gastrointestinal (GI) tract remodels to accommodate the carnivorous diet of the adult frog. Here we show that anorexigenic hypothalamic feeding controls are absent in the tadpole, but develop during metamorphosis concurrent with the production of the satiety signal leptin. Before metamorphosis there is a large increase in leptin mRNA in fat tissue. Leptin receptor mRNA increased during metamorphosis in the preoptic area/hypothalamus, the key brain region involved with the control of food intake and metabolism. This corresponded with an increase in functional leptin receptor, as evidenced by induction of socs3 mRNA and phosphorylated STAT3 immunoreactivity, and suppression of feeding behaviour after injection of recombinant frog leptin. Furthermore, we found that immunoneutralization of leptin in tadpoles at metamorphic climax caused them to resume feeding. The absence of negative regulation of food intake in the tadpole allows the animal to maximize growth prior to metamorphosis. Maturation of leptin-responsive neural circuits suppresses feeding during metamorphosis to facilitate remodelling of the GI tract.

Morphological and transcriptomic analyses reveal three discrete primary stages of postembryonic development in the common fire salamander, Salamandra salamandra.

The postembryonic development of amphibians has been characterized as divided into three predominant periods, hereafter named primary developmental stages: premetamorphosis (PreM), prometamorphosis (ProM), metamorphic climax (Meta), and completion of metamorphosis (PostM), largely based on examination of anuran development. Here, we categorized the postembryonic development of larvae of a poisonous fire salamander (Salamandra salamandra) by integrating morphology and gene expression (transcriptomic) data. Morphological analysis revealed three distinct clusters suggestive of PreM, ProM, and Meta, which were confirmed in parallel by microarray-derived gene expression analysis. In total, 3,510 probes targeted transcripts differentially expressed between the clusters we identified. Genes upregulated in PreM related to organogenesis, and those upregulated in Meta underlie structural proteins and related to development of anatomical structures and pigmentation. Biosynthesis pathways of pigments (pteridines and melanin) were upregulated during late ProM and Meta. Gas chromatographic analysis of alkaloids indicated the onset of steroidal alkaloid biosynthesis at ProM. When comparing gene expression in the fire salamander to that in other amphibians-three anurans, Xenopus laevis, X. tropicalis, and Michrohyla fissipes, and one caudate, Ambystoma mexicanum- we identified genes with conserved expression patterns involved in basic metamorphic processes such as skin restructuring and tail fin resorption. Our results support that primary stages of postembryonic development in caudates are homologous to those of anurans, and offer a baseline for the study of the evolution of developmental modes.

Structural and functional maturation of skin during metamorphosis in the Atlantic halibut (Hippoglossus hippoglossus).

To establish if the developmental changes in the primary barrier and osmoregulatory capacity of Atlantic halibut skin are modified during metamorphosis, histological, histochemical, gene expression and electrophysiological measurements were made. The morphology of the ocular and abocular skin started to diverge during the metamorphic climax and ocular skin appeared thicker and more stratified. Neutral mucins were the main glycoproteins produced by the goblet cells in skin during metamorphosis. Moreover, the number of goblet cells producing neutral mucins increased during metamorphosis and asymmetry in their abundance was observed between ocular and abocular skin. The increase in goblet cell number and their asymmetric abundance in skin was concomitant with the period that thyroid hormones (THs) increase and suggests that they may be under the control of these hormones. Several mucin transcripts were identified in metamorphosing halibut transcriptomes and Muc18 and Muc5AC were characteristic of the body skin. Na+, K+-ATPase positive (NKA) cells were observed in skin of all metamorphic stages but their number significantly decreased with the onset of metamorphosis. No asymmetry was observed between ocular and abocular skin in NKA cells. The morphological changes observed were linked to modified skin barrier function as revealed by modifications in its electrophysiological properties. However, the maturation of the skin functional characteristics preceded structural maturation and occurred at stage 8 prior to the metamorphic climax. Treatment of Atlantic halibut with the THs disrupter methimazole (MMI) affected the number of goblet cells producing neutral mucins and the NKA cells. The present study reveals that the asymmetric development of the skin in Atlantic halibut is TH sensitive and is associated with metamorphosis and that this barrier's functional properties mature earlier and are independent of metamorphosis.

Bisphenol F Disrupts Thyroid Hormone Signaling and Postembryonic Development in Xenopus laevis.

The safety of bisphenol A (BPA) alternatives has attracted much attention due to their wide use. In this study, we investigated the effects of bisphenol F (BPF), an alternative to BPA, on thyroid hormone (TH) signaling and postembryonic development in vertebrates using T3-induced and spontaneous Xenopus metamorphosis as models. We found that in the T3-induced metamorphosis assay, higher concentrations of BPF (100-10000 nM) antagonized T3-induced TH-response gene transcription and morphological changes including intestinal remodeling in a concentration-dependent manner, whereas 10 nM BPF exerted stimulatory effects on T3-induced integral metamorphosis when inhibited T3-induced TH-response gene transcription, demonstrating TH signaling disrupting effects of BPF. In the spontaneous metamorphosis assay, correspondingly, BPF inhibited development at metamorphic climax (with high endogenous TH levels), but promoted pre- and pro-metamorphic development (with low endogenous TH levels), displaying a developmental stage-dependent manner. Importantly, we observed agonistic actions of BPF on Notch signaling in intestines, showing that BPF disrupts vertebrate development possibly via multi pathways besides TH signaling. Thus, we infer the biphasic concentration-response relationship between BPF exposure and T3-induced metamorphosis could result from the interactions of TH signaling with other signaling pathways such as Notch signaling. Our study highlights the adverse influences of BPF on vertebrate development.

A novel stress hormone response gene in tadpoles of Xenopus tropicalis

Previous work identified a transcribed locus, Str. 34945, induced by the frog stress hormone corticosterone (CORT) in Xenopus tropicalis tails. Because thyroid hormone had no influence on its expression, Str. 34945 was dubbed the first “CORT-only” gene known from tadpoles. Here, we examine the genomic annotation for this transcript, hormone specificity, time course of induction, tissue distribution, and developmental expression profile. The location of Str. 34945 on the X. tropicalis genome lies between the genes ush1g (Usher syndrome 1G) and fads6 (fatty acid desaturase 6). A blast search showed that it maps to the same region on the X. laevis genome, but no hits were found in the human genome. Using RNA-seq data and conventional reverse transcriptase PCR and sequencing, we show that Str. 34945 is part of the 3′ untranslated region of ush1g. We find that CORT but not aldosterone or thyroid hormone treatment induces Str. 34945 in tadpole tails and that expression of Str. 34945 achieves maximal expression within 12–24 h of CORT treatment. Among tissues, Str. 34945 is induced to the highest degree in tail, with lesser induction in lungs, liver, and heart, and no induction in the brain or kidney. During natural metamorphosis, Str. 34945 expression in tails peaks at metamorphic climax. The role of ush1g in metamorphosis is not understood, but the specificity of its hormone response and its expression in tail make ush1g valuable as a marker of CORT-response gene induction independent of thyroid hormone.

Influence of desiccation threat on the metamorphic traits of the Asian common toad, Duttaphrynus melanostictus (Anura)

Phenotypic plasticity of metamorphic traits, in response to desiccation threat, was studied in Duttaphrynus melanostictus under laboratory conditions. Newly hatched Gosner stage 19 tadpoles were exposed to decreasing water levels (gradually or rapidly) up to the beginning of metamorphic climax (MC, Gosner stage 42). The control group was reared in unchanging water levels. The tadpoles experiencing desiccation threat reached MC earlier than those reared in constant water levels and metamorphosed (Gosner stage 46) at smaller body sizes. Time to reach MC was comparable between the groups of tadpoles experiencing a gradual or rapid decrease in water levels but their size at the completion of metamorphosis varied. They emerged at a significantly smaller size under rapid desiccation threat compared to the gradual desiccation threat. Impact on size at emergence was in proportion to the level of desiccation threat and this accelerated development and led to an early metamorphosis. The study shows the ability of D. melanostictus for developmental plasticity under adverse ecological conditions like the desiccation threat.

Organ Culture of the Xenopus Tadpole Intestine.

During Xenopus metamorphosis, most tadpole organs remodel from the larval to adult form to prepare for adaptation to terrestrial life. Organ culture serves as an important tool for studying larval-to-adult organ remodeling independent of the effects of other parts of the body. Here, I introduce a protocol for organ culture in vitro using the Xenopus laevis tadpole intestine before metamorphic climax. During culture in the absence of exogenous 3,3',5-triiodo-l-thyronine (T3), the most potent natural thyroid hormone, the intestine remains in its larval state without any metamorphic changes. In contrast, when T3 is added to the culture medium, the larval epithelium undergoes apoptosis, whereas adult stem cells appear, actively proliferate, and finally generate the differentiated adult epithelium within a week. At the same time, the surrounding nonepithelial tissues also develop. Thus, this culture model is useful for clarifying the control mechanisms of apoptosis in larval tissues, formation of adult stem cells, and cell proliferation and differentiation of adult tissues, all of which occur in harmony during natural metamorphosis. Moreover, a procedure for tissue recombination combined with organ culture provides a platform for investigating complex tissue interactions during organ remodeling. Such tissue recombination experiments will help to reveal the important role of nonepithelial tissues in larval epithelial apoptosis and/or adult stem cell development in the X. laevis intestine.

Chronic toxicity of 1,3,5-triazine herbicides in the postembryonic development of the western clawed frog Silurana tropicalis

Seven 1,3,5- triazine (s-triazine) herbicides (ametryn, prometryn, dimethametryn, simazine, atrazine, propazine, and cyanazine) were tested using an amphibian (Silurana tropicalis) metamorphosis assay focusing on morphometric, gravimetric, and thyroid-histological endpoints. Premetamorphic tadpoles were exposed to each s-triazine at 2 concentrations between 1/1000 and 1/10 of the 96-h acute toxicity values, until all tadpoles in the control group reached either the late prometamorphosic stages or the initial stage of metamorphic climax. All s-triazines tested induced significant retardation in growth and development at the higher concentrations (0.2–1.0mg/L), and some of them induced similar effects even at the lower concentrations (0.02–0.1mg/L) while each showing a linear dose-response. Total size of the thyroid glands tended to be reduced corresponding to the delayed development, but without showing histomorphological lesions typical of anti-thyroid chemicals. These consistent results suggest that the s-triazines can act as a chemical stressor inhibiting tadpole growth and development, possibly without disrupting the thyroid axis. In addition, tadpoles exhibiting spinal curvatures appeared in either one or both of the lower and higher concentration groups for each s-triazine tested. The incidence rate in the s-triazine exposure groups where tadpoles with scoliosis were observed ranged from 3.3% to 63.3%, some of which were significantly higher than that in the respective control groups (0–6.7%). It is speculated that the s-triazines may promote to occur axial malformations in developing tadpoles.

Locus Mapping, Molecular Cloning, and Expression Analysis of rps6kb2, a Novel Metamorphosis-Related Gene in Chinese Tongue Sole (Cynoglossus semilaevis).

Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. This unique process involves eye migration, a 90° rotation in posture, and asymmetrical pigmentation for adaptation to a benthic lifestyle. In the present study, we used genetics to map a metamorphosis-related locus (q-10M) in the male linkage group (LG10M), a small interval of 0.9cM corresponding to a 1.8M-bp physical area in chromosome 9 in the Chinese tongue sole (Cynoglossus semilaevis). Combined with single-marker analysis, ribosomal protein S6 kinase 2 (rps6kb2) a member of the family of AGC kinases was identified as a novel metamorphosis-related candidate gene. Its expression pattern during metamorphosis was determined by quantitative RT-PCR and whole-mount in situ hybridization analysis. rps6kb2 gene was significantly expressed in metamorphic climax stage larvae and distributed in all the tissues transforming during metamorphosis, including tail, jaw, eye and skin of larvae. The results suggest that rps6kb2 has a general role in tissue transformations during flatfish metamorphosis including tail changes, skull remodeling, eye migration, and asymmetrical pigmentation.

Genotoxic and mutagenic effects of Atrazine Atanor 50 SC on Dendropsophus minutus Peters, 1872 (Anura: Hylidae) developmental larval stages

The potential mutagenic and genotoxic effects of the herbicide atrazine were investigated in different developmental stages of Dendropsophus minutus tadpoles. These animals were exposed to 4 nominal concentrations of atrazine (2.25, 4.5, 9, and 18 mg/L) and 40 mg/L of Cyclophosphamide as a positive control, for 96 h. Negative controls were also added to the experiment. The tadpoles were divided into three groups according to Gosner's developmental stages, namely GS 25-33 as premetamorphic, GS 36-39 as prometamorphic, and GS 42-43 as metamorphic climax. Our results showed that the premetamorphic and metamorphic stages were more sensitive than the prometamorphic stage to the herbicide. A comet assay and micronucleus test for the sensitive stages demonstrated DNA damage in a concentration-dependent curve. Although a dose-response effect was not observed for the prometamorphic stage, a statistically significant difference was found between the treatment of 18 mg/L and the negative control. Moreover, the highest concentration of atrazine showed both the largest amount of DNA damage and the highest micronucleus frequency regardless of the developmental stage of D. minutus. In conclusion, atrazine was genotoxic and mutagenic for D. minutus in a dose-sensitive manner, dependent on larval developmental stages. Considering the prometamorphic stages showed no dose-response effect to atrazine, we suggest caution when using this stage in biomonitoring studies in order to avoid false negative results. Amphibians have been proven to be useful bioindicators, and we suggest replicating biomonitoring studies using different species to represent ecosystems' environmental impacts.

Leptin Induces Mitosis and Activates the Canonical Wnt/β-Catenin Signaling Pathway in Neurogenic Regions of Xenopus Tadpole Brain.

In addition to its well-known role as an adipostat in adult mammals, leptin has diverse physiological and developmental actions in vertebrates. Leptin has been shown to promote development of hypothalamic circuits and to induce mitosis in different brain areas of mammals. We investigated the ontogeny of leptin mRNA, leptin actions on cell proliferation in the brain, and gene expression in the preoptic area/hypothalamus of tadpoles of Xenopus laevis. The level of leptin mRNA was low in premetamorphic tadpoles, but increased strongly at the beginning of metamorphosis and peaked at metamorphic climax. This increase in leptin mRNA at the onset of metamorphosis correlated with increased cell proliferation in the neurogenic zones of tadpole brain. We found that intracerebroventricular (i.c.v.) injection of recombinant Xenopus leptin (rxLeptin) in premetamorphic tadpoles strongly increased cell proliferation in neurogenic zones throughout the tadpole brain. We conducted gene expression profiling of genes induced at 2 h following i.c.v. injection of rxLeptin. This analysis identified 2,322 genes induced and 1,493 genes repressed by rxLeptin. The most enriched Kyoto Encyclopedia of Genes and Genomes term was the canonical Wnt/β-catenin pathway. Using electroporation-mediated gene transfer into tadpole brain of a reporter vector responsive to the canonical Wnt/β-catenin signaling pathway, we found that i.c.v. rxLeptin injection activated Wnt/β-catenin-dependent transcriptional activity. Our findings show that leptin acts on the premetamorphic tadpole brain to induce cell proliferation, possibly acting via the Wnt/β-catenin signaling pathway.

Histopathological changes and lipid metabolism in the liver of Bufo gargarizans tadpoles exposed to Triclosan

In the current study, the adverse effects of TCS on liver health of B. gargarizans tadpoles were assessed. B. gargarizans larvae were exposed to TCS at 0, 10, 30, 60, and 150 μg L−1 from Gosner stage 3 until metamorphic climax. The hepatosomatic index (HSI), hepatic histological and ultrastructural features, and transcript levels of genes associated with detoxification and oxidative stress as well as lipid metabolism in the livers were determined. Exposure to 150 μg L−1 TCS resulted in increased HSI of tadpoles at metamorphic climax. Histological changes characterized by an increase in the number of melanomacrophage, nucleus pyknosis, and deposition of collagen fibers were observed in liver at 60 and 150 μg L−1 TCS. Moreover, marked ultrastructural alterations including high electron dense in mitochondrial matrix and lipid accumulation were also observed. In addition, abundances of transcripts of Cu/Zn superoxide dismutase (SOD), phospholipid hydroperoxide glutathione peroxidase (PHGPx), and heat shock protein 90 (HSP90) were decreased in larvae exposed to 60 and 150 μg L−1 TCS, while transcript level of HSP90 was increased at 30 μg L−1 TCS. Also, abundances of transcripts of acetyl-CoA carboxylase (ACC), carnitine palmitoyltransferase 2 (CPT2), peroxisome proliferator-activated receptor alpha (PPARa), fatty acid elongase 1 (FAE), sterol carrier protein 2 (SCP) were significantly lesser in larvae exposed to 60 and 150 μg L−1 TCS. Overall, TCS at high levels induced histopathological changes in the liver of B. gargarizans tadpoles. This might have been due to the alteration of oxidative stress–related genes and lipid metabolism-related genes expression levels.