Paper Biology 205 L The Teratogenic Effects Of Fipronil On Zebrafish Neurological Development

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 µM. In this study, the effect of increasing
concentrations (0.7 µM, 1.1 µM, 2.3 µM) 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

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 µM Fipronil – Touch Escape Response ....................................4
Movie 6: 36hrs Control – Notochord Morphology ...................................................5
Movie 7: 36hrs 1.1 µM Fipronil – Notochord Morphology......................................5
Movie 10: 48hrs Control – Touch Escape Response ...............................................5
Movie 11: 48hrs 0.7 µM Fipronil – Touch Escape Response ..................................5
Movie 13: 48 hrs 2.3 µM Fipronil – Touch Escape Response .................................5
Movie 12: 48 hrs1.1 µM Fipronil – Touch Escape Response ..................................5
Movie 14: 48hrs Control – Notochord Morphology .................................................6
Movie 15: 48hrs 0.7 µM – Notochord Morphology ..................................................6
Movie 16: 48hrs 1.1 µM Fipronil – Notochord Morphology....................................6
Movie 17: 48hrs 2.3 µM 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

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Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development

1 Introduction

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 µM [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 µM.
However, concentrations as high as 5.3 µg/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 µM 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].

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2  Materials and Methods

2.1 Zebrafish husbandry
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.

2.2 Chemicals
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 µM and 11 µM 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.

2.3 Fipronil Treatments
See Biology 205L protocol [12].
Zebrafish embryos were collected and exposed to three concentrations of Fipronil: 0.1%
acetone (control), 0.7 µM, 1.1 µM, and 2.3 µM 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).

2.4 Morphological and Behavioral Analysis
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.

2.5 Acridine Orange Staining for Necrosis
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 µg/ml Acridine Orange for 30 minutes. Embryos were then
washed 3 times in egg water and imaged using SPOT RT.

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3 Supplemental Movies

3.1 List Quicktime Movies Files (DV Digital Video H.264/AAC3 encoded)

! !

Movie 3: 36hrs 0.7 µM Fipronil – Touch Escape Response
Movie 2: 36hrs Control – Touch Escape Response

! !


!
!

Movie 6: 36hrs Control – Notochord Morphology Movie 7: 36hrs 1.1 µM Fipronil – Notochord Morphology

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! !

Movie 8: 36hrs 1.1 µM Fipronil – Notochord Morphology Movie 9: 36hrs 2.3 µM Fipronil – Notochord Morphology

!
!

Movie 10: 48hrs Control – Touch Escape Response Movie 11: 48hrs 0.7 µM Fipronil – Touch Escape Response

! !

Movie 12: 48 hrs 1.1 µM Fipronil – Touch Escape Response Movie 13: 48hrs 2.3 µM Fipronil – Touch Escape Response

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! !

Movie 15: 48hrs 0.7 µM – Notochord Morphology
Movie 14: 48hrs Control – Notochord Morphology

!
!

Movie 16: 48hrs 1.1 µM Fipronil – Notochord Morphology Movie 17: 2.3 µM 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

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 14-48hrs Control Notochord.dv
Movie 15-48hrs 0.7 mM Fipronil Notochord.dv

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4 Results

4.1 Behavioral Defects of Embryos Exposed to Fipronil

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%

4.2 Defects in Notochord Morphology, Body Length, and Circulation

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


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 µM 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


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 µM, 1.1 µM, 2.3 µM). 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


5 Discussion

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].

6 Acknowledgements

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.

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7 References

[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: 578–583.

[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
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microplate format. Environmental Toxicology and Chemistry. 23: 117–124.

[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:
522–528.

[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: 399–413.

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[14] Saint-Amant, L., and Drapeau, P. (1998). Time course of the development of motor
behaviors in the zebrafish embryo. Journal of Neurobiol. 37: 622–632.

[15] Bueno, O. F., and Leidenheimer, N. J. (1998). Colchicine inhibits GABAA receptors
independently of microtubule depolymerization. Neuropharmacology. 37: 383–390.

[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: 391–396.

[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: 522–528.

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