The plant-derived triterpenoid, cucurbitacin B, but not cucurbitacin E, inhibits the developmental transition associated with ecdysone biosynthesis in Drosophila melanogaster.

In insects, some sterols are essential not only for cell membrane homeostasis, but for biosynthesis of the steroid hormone ecdysone. Dietary sterols are required for insect development because insects cannot synthesize sterols de novo. Therefore, sterol-like compounds that can compete with essential sterols are good candidates for insect growth regulators. In this study, we investigated the effects of the plant-derived triterpenoids, cucurbitacin B and E (CucB and CucE) on the development of the fruit fly, Drosophila melanogaster. To reduce the effects of supply with an excess of sterols contained in food, we reared D. melanogaster larvae on low sterol food (LSF) with or without cucurbitacins. Most larvae raised on LSF without supplementation or with CucE died at the second or third larval instar (L2 or L3) stages, whereas CucB-administered larvae mostly died without molting. The developmental arrest caused by CucB was partially rescued by ecdysone supplementation. Furthermore, we examined the effects of CucB on larval-prepupal transition by transferring larvae from LSF supplemented with cholesterol to that with CucB just after the L2/L3 molt. L3 larvae raised on LSF with CucB failed to pupariate, with a remarkable developmental delay. Ecdysone supplementation rescued the developmental delay but did not rescue the pupariation defect. Furthermore, we cultured the steroidogenic organ, the prothoracic gland (PG) of the silkworm Bombyx mori, with or without cucurbitacin. Ecdysone production in the PG was reduced by incubation with CucB, but not with CucE. These results suggest that CucB acts not only as an antagonist of the ecdysone receptor as previously reported, but also acts as an inhibitor of ecdysone biosynthesis.

Metamorphic acceleration following the exposure to lithium and selenium on American bullfrog tadpoles (Lithobates catesbeianus).

To regulate the presence of contaminants in Brazilian water, the Brazilian Environmental Council (CONAMA) promulgates regulations regarding the concentrations of given compounds that are supposed to be safe for aquatic life. Considering these regulations, this study tested the effects of considered safe levels of lithium (2.5 mgL-1) and selenium (0.01 mgL-1), isolated and mixed, on the American bullfrog (Lithobates catesbeianus) tadpoles. The evaluation was done through the use of biomarkers of larval development as total wet weight (TWW), snout-vent-length (SVL), hind-limb-length (HLL), activity level (AL), histologic evaluation of the thyroid gland and the mortality rate. The tadpoles were allocated into four groups (n = 20 each): a control group (CT); a group exposed to lithium (LI), a group exposed to selenium (SE), and a group exposed to both lithium and selenium (SELI). The whole assay was carried out over 21 days, with two rounds of data collection (on 7th and 21st day) to evaluate the responses over time. A statistical reduction in the AL was observed in the tadpoles from the LI and SELI groups after 7 days of exposure, the same pattern was observed after 21 days. Histological analyses of the thyroid gland showed signs of up-regulation (i.e. statistic reduction in number and area of the follicles, as well a significant reduction in the area of the gland) in all exposed groups, which represents an endocrine response as an adaptative strategy to deal with polluted aquatic environment. The stress triggered by the polluted medium is discussed.

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.

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 ß (TRα and TRß) 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.

Larvicidal efficacy of Adiantobischrysene from Adiantum latifolium against Oryctes rhinoceros through disrupting metamorphosis and impeding microbial mediated digestion.

BACKGROUND: Oryctes rhinoceros Linn. (Coleoptera: Scarabaeidae) is a serious pest of coconuts and other palms. Symbiotic gut bacteria play significant roles in the digestion of cellulosic materials as well as in some other physiological processes essential for the existence of O. rhinoceros larvae. The study was undertaken to isolate a compound with antibacterial and larvicidal activities from the leaves of Adiantum latifolium Lam. following a bioassay-guided method. RESULTS: Methanol extract (ME) of dry leaf powder of A. latifolium showed larvicidal activity against third-instar O. rhinoceros (LD50 , 5018 mg/kg) with antibacterial activity on its gut microbiota. An in vitro study showed the bacteria Bacillus cereus, Micrococcus lylae, Stenotrophomonas maltophilia, Kocuria rosea, Burkholderia mallei, Staphylococcus epidermidis, S. arlettae and Corynebacterium afermentans identified from the larval gut were sensitive to ME. Bioactivity-guided isolation of the compound by liquid-liquid extraction and column chromatography resulted in Adiantobischrysene which showed antibacterial and larvicidal activity (LD50 , 8.4 mg/kg) and led to weight loss and precocious metamorphosis in larvae. An enzyme immunoassay showed a large peak in 20-hydroxyecdysone that commits larvae to precocious metamorphosis. CONCLUSION: This study demonstrated that the antibacterial and metamorphosis disrupting activity of Adiantobischrysene make it a natural pesticidal compound against O. rhinoceros. © 2018 Society of Chemical Industry.

Dietary L-arginine accelerates pupation and promotes high protein levels but induces oxidative stress and reduces fecundity and life span in Drosophila melanogaster.

L-Arginine, a precursor of many amino acids and of nitric oxide, plays multiple important roles in nutrient metabolism and regulation of physiological functions. In this study, the effects of L-arginine-enriched diets on selected physiological responses and metabolic processes were assessed in Drosophila melanogaster. Dietary L-arginine at concentrations 5-20 mM accelerated larval development and increased body mass, and total protein concentrations in third instar larvae, but did not affect these parameters when diets contained 100 mM arginine. Young (2 days old) adult flies of both sexes reared on food supplemented with 20 and 100 mM L-arginine possessed higher total protein concentrations and lower glucose and triacylglycerol concentrations than controls. Additionally, flies fed 20 mM L-arginine had higher proline and uric acid concentrations. L-Arginine concentration in the diet also affected oxidative stress intensity in adult flies. Food with 20 mM L-arginine promoted lower protein thiol concentrations and higher catalase activity in flies of both sexes and higher concentrations of low molecular mass thiols in males. When flies were fed on a diet with 100 mM L-arginine, lower catalase activities and concentrations of protein thiols were found in both sexes as well as lower low molecular mass thiols in females. L-Arginine-fed males demonstrated higher climbing activity, whereas females showed higher cold tolerance and lower fecundity, compared with controls. Food containing 20 mM L-arginine shortened life span in both males and females. The results suggest that dietary L-arginine shows certain beneficial effects at the larval stage and in young adults. However, the long-term consumption of L-arginine-enriched food had unfavorable effects on D. melanogaster due to decreasing fecundity and life span.

Indoles induce metamorphosis in a broad diversity of jellyfish, but not in a crown jelly (Coronatae).

Many animals go through one or more metamorphoses during their lives, however, the molecular underpinnings of metamorphosis across diverse species are not well understood. Medusozoa (Cnidaria) is a clade of animals with complex life cycles, these life cycles can include a polyp stage that metamorphoses into a medusa (jellyfish). Medusae are produced through a variety of different developmental mechanisms-in some species polyps bud medusae (Hydrozoa), in others medusae are formed through polyp fission (Scyphozoa), while in others medusae are formed through direct transformation of the polyp (Cubozoa). To better understand the molecular mechanisms that may coordinate these different forms of metamorphosis, we tested two compounds first identified to induce metamorphosis in the moon jellyfish Aurelia aurita (indomethacin and 5-methoxy-2-methylindole) on a broad diversity of medusozoan polyps. We discovered that indole-containing compounds trigger metamorphosis across a broad diversity of species. All tested discomedusan polyps metamorphosed in the presence of both compounds, including species representatives of several major lineages within the clade (Pelagiidae, Cyaneidae, both clades of Rhizostomeae). In a cubozoan, low levels of 5-methoxy-2-methylindole reliably induced complete and healthy metamorphosis. In contrast, neither compound induced medusa metamorphosis in a coronate scyphozoan, or medusa production in either hydrozoan tested. Our results support the hypothesis that metamorphosis is mediated by a conserved induction pathway within discomedusan scyphozoans, and possibly cubozoans. However, failure of these compounds to induce metamorphosis in a coronate suggests this induction mechanism may have been lost in this clade, or is convergent between Scyphozoa and Cubozoa.

Dietary Accumulation of Inorganic Selenium by a Larval Amphibian (Hyla chrysoscelis) and Influence on Accumulation of Background Mercury.

Larval gray tree frogs (Hyla chrysoscelis) were exposed to inorganic Se (SeO2) added to the diet (10.2 and 86.3 ug/g dw) and monitored for accumulation and effect. Background concentrations of Hg were also measured in food and carcasses to assess possible effects of Se on Hg accumulation. Selenium was accumulated in a dose dependent manner, and life stages did not differ. No effects of Se exposure were observed on survival, growth, or time to metamorphosis. Mercury concentrations in carcasses, resulting from background concentrations in food, were significantly affected by the presence of Se. In the high Se treatment, Hg concentrations were significantly decreased relative to those in the low Se treatment and the control. Our study suggests that exposure to inorganic Se as SeO2 at the concentrations tested do not elicit adverse biological effects, but exposure to relatively high concentrations of Se may reduce accumulation of Hg from food.

Bioaccumulation and Biodistribution of Selenium in Metamorphosing Tadpoles.

Selenium is an important macronutrient with a very narrow margin between essentiality and toxicity. Amphibians are hypothesized to be particularly sensitive due to the potential for metamorphosis-driven mobilization, which could transfer or concentrate contaminant burdens within specific organs. We explored the potential role of tissue degeneration and remodeling during anuran metamorphosis as a mechanism for altering tissue-specific Se burdens. Limnodynastes peronii tadpoles were exposed to dissolved 75Se (as selenite) for 7 days and depurated until completion of metamorphosis. Bioaccumulation and retention kinetics were assessed in whole tadpoles and excised tissues using gamma spectroscopy, and temporal changes in biodistribution were assessed using autoradiography. Tadpoles retained Se throughout metamorphosis, and partitioned the element predominantly within digestive and excretory tissues, including livers > mesonephros > guts > gallbladder. Importantly, our results demonstrate that Se biodistribution varies significantly throughout development. This is indicative of tissue transference, and particularly in tissues developing de novo after depuration. To the best of our knowledge, this is the first study demonstrating Se transference during metamorphic tissue remodelling. Further research is warranted to explore the fate and metabolism of Se (and other metal and metalloids) during anuran development and the implications of transference for influencing toxicity.

Effects of the herbicide triclopyr on metamorphic northern red-legged frogs.

Aquatic herbicides are used to manage invasive emergent plants in and around wetlands. Metamorphic frogs that emerge during the aquatic weed management season may be at risk of herbicide exposure. Metamorphic northern red-legged frogs (Rana aurora) were exposed to a triclopyr tank mix labeled for control of broadleaf emergent aquatic weeds such as invasive purple loosestrife (Lythrum salicaria). The tank mix consisted of Renovate® 3 (triclopyr triethylamine salt 44.4%), the modified vegetable oil surfactant Competitor®, and the marker dye Hi-Light® . Metamorphs were exposed to the tank mix and a clean-water control for 96 h, and then reared in clean water for 60 d. Exposure to the tank mix resulted in no treatment-related mortalities, no effects on behavior immediately post exposure, and no effects on body or liver condition indices. Exposure to the tank mix resulted in lethargy during exposure and a 1-d delay in completion of metamorphosis. Deformities present in the rearing population confounded results for some endpoints. Observed effects were minimal, especially compared with the potential for ecological impacts from unmanaged invasive plants. Environ Toxicol Chem 2017;36:2316-2326. © 2017 SETAC.

A morphometric and molecular study of the apoptosis observed on tadpoles' tail explants under the exposition of triiodothyronine in different homeopathic dilutions.

BACKGROUND: As a therapeutic system, homeopathy is supported by: i) similitude and experimentation in healthy individuals, ii) potentization. A challenge for researchers consists in looking for signals in water (or vehicle) to explain the storage of information in extremely high dilutions and the transfer of such information to the living systems. Anuran amphibian metamorphosis is controlled by thyroid hormones (TH), including the resorption of the tadpole tail. Apoptosis is a genetically regulated form of cell death that can be triggered by various extracellular and intracellular stimuli resulting in coordinated activation of a family of cysteine proteases called caspases. METHODS: This study was blind and randomized. It performed in three stages: I) the identification of the most effective T3 homeopathic dilution to induce apoptotic reactions in Rana (Lithobates) catesbeianus tadpole tail explants stimulated by T3 in substantial, II) study of different controls and III) detection in explants under the action of the most effective dilution of T3, as established in Stage I. RESULTS: There was no statistically significant difference between tail macroscopic dimensions between the groups. T3 10cH decreased the expression of caspase 3/7 mRNA, in explants treated with T3 20 nM. CONCLUSION: The present experiment is in agreement with the hypothesis that T3, at a 10cH homeopathic dilution, changes the metamorphosis molecular network.

Effects of Elevated In Ovo Selenium Exposure on Late Stage Development of Xenopus laevis Tadpoles.

The effects of elevated in ovo selenium (Se) exposure on late stage larval Xenopus laevis development were investigated. Adult females were fed diets augmented with selenomethionine for 68 days and bred with untreated males to obtain embryos with measured concentrations of 1.6 (control), 10.8, 28.1 and 81.7 µg Se/g dry mass. Embryos were reared under uncontaminated conditions until 50 % of individuals within an aquarium had completed metamorphosis. The highest in ovo Se exposure group exhibited greater froglet body mass and snout to vent length while having a higher proportion of tadpoles at earlier stages of development. No differences were detected among treatment groups for mortality or metamorphic timing during the rearing period. This research suggests that in ovo Se exposure has minimal effect on the survival and development of late stage larval X. laevis in a controlled laboratory environment with adequate food availability.

Amphibians and ultra high diluted thyroxine--further experiments and re-analysis of data.

BACKGROUND: A model of thyroxine and metamorphosis of highland amphibians is frequently mentioned as an example of experiments on extremely diluted substances in discussions around 'homeopathy'. METHODS: The model was scrutinized by reanalysing the results of the initial researcher A and a second researcher B as well as of 5 external researchers C between 1990 and 2013. Rana temporaria larvae were taken from an alpine highland biotope. The test solution was thyroxine 10(-30) (T30x), tetra-iodo-thyronine sodium pentahydrate diluted with pure water in 26 steps of 1:10, being agitated after each step. Analogously prepared water (W30x) was used for control. Tadpoles were observed from the 2-legged to the 4-legged stage. Experiments were performed in different years, at different times of season, and their duration could vary. Frequencies of 4-legged animals, effect sizes and areas under the curves (AUCs) were calculated and regression analyses were performed to investigate possible correlations between year, season, duration etc. Experiments were in line with animal protection guidelines. RESULTS: The total set of data A + B + C as well as subsets A (initial researcher, N=286+293), B (second centre, 965 + 965) and C (5 external researchers, 690 + 690) showed an effect of extremely diluted agitated thyroxine reverse to that known of molecular thyroxin, i.e. test values were below control by 11.4% for A, 9.5% for B and 7.0% for C (p<0.001 for each of the subsets). The effect size (Cohen's d) was >0.8 (large) for both A and B and 0.74 (medium) for C. CONCLUSION: Although a perfect reproducibility was not obtained, this paradoxical phenomenon was generally consistent in different observations. Correlations were found between details of laboratory handling, as well as environment temperature, and the size of the results.

Bisphenol A affects larval growth and advances the onset of metamorphosis in Drosophila melanogaster.

Exposure to Bisphenol A (BPA) has been reported to dysregulate endocrine pathways in a wide array of vertebrate species. The effects of BPA on invertebrate species are less well understood. We tested the effects of BPA on growth and development in Drosophila as these processes are governed by well-studied endocrine pathways. In this study, we tested the effects of three concentrations of BPA (0.1mg/L, 1mg/L or 10mg/L) and found a statistically significant increase in larval growth for the low dose treatment group (0.1mg/L), but not statistically significant for the high dose treatment group (10mg/L). BPA exposure resulted in an increased body size in treated animals at 48, 72 and 96h after egg laying (AEL). This finding reflects a non-monotonic dose-response that has been observed for an increasing number of endocrine disrupting compounds. The increase in growth rate found for all treatment groups was associated with a statistically significant increase in food intake observed at 72h AEL. Furthermore, we observed that the increased growth rate was coupled with an earlier onset of pupariation consistent with previously reported phenotypes resulting from increased activity of insulin/insulin growth factor signaling (IIS) in Drosophila. Since the timing of the onset of pupariation in Drosophila is controlled through the complex interaction of the IIS and the ecdysone signaling pathways, our findings suggest that BPA exerts its effects through disruption of endocrine signaling in Drosophila.

Analysis of gene expression in Homarus americanus larvae exposed to sublethal concentrations of endosulfan during metamorphosis.

Agricultural pesticide runoff has been suspected as the cause of numerous fish kills in rivers throughout Prince Edward Island but the impact on the surrounding marine environment is unknown. Endosulfan, an organochlorine pesticide, is a potent neurotoxin and molt inhibitor used to combat the Colorado potato beetle however it has the potential to affect non-target organisms including the American lobster (Homarus americanus). Metamorphosis is a critical stage of development and the effects of contaminant exposure during this time are largely unknown in lobster. A 14day endosulfan exposure was performed to identify the effects on survival, development and gene expression in lobster larvae during metamorphosis; all of which were predicted to be negatively impacted. The higher endosulfan concentrations resulted in greater mortality and a significant increase in the number of days required to reach metamorphosis in surviving animals. A custom made H. americanus microarray was used for monitoring the changes in expression of 14,592 genes at the termination of the exposure. Genes with >1.5 fold change and identified as being significant at p<0.05 using one-way ANOVA were selected for further analysis. A total of 707 genes were identified as being significantly differentiated, however with only ~40% annotation of the array, the majority of these genes were unknown. Annotated genes of interest were involved in many processes: development, metabolism, immunity and oxidative stress response and gene regulation. Nine genes of interest (histone H1, farnesoic acid O-methyltransferase, cuticle protein, glutathione S-transferase, thioredoxin, NADH dehydrogenase, ecdysone nuclear receptor Fushi tarazu F1 (FTZ-F1), ferritin and ecdysone inducible protein E75 (EIP-E75)) were selected for RT-qPCR validation of the microarray results. The RT-qPCR method was more sensitive than the microarray yet detected similar expression patterns. The two highest endosulfan concentrations resulted in increased mortalities, developmental delays in reaching metamorphosis and significant changes in gene expression. This research provides a foundation for using microarray gene expression profiles as screening tools for exploring the impact of environmental contaminants on lobster.

Taurine and fish development: insights for the aquaculture industry.

Expansion of the aquaculture industry is limited by incomplete knowledge on fish larval nutritional requirements. Nevertheless, it is believed that dietary taurine deficiencies may be particularly critical for fish larvae. The reasons include the high taurine levels found during egg and yolk-sac stages of fish, suggesting that taurine may be of pivotal importance for larval development. Moreover, unlike aquaculture feeds, natural preys of fish larvae contain high taurine levels, and dietary taurine supplementation has been shown to increase larval growth in several fish species. This study aimed to further explore the physiological role of taurine during fish development. Firstly, the effect of dietary taurine supplementation was assessed on growth of gilthead sea bream (Sparus aurata) larvae and growth, metamorphosis success and amino acid metabolism of Senegalese sole (Solea senegalensis) larvae. Secondly, the expression of taurine transporter (TauT) was characterised by qPCR in sole larvae and juveniles. Results showed that dietary taurine supplementation did not increase sea bream growth. However, dietary taurine supplementation significantly increased sole larval growth, metamorphosis success and amino acid retention. Metamorphosis was also shown to be an important developmental trigger to promote taurine transport in sole tissues, while evidence for an enterohepatic recycling pathway for taurine was found in sole at least from juvenile stage. Taken together, our studies showed that the dependence of dietary taurine supplementation differs among fish species and that taurine has a vital role during the ontogenetic development of flatfish, an extremely valuable group targeted for aquaculture production.

Developmental exposure to fluoxetine modulates the serotonin system in hypothalamus.

The selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLU, Prozac®) is commonly prescribed for depression in pregnant women. This results in SSRI exposure of the developing fetus. However, there are knowledge gaps regarding the impact of SSRI exposure during development. Given the role of serotonin in brain development and its cross-talk with sex hormone function, we investigated effects of developmental exposure to pharmacologically relevant concentrations of FLU (3 and 30 nM (measured)) on brain neurotransmitter levels, gonadal differentiation, aromatase activity in brain and gonads, and the thyroid system, using the Xenopus tropicalis model. Tadpoles were chronically exposed (8 weeks) until metamorphosis. At metamorphosis brains were cryosectioned and levels of serotonin, dopamine, norepinephrine, and their metabolites 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were measured in discrete regions (telencephalon, hypothalamus and the reticular formation) of the cryosections using high-performance liquid chromatography. Exposure to 30 nM FLU increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus compared with controls. FLU exposure did not affect survival, time to metamorphosis, thyroid histology, gonadal sex differentiation, or aromatase activity implying that the effect on the serotonergic neurotransmitter system in the hypothalamus region was specific. The FLU concentration that impacted the serotonin system is lower than the concentration measured in umbilical cord serum, suggesting that the serotonin system of the developing brain is highly sensitive to in utero exposure to FLU. To our knowledge this is the first study showing effects of developmental FLU exposure on brain neurochemistry. Given that SSRIs are present in the aquatic environment the current results warrant further investigation into the neurobehavioral effects of SSRIs in aquatic wildlife.

Specific time of exposure during tadpole development influences biological effects of the insecticide carbaryl in green frogs (Lithobates clamitans).

The orchestration of anuran metamorphosis is initiated and integrated by thyroid hormones, which change dynamically during larval development and which may represent a target of disruption by environmental contaminants. Studies have found that some anurans experience increased rates of development when exposed to the insecticide carbaryl later in larval development, suggesting that this insecticide could affect thyroid hormone-associated biological pathways. However, the time in development when tadpoles are sensitive to insecticide exposure has not been clearly defined nor has the mechanism been tested. In two separate studies, we exposed recently hatched green frog (Lithobates clamitans) tadpoles to a single, three day carbaryl exposure in the laboratory at either 2, 4, 8, or 16 weeks post-hatching. We examined the impact of carbaryl exposure on mRNA abundance patterns in the brains of frogs following metamorphosis months after a single three day exposure (experiment 1) and in tadpole tails three days after exposure (experiment 2) using cDNA microarrays and quantitative real time polymerase chain reaction (QPCR) analyses. For tadpoles reared through metamorphosis, we measured tadpole growth and development, as well as time to, mass at, and survival to metamorphosis. Although carbaryl did not significantly impact tadpole development, metamorphosis, or survival, clear exposure-related alterations in both tail and brain transcript levels were evident when tadpoles were exposed to carbaryl, particularly in tadpoles exposed at weeks 8 and 16 post-hatching, indicating both short-term and long-term alterations in mRNA expression. These results indicate that carbaryl can have long-lasting effects on brain development when exposure occurs at sensitive developmental stages, which may have implications for animal fitness and function later in the life cycle.

Replication of an experiment on extremely diluted thyroxine and highland amphibians.

INTRODUCTION: The purpose of this study was to reproduce an experiment with diluted thyroxine and amphibians. A detailed account of the difficulties of this line of research has been published by the initial team (Endler and Scherer-Pongratz). One experiment which has been reported reproducible by the initial team and independent researchers is the effect of extremely diluted agitated thyroxine (T30x) versus analogously prepared water (W30x) in amphibians from biotopes above the tree line (highland amphibians). METHODS: (A) The author replicated the experiment. Rana temporaria were taken from an alpine biotope and the methods given in the original protocols were followed. Animals were treated from the 2-legged stage on. (B), the author reanalyzed the results reported by the initial team and by independent researchers (van Wijk, Lassnig, Zausner-Lukitsch, Bach, Harrer). RESULTS: (A) In the author's own experiment, there was a clear trend of T30x animals developing more slowly (i.e. up to 6 h within 3 days) than W30x animals. This is in line with the previous experiments. Due to small numbers of animals, the differences in the frequency of larvae reaching the 4-legged stage and the stage with reduced tail were not statistically significant (p > 0.05). The effect size was large (d > 0.08). (B) In the analysis of all available data with regard to the 4-legged stage, pooled T30x values from the initial team were 10.1% smaller than W30x values (100%) and pooled T30x values from the 5 independent researchers were 12.4% smaller (p < 0.01 and d > 0.08). Analogously, the number of animals entering the juvenile stage with reduced tail was smaller for T30x than for W30x.