1. The Teratogenic Effects of Fipronil on the Neurological Development of Zebrafish Embryos Joshua E. Mendoza-Elias Biology 205L: Experiments in Developmental Biology and Molecular Genetics Fall 2007 Duke University Dr. Alyssa Perz-Edwards, Ph.D. Trinity School of Arts and Sciences, Biology Department Submitted: Wednesday, December 13, 2006 Abstract Growing research has implicated Fipronil as a teratogen in the development of chordates. Fipronil is a pesticide that acts to disrupt and inhibit normal nerve activity. The result is excessive nerve stimulation caused by overstimulation of gamman-aminobuytric (GABA) gated chloride ion channels. Experiments using zebrafish (Danio rerio) indicate Fipronil is teratogenic at concentrations above 0.7 M. In this study, the effect of increasing concentrations (0.7 M, 1.1 M, 2.3 M) of Fipronil on neurological development were examined. Our results showed that within six hours of GABA-receptor expression (36 hours- high pec) there was neurological damage as indicated by an abnormal behavioral escape touch response (resembling the accordion class phenotype), abnormal morphology (ventrally curved long fin), and damage to neurological tissue (notochord damage concentrated in the dorsal area of the trunk-tail interface). Within 48 hours (long-pec) damage was more extreme as observed in behavior, long tail morphology, and damage to neurological tissue. Furthermore, distinct regions of necrotic tissue were visible, accompanied by aberrant circulation. This data demonstrates Fipronil teratogenicity increases with exposure and concentration and is correlated with increasing damage to the notochord during development. This vertebrate study may have implications for diseases such as fetal alcohol syndrome (FAS) and autism on the phenotypic impact of Fipronil on the development of GABAergic neural pathways. Keywords: zebrafish, fipronil, teratogen, GABA channels, notochord, necrosis. ________________________________________________________________________ Joshua Mendoza-Elias, Trinity College of Arts and Sciences, Department of Biology. E-mail: joshua.mendozaelias@duke.edu. Website: http://www.duke.edu/~jme17

2. Teratogenic Effects of Fipronil on Zebrafish Neurological Devlopment Mendoza-Elias, Joshua E. Table of Contents: 1 Introduction ........................................................................... 3 2.1 Zebrafish husbandry .................................................................. 4 2.2 Chem icals ................................................................................. 4 2.3 Fipronil Treatm ents .................................................................. 4 Movie 1: Accordion Class Phenotype Touch Escape Response. ............................3 2.4 M orphological and Behavioral Analysis ...................................... 4 2.5 Acridine Orange Staining for Necrosis ....................................... 4 3 Supplemental Movies .............................................................. 5 3.1 List Quicktim e M ovies Files (DV Digital Video H .264/AAC -3 encoded) ......................................................................................... 5 Movie 2: 36hrs Control Touch Escape Response .................................................4 Movie 3: 36hrs 0.7 M Fipronil Touch Escape Response ....................................4 Movie 4: 36hrs 1.1 M Fipronil Touch Escape Response ....................................4 Movie 5: 36hrs 2.3 M Fipronil Touch Escape Response ....................................4 Movie 6: 36hrs Control Notochord Morphology ...................................................5 Movie 7: 36hrs 1.1 M Fipronil Notochord Morphology......................................5 Movie 8: 36hrs 1.1 M Fipronil Notochord Morphology......................................5 Movie 9: 36hrs 2.3 M Fipronil Notochord Morphology......................................5 Movie 10: 48hrs Control Touch Escape Response ...............................................5 Movie 11: 48hrs 0.7 M Fipronil Touch Escape Response ..................................5 Movie 13: 48 hrs 2.3 M Fipronil Touch Escape Response .................................5 Movie 12: 48 hrs1.1 M Fipronil Touch Escape Response ..................................5 Movie 14: 48hrs Control Notochord Morphology .................................................6 Movie 15: 48hrs 0.7 M Notochord Morphology ..................................................6 Movie 16: 48hrs 1.1 M Fipronil Notochord Morphology....................................6 Movie 17: 48hrs 2.3 M Fipronil Notochord Morphology....................................6 4 Results ................................................................................... 8 4.1 Behavioral Defects of Em bryos Exposed to Fipronil .................... 8 Table 1: Scoring Zebrafish Touch Escape Response Behavioral Test ...................8 4.2 Defects in N otochord M orphology, Body Length, and Circulation . 8 Figure 1: Fipronil causes abnormal notochord morphology at 36 hrs hpf. . ........9 Figure 2: Fipronil causes abnormal notochord morphology at 48 hrs hpf. . .........9 Figure 3: Anterior section of long-fin shows suggests a higher incidence of cell death. .....................................................................................................................10 5 Discussion ............................................................................ 11 6 Acknowledgements ................................................................ 11 7 References ............................................................................ 12 Page 2 of 13

3. Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development 1Introduction Fipronil is a commercial pesticide designed to kill pests (fleas, cockroaches, ants, termites). Common usage includes agricultural pesticide and a topical application for household pets. Fipronil functions by disrupting insect gamma-aminobutyric acid (GABA) gated chloride channels by blocking the inhibitory action of GABA receptors in the central nervous system (CNS) [1, 2, 3]. The result is disruption of GABA inhibitory action. Physiologically, fipronil exposure causes hyperexcitation at low doses and death at high doses. More importantly, insect and vertebrate GABA receptors have common structural features as they belong to the ligand-gated ion channel superfamily [5]. However, fipronil has a higher binding affinity for insect GABA receptors than vertebrate GABA receptors [6]. This physical property is believed to account for the lower toxicity of Fipronil in mammals [5]. The result is excessive nerve stimulation caused by overstimulation of gamman-aminobutyric (GABA) gated chloride ion channels [1, 2, 4, 6]. However, experiments using the zebrafish (Danio rerio) indicate that it is a teratogen when concentrations are above 0.07 M [7]. In light of this, commercial uses of Fipronil have demonstrated that pollution of water can impact non-target aquatic life [8, 9]. This indicates that organisms that are exposed to water contaminated with fipronil may suffer neurotoxicological effects [9]. The minimum concentration (toxicity floor) causing abnormality in zebrafish is 0.7 M. However, concentrations as high as 5.3 g/l (Cary et al) have been observed [16]. This data implies toxic effects are associated with fipronil and need to be further characterized in vertebrate models. Using the zebrafish model, the effects of increasing concentrations of Fipronil on neurological development will be examined. The results of the experiment will show that within six hours of GABA-receptor expression (36 hours-high pec) there is neurological damage as indicated by an abnormal behavioral response (accordion class phenotype) (see: Movie 1), and an abnormal morphology in the tail. Within 48 hours (long-pec), the damage to neural tissue is more extreme and results in areas of necrotic tissue and disrupted circulation. Loss of behavioral touch- response (stimulation of the Mauthner neuron) was shown to coincide with a rise in fipronil concentration. Brightfield microscopy demonstrated damage to the long-fin morphology in the form of shortening of the rostral-caudal body length as well as necrotic lesions and aberrant systemic blood flow. Preliminary acridine orange stainings supported that the regions of necrotic tissue coincided with cell death. These data show the anterior end of the notochord is the Movie 1: Accordion Class Phenotype Touch Escape Response. Zebrafish treated with 3.0 M Fipronil to generate the accordion class focal neural damage. phenotype. Brightfield microscopy 20x. Click image to play. Video loops 3 times Furthermore, this data full speed, 1 time 20% speed. 3 tones indicate: beginning of clip, repeated clips, suggests fipronil acts by a slow-motion clips, end of video file. unique mechanism [7]. Page 3 of 13

4. Teratogenic Effects of Fipronil on Zebrafish Neurological Devlopment Mendoza-Elias, Joshua E. 2MaterialsandMethods 2.1Zebrafishhusbandry Wild-type zebrafish (AB strain) were maintained according to standard husbandry procedures [10] at 26 C on a light-dark (14-10 hour) cycle. Artificial system water (egg water) was prepared by mixing purified water with Instant Ocean Sea Salt (Aquatic Ecosystems Inc., Apopka, FL). System water pH was maintained between 7.0-7.4. Adult male and female zebrafish spawned using conventional procedures [12]. Fertilized eggs were collected, cleaned and staged [11] and then transferred to plastic Petri dishes containing fresh system water. 2.2Chemicals Fipronil (5- amino-1-[2,6-dichloro-4-(trifluromethyl)phenyl]-4- [(trifluromethyl)sulfinyl]-1H-pyrazole) (C12H4Cl2F6N4OS) (98.2% purity) was dissolved in acetone and stock solution of 1.1 M and 11 M was prepared in system water with a final acetone concentration of 0.1%. Stocks were stored at 4 C in the dark. Dilutions were prepared daily from stock solutions. PTU egg water was made by dissolving 1-phenyl-2-thiourea to 0.003% with egg water. This resulted in the blockage of the melanin biosynthetic pathway and loss of pigmentation in melanophores resulting in greater visibility. 2.3FipronilTreatments See Biology 205L protocol [12]. Zebrafish embryos were collected and exposed to three concentrations of Fipronil: 0.1% acetone (control), 0.7 M, 1.1 M, and 2.3 M dissolved in PTU-egg water. Each exposure contained 10 embryos. Exposures were initiated before 30 hours post fertilization (hpf). The embryos were then dechorionated at 34 hours post fertilization (hpf). 2.4MorphologicalandBehavioralAnalysis Two time points were observed: 36 hrs of development (high-pec) and 48 hours (long-fin). Embryos were examined using brightfield microscopy for live video and micrographs. Data on touch response, notochord morphology and circulation were collected. Video was collected at 20x magnification. Three different tones indicate: beginning of video, repeated clips, slow motion clips and end of video file. Notochord morphology was recorded at 100x magnification. Digital light micrographs were obtained using SPOT RT cooled CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Animals were anesthetized with 0.016 mg/ml tricaine and immobilized with 3% methyl cellulose for imaging as necessary [12]. The response to mechanical stimulus (touch) was used as a measure of sensorimotor integration [13]. Dechorionated fish were gently touched on the head with a probe (needle). Escape response was then compared to control. 2.5AcridineOrangeStainingforNecrosis See Biology 205L protocol [12]. Acridine orange staining was then used to examine lesions of necrotized cells in the long fin. Embryos were soaked in 5 g/ml Acridine Orange for 30 minutes. Embryos were then washed 3 times in egg water and imaged using SPOT RT. Page 4 of 13

5. Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development 3SupplementalMovies 3.1ListQuicktimeMoviesFiles(DVDigitalVideoH.264/AAC3encoded) ! ! Movie 3: 36hrs 0.7 M Fipronil Touch Escape Response Movie 2: 36hrs Control Touch Escape Response ! ! Movie 5: 36hrs 2.3 M Fipronil Touch Escape Response Movie 4: 36hrs 1.1 M Fipronil Touch Escape Response ! ! Movie 6: 36hrs Control Notochord Morphology Movie 7: 36hrs 1.1 M Fipronil Notochord Morphology Page 5 of 13

6. Teratogenic Effects of Fipronil on Zebrafish Neurological Devlopment Mendoza-Elias, Joshua E. ! ! Movie 8: 36hrs 1.1 M Fipronil Notochord Morphology Movie 9: 36hrs 2.3 M Fipronil Notochord Morphology ! ! Movie 10: 48hrs Control Touch Escape Response Movie 11: 48hrs 0.7 M Fipronil Touch Escape Response ! ! Movie 12: 48 hrs 1.1 M Fipronil Touch Escape Response Movie 13: 48hrs 2.3 M Fipronil Touch Escape Response Page 6 of 13

7. Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development ! ! Movie 15: 48hrs 0.7 M Notochord Morphology Movie 14: 48hrs Control Notochord Morphology ! ! Movie 16: 48hrs 1.1 M Fipronil Notochord Morphology Movie 17: 2.3 M Fipronil Notochord Morphology Filenames: Movie files can be accessed through the M edical School Supplement Portfolio DVD. Movie 1- Accordion Phenotype.dv 36 hrs (high-pec) Videos: Movie 2-36hrs Control Touch Escape Response.dv Movie 3-36hrs 0.7mM Fipronil Touch Escape Response.dv Movie 4-36hrs 1.1mM Fipronil Touch Escape Repsonse.dv Movie 5-36hrs 2.3mM Fipronil Touch Escape Response.dv Movie 6-36hrs Control Notochord.dv Movie 7-36hrs 0.7mM Fipronil Notochord.dv Movie 8-36hrs 1.1mM Fipronil Notochord.dv Movie 9-36hrs 2.3mM Fipronil Notochord.dv 48 hrs (long-fin) Videos: Movie 10-48hrs Control Touch Escape Response.dv Movie 11-48hrs 0.7 mM Fipronil Touch Escape Response.dv Movie 12-48hrs 1.1 mM Fipronil Touch Escape Response.dv Movie 13-48hrs 2.3 mM Fipronil Touch Escape Response.dv Movie 14-48hrs Control Notochord.dv Movie 15-48hrs 0.7 mM Fipronil Notochord.dv Movie 16-48hrs 1.1 mM Fipronil Notochord.dv Movie 17-48hrs 2.3 mM Fipronil Notochord.dv Page 7 of 13

8. Teratogenic Effects of Fipronil on Zebrafish Neurological Devlopment Mendoza-Elias, Joshua E. 4Results 4.1BehavioralDefectsofEmbryosExposedtoFipronil Increasing concentration of fipronil was correlated with a loss of the wild-type touch response (See supplemental QuickTime Movie video files). Degrees of severity were observed: wild- type, mild, intermediate and severe. Wild-type responses were used as a basis of comparison. Mild responses were characterized by extended duration of the escape response behavior. Intermediate responses were characterized by delayed reaction to touch stimuli and a more sustained duration of the escape response behavior. Severe responses were characterized by a sustained duration of the escape response behavior and uncontrolled motion seizure like motion. Increasing defects in touch response were observed for increasing concentration of fipronil (Table 1). Likewise, increasing defects in touch response were observed for increased exposure time to fipronil (Table 1). Table 1: Scoring Zebrafish Touch Escape Response Behavioral Test 36 hrs Raw data % Sample 48 hrs Raw Data % Sample (high pec) (N) (long fin) (N) Wild-Type 15 24.59% W ild-Type 23 35.93% Moderate 39 69.93% M oderate 31 48.83% Severe 7 11.47 Severe 10 15.62% 4.2DefectsinNotochordMorphology,BodyLength,andCirculation Damage was observed to occur in long fin. Zebrafish embryos display a reduction in body length and an increase in ventral curvature of the long-fin. More specifically, damage was most pronounced in the ventral rostral region of the notochord in proximity to the extended yolk sac. Notochord lesions displayed fragmentation of the vacuoles, lesions suggesting necrosis, and a loss of systemic circulation in the regions containing lesions. Increasing notochord degeneration was correlated to increasing fipronil concentration and exposure time (see: Figure 1, see: Figure 2). Acridine orange staining was performed to probe lesions of necrotized tissue to reveal the extent of necrosis. Once again, increasing necrotized regions were correlated to increasing fipronil concentrations (see: Figure 3). Page 8 of 13

9. Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development Figure 1: Fipronil causes abnormal notochord morphology at 36 hrs hpf. Zebrafish notochord morphology at 36 hours high-pec at 100x. Larger necrotic tissue domains are observed with increasing concentrations of fipronil. Increasing curvature of the long-fin and abnormal circulation are also observed. Signs of mechanical stress are most exhibited in the clean break of the 1.1 M treatment. Embryos are orientated anterior left and dorsal up. Figure 2: Fipronil causes abnormal notochord morphology at 48 hrs hpf. Zebrafish notochord morphology at 48 hours long-pec at 100x. Larger necrotic tissue domains are observed with increasing concentrations of fipronil. Increasing curvature of the long-fin and abnormal circulation are also observed. Arrows indicate necrotic tissue damage. Embryos are orientated anterior left and dorsal up. Page 9 of 13

10. Teratogenic Effects of Fipronil on Zebrafish Neurological Devlopment Mendoza-Elias, Joshua E. Figure 3: Anterior section of long-fin shows suggests a higher incidence of cell death. Comparison of cell death from fluorescent microscopy. Acridine orange staining was performed to examine the extent of necrotic tissue resulting from increasing fipronil treatments (0.7 M, 1.1 M, 2.3 M). The figure shows fish at magnification 50x (A) and at magnification 100x (B). Fish are orientated anterior left and dorsal up. Page 10 of 13

11. Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development 5Discussion The teratogenic effects of fipronil on zebrafish embryos included a decrease of rostral-caudal body length, an impaired motor response, notochord degeneration, necrosis of the anterior notochord and an abnormal circulatory system in damaged regions of the notochord. These defects are found to arise at 30 hpf when GABA receptors are expressed and coincide with the development of the touch-mediated response and the capacity of sustained swimming movements [14]. The reduced fipronil-exposed embryos reflected damage consistent with sustained bilateral contractions resulting in notochord degeneration. Although the developmental defects caused by fipronil are consistent with effects on neuromuscular physiology, the precise targets of fipronil remain uncharacterized. As with fipronil exposure, accordion class mutants have disorganized axial muscle fibers and notochord degeneration. This has been shown to be prevented in fipronil treated zebrafish embryos and accordion class mutants by the pharmacological paralytic MS-222 during development [7]. Fipronil is designed as a selective antagonist of GABA receptors. As a member of the ligand-gated ion channel superfamily, this suggests that the activity of fipronil may affect other targets that possess homologous sequences and/or structures [15]. Evidence for this is supported by treatment with the potent GABAA receptor antagonist, gabazine. The phenotype associated with gabazine exposure does not mimic the effects of fipronil exposure in zebrafish embryos [7]. Experiments with the GlyR antagonist strychnine produced a phenotype almost identical to the phenotype associated with fipronil exposure [7]. The target of these treatments, combined with the accordion phenotype, suggests that affected loci of the accordion class are potential targets for the site of fipronil action. More specifically, the data point to the glycinergic reciprocal inhibitory pathway in the hindbrain and spinal cord [7] as a likely place to search for fipronil targets of action. In studies on prenatal mice, glycine mediated inhibition resulted in reorganization of motor neurons [16]. In fipronil treatments, zebrafish displayed signs of abnormal morphology; however, given that fipronil treated zebrafish can be rescued by removing them from fipronil exposure. Combined with the data, this seems to suggest that damage to the notochord may be due to mechanical stress from the uncontrolled depolarization of GABA receptors. The two wave-like motions from opposite ends of the zebrafish embryos appear to interfere constructively resulting in damage to the anterior region of the dorsal notochord. Currently, the effects between the CNS and the peripheral nervous system (PNS) at the site of notochord damage cannot be determined as coming from either system. But, data and literature implicate the source damage to the zebrafish stems from the disruption of GABA and glycine receptors. This is supported from sequence and similarity among member of the ligand-gated ion channel superfamily [7, 17]. As such, cross reactions are selective between both types of receptors is expected and probably responsible for the fipronil phenotype [17]. 6Acknowledgements This study was supported by the Biology 205L course (Experiments in Developmental Biology and Molecular Genetics) and a grant from the Howard Hughes Institute. I would like to thank Dr. Alyssa Perz-Edwards, Dr. Nicole Roy, Dr. Amy Bejsovec, Wendy Beane, and Monica Zhang for all their technical expertise and assistance. Page 11 of 13

12. Teratogenic Effects of Fipronil on Zebrafish Neurological Devlopment Mendoza-Elias, Joshua E. 7References [1] Cole, Loretta M., Nicholson; Russel A., and Casida, John E.. 1993. Action of phenylpyrazole insecticides at the GABA-gated chloride channel. Pest. Biochemical Physiology. 46: 47-54. [2] Hamon, Nicholas; Shaw, Richard, and Yang, Henry. 1996. Worldwide development of fipronil insecticide. Conference Proceedings of the Beltwide Cotton Conference Pests and Disease. 2: 759-765. [3] Connelly, Pete. 2001. Environmental fate of fipronil. California Environmental Protection Agency. Sacramento, CA. [4] Colliot, F.; Kukorowski, K.A.; Hawkins, P.W. and Roberts, D.A. 1992. Fipronil: a new soil and foliar broad spectrum insecticide. Brighton Crop Proteonomics. Conference Proceedings of the Beltwide Cotton Conference Pests and Disease. 1:29-34. [5] Hosie, Alastair; Sattelle, David; Aronstein, Kate and ffrench-Constant, Richard. 1997. Molecular biology of insect neuronal GABA receptors. Trends in Neuroscience. 20: 578583. [6] Jentsch, TJ; Valentin; Stein, V.; Frank; Weinreich, F. and Zdebik, Anselm A. (2002). Molecular structure and physiological function of chloride channels. Physiol. Rev. 82: 503 568. [7] Stehr, Carla; Linbo, Tiffany L.; Incardona, John P.; Scholz, Nathaniel L. 2006. The developmental neurotoxicity of fipronil: notochord degeneration and locomotor defects in zebrafish embryos and larvae. Toxicological Sciences. 92 (1): 270 -27. [8] Chandler, G. Thomas; Cary, Tawnya L.; Volz, David C.; Walse, Spencer S.; Ferry, John L.; Klosterhaus, Susan L. (2004). Fipronil effects on estuarine copepod (Amphiascus tenuiremis) development, fertility, and reproduction: A rapid life-cycle assay in 96-well microplate format. Environmental Toxicology and Chemistry. 23: 117124. [9] Cary, Tawnya L; Chandler, G. Thomas; Volz, David C; Walse, Spencer S and Ferry, John L. 2004. Phenylpyrazole insecticide fipronil induces male infertility in the estuarine meiobenthic crustacean Amphiascus tenuiremis. Environmental Science and Technology. 38: 522528. [10] Westerfield, M. (2000). The Zebrafish Book. University of Oregon Press, Eugene, OR. [11] Kimmel, C.B.; Ballard, W.W.; Kimmel, S.R.; Ulman, B., and Schilling, T.F. 1995. Stages of embryonic development of the zebrafish. Developmental Dynamics. 203: 253-310. [12] Perz-Edwards, A.; Roy, N. 2006. Biology 205L Zebrafish Protocol Manual. Durham, NC. Duke University. [13] Granato, M; van Eeden, FJ; Schach, U; Trowe, T; Brand, M; Furutani-Seiki, M; Haffter, P; Hammerschmidt, M; Heisenberg, CP; Jiang, YJ; Kane, DA; Kelsh, RN; Mullins, MC; Odenthal, J and Nusslein-Volhard, C. (1996). Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva. Development. 123: 399413. Page 12 of 13

13. Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development [14] Saint-Amant, L., and Drapeau, P. (1998). Time course of the development of motor behaviors in the zebrafish embryo. Journal of Neurobiol. 37: 622632. [15] Bueno, O. F., and Leidenheimer, N. J. (1998). Colchicine inhibits GABAA receptors independently of microtubule depolymerization. Neuropharmacology. 37: 383390. [16] Kudo, N., Nishimaru, H. 2005. Reorganization of locomotor activity during development in the prenatal rat. Department of Physiology, Institute of Basic Medical Sciences, University of Tsukaba, Ibaraki. 305-8575. [17] Machu, T. K. (1998). Colchicine competitively antagonizes glycine receptors expressed in Xenopus oocytes. Neuropharmacology. 37: 391396. [18] Cary, T. L.; Chandler, G. T.; Volz, D. C.; Walse, S. S., and Ferry, J. L. 2004. Phenylpyrazole insecticide fipronil induces male infertility in the estuarine meiobenthic crustacean Amphiascus tenuiremis. Environmental. Science and Technology. 38: 522528. Page 13 of 13