Effects of atrazine on thyroid hormone induction of metamorphosis in the axolotl (Ambystoma mexicanum) Pam Clarkson My name is Pam Clarkson and I’m a sophomore at Minot State and have been involved with the AGP (or “Amphibian Growth Project”) for three semesters. The AGP is devoted (1) to understanding the factors that underlie and influence amphibian growth and development, and (2) developing amphibians as model organisms in environmental and biomedical research.   Specifically, I’ll be discussing data that we collected in order to test the hypothesis that atrazine, perhaps the world’s most commonly used herbicide, influences the development of the salamander.

ENDOCRINE DISRUPTION ATRAZINE -- Post application (Spring) runoff can be > 20 ppb -- typical surface and groundwater concentrations at other times during other times of the year, 0.001 to 1.0 ppb -- 3 ppb EPA standard for drinking water Atrazine is a known endocrine disruptive agent. It is applied to control weeds in farm fields, especially in corn, sorghum and soybean plots. At application, the dosage levels are very concentrated, with the result that the source of water for amphibian breeding ponds, spring runoff, can have very high concentrations of atrazine.   These high concentrations are usually far in excess of EPA standards for drinking water. In what has become a famous, and controversial, result, Tyrone Hayes and his lab found that tadpole (or larval) Xenopus immersed in atrazine solution can develop hermaphroditism at concentrations as low of 0.1 ppb. This is 30 times lower than the EPA drinking water standard. As seen in the figure on the right, serial section histological examination of the gonads of a recently metamorphosed Xenopus at 0.1 ppb atrazine exposure show both development of ovaries and testes, both of which turn out to be non-functional by later histological inspection. These effects appear to be restricted to the males; it has since been hypothesized that atrazine facilitates the conversion of testosterone to estradiol. Subsequent analyses in other labs using Xenopus and other species of frog have been mixed. Because sexual differentiation is under endocrine control, we hypothesized that atrazine will affect metamorphic development and larval growth, two systems that are also under endocrine control. Males exposed to 0.1 ppb have lowered testosterone, elevated estrogen, and intersex gonads Xenopus laevis Hayes et al, 2002, PNAS

Introduction Thyroid hormones induce metamorphosis in the axolotl We investigate affects of atrazine on TH-induction of metamorphosis in the axolotl We investigate affects of atrazine on growth The purpose of the experiment is threefold: Through exposure to thyroid hormones, we induce metamorphosis in axolotls. To determine whether or not atrazine affects metamorphosis in the axolotl, we expose the animals to TH and atrazine. We also want to know if atrazine affects growth and gonadal development.

Big Picture To begin characterization of gene expression during salamander metamorphosis Hypothesis: no variation in gene expression during metamorphosis For comparison to endocrine disrupted metamorphosis To correlate patterns of gene expression with morphological, developmental, and life history malformations during endocrine disruption Placed in a larger context, our work with salamanders is devoted to more deeply understanding how endocrine disruption works: first, to identify loci that are differentially expressed during xenobiotic exposure; and second, to correlate these genomic responses with the morphological and life history results, for example, feminization and delayed metamorphosis.   The genomic portion of this program is conducted with our collaborators at the University of Kentucky. The morphological and life history results are examined in our lab and this is the content of my talk: how atrazine influences the morphological expression of metamorphosis. To identify loci that will be useful in field diagnostics of normal and endocrine disrupted metamorphosis

paedomorphosis metamorphosis The tiger salamander is the only species native to ND. They can chose whether or not to undergo metamorphosis. Our laboratory model, the axolotl, are obligated to be paedomorphic, remaining in the larval state while attaining sexual maturity. However, metamorphosis can be induced by exposure to thyroid hormones. We use axolotls because they are the only species of salamander that reproduce in the lab and we have complete control over metamorphic timing. Conveniently, the axolotl and tiger salamanders are the nearest sister species to one another. The eggs hatch into larvae, which grow to sexually mature paedomorphs, which can then reproduce, or undergo metamorphosis, and then reproduce. metamorphosis

This is the design of a previously completed experiment This is the design of a previously completed experiment. The intent of this experiment was to use microarrays to provide a gene expression profile during TH-induction of metamorphosis in the axolotl. In this experiment, animals are grown for 200 days and then tissues were harvested after 0, 2, 12, and 24 days of exposure to 0, 5, or 50 nano Molar concentrations of thyroid hormone. We used this data, in collaboration with our partners at the University of Kentucky, to make a (the first and only salamander) microarray chip. After animation: This is the more complete design of the subsequent experiment. This experiment mirrored Page et al. while crossing with three levels of atrazine exposure. Atrazine exposure was for the full duration of the experiment. Sample sizes were seven per treatment with thirty treatments for a total of 210 animals. Tissues (tail, skin, brain, liver) were harvested and genomic analysis is being conducted with our collaborators at the University of Kentucky. I’m going to discuss the morphological results of this experiment. Page et al., 2007

Results Summary - Effect of atrazine on growth - Confirm standrard TH-induction - Effect of atrazine on metamorphosis Our results fall into three categories.   First, we tested the effect of atrazine on larval growth by examining axolotl mass, prior to thyroid hormone treatment. As you will see, larval growth is accelerated at intermediate levels of atrazine, a surprising result that we have seen in several related AGP experiments. Second, we confirmed the expected result of TH-application: that is that axololt treated with TH will undergo complete and normal metamorphosis. “Normal,” meaning when compared to the metamorphosis of the sister species the tiger salamander. Third, we examined the effect of atrazine on metamorphic development in the TH treated salamander. As you will see, we failed to find any significant influence of atrazine on metamorphosis.

post-TH length (mm SVL) pre-TH mass (g) post-TH length (mm SVL) 7 7 ) 35 ) 35 These are the summary mean responses, along with Standard Deviations, corresponding to atrazine dosage. The figure in the upper left indicates size of the axolotl after 200 days of atrazine exposure: note that the axolotls at 7 ppb (or ug/L) are significantly heavier than control or those at 35 ppb. We have seen this effect in prior experiments. We don’t have an explanation for this at present, but it does indicate an endocrine controlled system that is affected by atrazine exposure, albeit in a complicated manner.   The remaining three figures indicate that 7ppb axolotls are longer and have longer gills and longer fins suggesting that these axolotls have depressed metamorphic development. However, when these responses are compensated for the fact that these axolotls were already larger, these responses then are not significantly different. gill length (mm) fin length (mm) 7 35 7 35 ) )

post-TH length (mm SVL) pre-TH mass (g) post-TH length (mm SVL) 5 5 50 50 In terms of the TH-induction of metamorphosis, the two figures along the bottom indicate that those axolotls exposed to 5 and 50 nM thyroid hormone exhibit progressively accelerated metamorphosis: the shorter gill and shorter tail fins indicated the reabsorption of these tissues that occurs during metamorphosis.   These result simply confirm the expected TH-induction of metamorphosis. We have not attempted to visually demonstrate any interaction between TH and Atrazine dosage, because this interaction was not significant. What this means is that we have failed to demonstrate that atrazine affects metamorphic development. So, we see that atrazine influence one of two of the endocrine controlled systems we investigated. It influenced larval growth, but it did not affect metamorphic development. gill length (mm) fin length (mm) 5 5 50 50

Amanda Lafountain (MSU) Sharon Denks (FBCC) Dwight Blackhawk (FBCC) ACKNOWLEDGMENTS Chris Beachy (MSU) Heather Modrow (MSU) Leah Crites (MSU) Charles Crites (MSU) Amanda Lafountain (MSU) Sharon Denks (FBCC) Dwight Blackhawk (FBCC) Erica Goodbear (FBCC) Janel Richter (MSU) Claude Ouedraego (MSU) Karen Pocha-Melby (MSU) It needs to be said that this kind of work requires a lot of hands and a lot of time and a lot of care. As such, it can’t be done by a single individual, and the persons listed here were involved in many stages of the work, from obtaining embryos, to husbandry, to dissection, experimental design and statistical analysis.   Because this work requires a lot of bodies, it also can require money, and this work benefits greatly from the financial support of the North Dakota INBRE.

Summation - Accelerated growth at intermediate atrazine dosage; effect is corroborated in our lab in other experiments Axolotls undergo metamorphosis after TH-induction - Failed to find any effect of atrazine on metamorphosis We’ve confirmed accelerated growth at intermediate atrazine dosages in 4 other experiments and do not have a reasonable explanation. However, it clearly indicates that atrazine influences the endocrine system in axolotls. We can induce metamorphosis in the axolotl by TH-induction. However, we did not find any influence of atrazine on metamorphosis. CONCLUSION: To sum up, it appears that atrazine influences development differently in salamanders compared to frogs. Thank you, and I will take any questions.