TOXICOLOGICAL SCIENCES 92(1), 270–278 (2006)
doi:10.1093/toxsci/kfj185
Advance Access publication April 12, 2006
The Developmental Neurotoxicity of Fipronil: Notochord Degeneration
and Locomotor Defects in Zebrafish Embryos and Larvae
Carla M. Stehr, Tiffany L. Linbo, John P. Incardona, and Nathaniel L. Scholz*
Ecotoxicology and Environmental Fish Health Program, Northwest Fisheries Science Center, National Oceanic and
Atmospheric Administration Fisheries, 2725 Montlake Boulevard East, Seattle, Washington 98112
Received February 6, 2006; accepted March 31, 2006
Fipronil is a phenylpyrazole insecticide designed to selectively
inhibit insect gamma-aminobutyric acid (GABA) receptors. Although fipronil is often used in or near aquatic environments, few
studies have assessed the effects of this neurotoxicant on aquatic
vertebrates at sensitive life stages. We explored the toxicological
effects of fipronil on embryos and larvae using the zebrafish (Danio
rerio) experimental model system. Embryos exposed to fipronil at
nominal concentrations at or above 0.7mM (333 mg/l) displayed
notochord degeneration, shortening along the rostral-caudal body
axis, and ineffective tail flips and uncoordinated muscle contractions along the body axis in response to touch. This phenotype
closely resembles zebrafish locomotor mutants of the accordion
class and is consistent with loss of reciprocal inhibitory neurotransmission by glycinergic commissural interneurons in the spinal
cord. Consistent with the hypothesis that notochord degeneration
may be due to abnormal mechanical stress from muscle tetany, the
expression patterns of gene and protein markers specific to
notochord development were unaffected by fipronil. Moreover,
the degenerative effects of fipronil (1.1mM) were reversed by
coexposure to the sodium channel blocker MS-222 (0.6mM). The
notochord effects of fipronil were phenocopied by exposure to
70mM strychnine, a glycinergic receptor antagonist. In contrast,
exposure to gabazine, a potent vertebrate GABAA antagonist,
resulted in a hyperactive touch response but did not cause
notochord degeneration. Although specifically developed to target
insect GABA receptors with low vertebrate toxicity, our results
suggest that fipronil impairs the development of spinal locomotor
pathways in fish by inhibiting a structurally related glycine receptor
subtype. This represents an unanticipated and potentially novel
mechanism for fipronil toxicity in vertebrates.
Key Words: zebrafish; fipronil; neurotoxicity; pesticide; GABA;
glycine.
Fipronil is a phenylpyrazole insecticide that is widely used to
control pests such as cockroaches, ants, termites, rice weevils,
mole crickets, and fleas. Fipronil selectively binds to insect
gamma-aminobutyric acid (GABA)–gated chloride channels,
*
For correspondence via fax: (206) 860-3335. E-mail: Nathaniel.Scholz@
noaa.gov.
Published by Oxford University Press 2006.
blocking the inhibitory action of GABA in the central nervous
system (CNS). Physiologically, fipronil exposure produces
hyperexcitation at low doses and convulsions and death at
high doses. Insect GABA receptors are structurally similar to
vertebrate GABAA and GABAC receptors (Hosie et al., 1997),
which together with glycine receptors (GlyRs) are members of
the ligand-gated chloride channel family (Jentsch et al., 2002).
These receptors have structural features that are common
among all members of the ligand-gated ion channel superfamily. Although GABA-gated chloride channels are expressed in
the CNS of both vertebrates and invertebrates, fipronil has
a considerably higher affinity for insect GABA receptors
relative to vertebrate GABAA receptors and little affinity for
vertebrate GABAC receptors. This property is thought to
account for the low toxicity of fipronil in mammals relative
to arthropods. (Chandler et al., 2004; Gant et al., 1998; Hainzl
et al., 1998; Ratra et al., 2001, 2002; Tingle et al., 2003).
Recent investigations, primarily on crustaceans, have shown
that fipronil can reach environmental levels that impact nontarget aquatic life (Cary et al., 2004; Chandler et al., 2004; Key
et al., 2003; Schlenk et al., 2001; Wirth et al., 2004). However,
the toxicity of fipronil to fish and other vertebrates is not well
characterized. This is particularly true for sublethal effects on
sensitive life stages. For example, the potential neurotoxicological impact of fipronil on fish during early developmental
stages is poorly understood.
GABA-mediated neurotransmission is involved in the modulation of several neural pathways that arise relatively early in
fish development. For example, the circuitry underlying locomotor behaviors such as the escape response and rhythmic
swimming are established soon after patterning of the hindbrain and spinal cord (Saint-Amant and Drapeau, 1998, 2000),
respectively, and GABAergic interneurons are integral to these
systems (Higashijima et al., 2004; Lee et al., 1993; Tegner
et al., 1993; Triller et al., 1997). However, little is known about
the potential effects of GABA receptor blockade during CNS
development.
The zebrafish (Danio rerio) has become an important tool in
developmental neurobiology, and simple behavioral screens
have identified mutants defective in sensory and locomotor
SPINAL DEFECTS IN FIPRONIL-EXPOSED ZEBRA FISH
functions (Granato et al., 1996). Zebrafish mutant lines
provide considerable insight into mechanisms of developmental toxicity, via the comparison of mutant phenotypes with
those arising from exposure to contaminants or other small
molecules (Hill et al., 2005; Incardona et al., 2004; Peterson
et al., 2000). Genetic dissection of sensorimotor functions in
zebrafish has converged with neurophysiological and neuropharmacological studies in larger fish species to provide a
detailed picture of the cellular and molecular underpinnings of
fundamental locomotor behaviors (Grillner, 2003; Kullander,
2005). Functional roles for both excitatory (glutamate) and
inhibitory (glycine and GABA) neurotransmitters are thus well
known in zebrafish.
Here we describe a novel neurotoxicological effect of
fipronil during zebrafish development. Zebrafish embryos
exposed to fipronil showed a dose-dependent loss of touchinduced escape response, a shortening of the longitudinal body
axis, and notochord degeneration. The abnormal motility and
morphological defects observed in fipronil-exposed larvae are
very similar to accordion class zebrafish mutants, which are
proposed to affect reciprocal inhibition in the spinal cord
mediated by glycinergic commissural interneurons (Granato
et al., 1996; Hirata et al., 2005). Consistent with this, the
effects of fipronil were mimicked by exposure to strychnine,
a GlyR antagonist, but not by exposure to gabazine, a selective
antagonist of vertebrate GABAA receptors. These findings
suggest that rather than binding zebrafish GABA receptors,
fipronil may inhibit a structurally related GlyR subtype
expressed during development of spinal locomotor pathways.
Alternatively, these data may implicate a pharmacologically
novel GABA component of the developing spinal circuitry.
MATERIALS AND METHODS
Zebrafish husbandry. Wild-type zebrafish (AB strain) were maintained
according to standard husbandry procedures (Westerfield, 2000) in a recirculating ZMod system (Marine Biotech, Inc., Beverly, MA) at 26C on a lightdark (14-10 h) cycle. Artificial ‘‘system’’ water was prepared by passing
municipal city water through a reverse osmosis system (Aqua Engineering and
Equipment Inc., Winter Park, FL) with the subsequent addition of Instant
Ocean salts (Aquatic Ecosystems Inc., Apopka, FL) to bring the water
conductivity to 1500 lS/cm. System water was maintained at an average pH
of 7.0–7.4. Adult male and female zebrafish were spawned using conventional
procedures (Westerfield, 2000). Fertilized eggs were collected, cleaned, and
staged (Kimmel et al., 1995) and then transferred to glass petri dishes
containing fresh system water.
Chemicals. Fipronil (98.2% purity; Chem Services, Inc., West Chester,
PA) was dissolved in acetone and stock solutions of 4.6lM or 11lM were
prepared in system water with a final acetone concentration of 0.2%. Stocks
were stored at 4C in the dark. Dilutions were prepared daily from stock
solutions. A 10mM stock solution of gabazine (SR95531; Tocris Bioscience,
Ellisville, MO) was prepared in sterile distilled water. A 6mM stock solution of
strychnine (Sigma-Aldrich, St Louis, MO) was prepared in dimethyl sulfoxide,
and a 20mM stock of MS-222 (tricaine methanesulfonate ethyl-3 aminobenzoate; Sigma-Aldrich) was prepared in system water.
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Exposures. Waterborne exposures to fipronil, strychnine, and MS-222 were
conducted in 60-mm glass petri dishes. Each exposure contained 15 embryos
and was each exposure performed in triplicate. Exposures were static and solutions were renewed every 24 h. Exposures were initiated at ~ 1–2 h postfertilization (hpf; 8–64 cell stage). Embryos were maintained in the dark at 28.5C in
a temperature-controlled incubator. For the dose-response experiment, embryos
were exposed to fipronil continuously through 5 days postfertilization (dpf) at
nominal concentrations of 0.007, 0.023, 0.07, 0.23, 0.7, 2.3, and 11lM
(corresponding to 0.003, 0.01, 0.033, 0.1, 0.333, 1.0, and 5.0 mg/l, respectively).
To determine the temporal window for fipronil-induced developmental
defects, embryos were exposed to 1.1lM (0.5 mg/l) fipronil in 8-h pulses
spanning the 0- to 8-, 8- to 16-, 16- to 24-, 24- to 32-, 32- to 40-, or 40- to 48-h
interval postfertilization. Embryos were then transferred to clean system water
and maintained until 72 hpf.
The effects of fipronil on functional, histological, and morphological aspects
of zebrafish development were determined by continuously exposing embryos
to 1.1lM fipronil beginning at 1–2 hpf. Animals were then observed or transferred to fixative at the appropriate developmental stage between 24 and 72 h.
Embryos (n ¼ 45) were similarly exposed to 70lM strychnine, a compound
known to induce locomotor impairment and notochord degeneration in
zebrafish (Granato et al., 1996; Hirata et al., 2004).
To overcome the hydrophilicity of gabazine, embryos at one to eight cells
were microinjected with gabazine (n > 34 per dose) or sterile distilled water
(n > 28) using a PLI-90 picoinjector (Harvard Apparatus, Holliston, MA) and
borosilicate glass micropipettes (1.0-mm diameter) fabricated with a P-30
vertical puller (Sutter Instruments, Novato, CA). To determine dose response,
4 nl gabazine was injected at concentrations of 1.25, 2.5, 5, 10, and 20mM.
Each concentration was injected with a separate micropipette, and injection
volume was established by setting the PLI-90 to deliver a droplet of 100-lm
diameter (measured with an eyepiece reticle) into heavy oil. Based on a wet
weight estimate of 240 lg for zebrafish embryos (Hagedorn et al., 1997;
Petersen and Kristensen, 1998), this resulted in doses of ~ 21, 42, 84, 167, and
334 nmol/g, respectively. Gabazine-injected embryos were subsequently reared
in clean system water.
The potential contribution of tetanic muscle contractions to fipronil-induced
spinal defects was evaluated by treating embryos with 1.1lM fipronil for the
first 24 h of development and then coexposing embryos to fipronil and 0.6mM
MS-222 from 24 to 48 hpf (Hirata et al., 2004; Teraoka et al., 2006).
Morphological and behavioral analyses. Embryos and larvae were
examined with a Nikon SMZ 800 stereomicroscope and a Nikon Eclipse
E600 compound microscope equipped for differential interference contrast
(DIC) microscopy. Digital light micrographs were obtained with a SPOT RT
cooled CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Animals were anesthetized with ~ 2mM MS-222 for imaging as necessary.
Embryos were screened for anatomical defects daily. During pulse-chase
experiments (8-h exposure windows), animals were observed every 8 h during
the first 48 h of the experiment.
The lengths of individual animals were measured at the end of the doseresponse experiment (5 dpf), the pulse-chase experiment (3 dpf), and the
gabazine, strychnine, and fipronil/MS-222 coexposure experiments (48 hpf).
Lengths were measured from digital micrographs (acquired using SPOT
imaging software) as the distance from the anterior end of the mouth to the
end of the caudal peduncle along the notochord.
The response to a mechanical stimulus (touch) was used as a measure of
sensorimotor integration (Granato et al., 1996; Saint-Amant and Drapeau,
1998). Hatched or dechorionated fish were gently touched on the head with
a probe (nylon monofilament inserted into a probe handle). Animals that swam
away after one, two, or three repeated stimuli were scored as responders. All
others were scored as nonresponders. Selected examples were recorded with
digital video microscopy using a Nikon SMZ800 stereomicroscope equipped
with a Unibrain iFire 400 camera and BTV Carbon Pro software.
Histology, immunohistochemistry, and in situ hybridization. For general histology, embryos were fixed in 3% paraformaldehyde and 0.75%
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STEHR ET AL.
glutaraldehyde in a sodium cacodylate buffer (pH 7.4; Electron Microscopy
Sciences, Hatfield, PA) and embedded in Spurr’s low-viscosity embedding
medium (Pelco International, Redding, CA). Sections (~ 1 lm) were cut with
glass knives, adhered to glass slides, and stained with Richardson’s solution
(2% methylene blue, 1% azure II, 2% sodium borate; Mallinckrodt Baker,
Phillipsburg, NJ). Sections of the notochord were evaluated in 8–12 embryos for
each treatment (1.1lM fipronil, 0.2% acetone, or control) at 24, 30, and 48 hpf.
Whole-mount immunolocalization was carried out as described previously
(Incardona et al., 2004). Primary antibodies included antiengrailed 4D9,
antimyosin heavy chain mouse monoclonal F59 (both from Developmental
Studies Hybridoma Bank, Iowa City, IA), and rabbit polyclonal antilaminin
(Sigma-Aldrich). For laminin and engrailed immunocytochemistry, embryos
were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in 0.1M
sodium phosphate buffer at 24 and 30 hpf. For myosin (F59) immunofluorescence, embryos were fixed at 48 hpf in a graded methanol series followed by
rehydration. Secondary antibodies were donkey anti-rabbit IgG (antilaminin) or
donkey anti-mouse IgG (antiengrailed) conjugated to horseradish peroxidase
(both from Jackson ImmunoResearch, West Grove, PA) or Alexa Fluor 488–
conjugated goat anti-mouse IgG (F59; Molecular Probes/Invitrogen, Carlsbad,
CA). Peroxidase-conjugated antibodies were visualized with 0.5 mg/ml
diaminobenzidine and 0.003% hydrogen peroxide in tris-buffered saline.
Immunoperoxidase-labeled embryos were imaged with DIC optics, and F59
immunofluorescence was imaged using a Zeiss LSM 5 Pascal confocal system
(Carl Zeiss Advanced Imaging Microscopy, Jena, Germany). At least 10
embryos were examined for each time point and immunochemical marker.
For in situ hybridizations, embryos were fixed overnight in 4% paraformaldehyde in 0.1M phosphate buffer. Hybridization with digoxigeninlabeled riboprobes was carried out using standard protocols (Thisse et al., 1993;
Westerfield, 2000). Genes analyzed included collagen 2a1 (col2a), tiggy winkle
hedgehog (twhh), no tail (ntl), one-eyed pinhead (oep), and floating head (flt).
Bound riboprobes were detected with alkaline phosphatase–conjugated antidigoxigenin antibody (Roche Diagnostics, Indianapolis, IN) with nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics).
Embryos were imaged using DIC microscopy and SPOT digital camera
(Diagnostic Instruments) commercial software. Embryos were fixed from each
control and fipronil treatment group (n ¼ 12–15 animals) at 24 and 30 hpf for
each in situ marker.
Statistics. Statistical analyses of individual fish lengths were performed
with one-way ANOVA and Tukey’s honestly significant difference (HSD)
post hoc test (Kaleidograph, Syngery Software Technologies, Inc., Essex
Junction, VT). To evaluate the dose-dependent effects of fipronil on fish
lengths, data were fit with a logistic regression using the formula:
y ¼ ½m1 þ ðm2 m1 Þ=½1 þ em3 ðxm4 Þ ;
where m1 ¼ minimum length, m2 ¼ maximum length, m3 ¼ slope, m4 ¼
concentration halfway between minimum and maximum length, y ¼ length,
and x ¼ exposure concentration. A nonparametric Kruskal-Wallis rank sum test
(Kaleidograph) was used to compare touch response and the prevalence of
anatomical abnormalities between exposed and control fish.
RESULTS
Locomotor Defects in Fipronil-Exposed Embryos and Larvae
The locomotor behavior of zebrafish embryos develops in
a stereotypical manner (Saint-Amant and Drapeau, 1998). At
17 hpf, embryos show spontaneous contractions of trunk
muscles that are independent of sensory stimuli. At 21 hpf,
the embryo responds to a touch on the head with rapid tail coils,
and at 27 hpf the same mechanical stimulus produces bursts of
swimming. The rate of swimming in response to touch be-
comes maximal by 36 hpf (Saint-Amant and Drapeau, 1998).
Hatching-stage larvae (post 48 hpf) thus show a mature escape
reflex in response to tactile stimulation, and this serves as
a simple assay for sensorimotor integration. Unexposed fish
showed a normal touch response at 30, 48, or 72 hpf as
indicated by effective swimming movements of the tail (Fig.
1A). In contrast, embryos continuously exposed to fipronil 0.7lM (0.333 mg/l) responded to a head touch with wavelike
contractions of the body axis but were incapable of swimming
(Fig. 1B and 1C and Supplemental Movie 1). The impairment
of touch-induced locomotion was first evident at ~ 30 hpf and
continued through to the end of the 5-day exposure. Impaired
motor response was dose dependent (Fig. 2), and the prevalence of larvae with an abnormal touch response was significantly higher in fish exposed to 0.7lM fipronil (p < 0.001,
Kruskal-Wallis rank sum).
Pulsed fipronil exposures revealed a sensitive period of locomotor deficiency corresponding to the onset and maturation of
the touch response. Embryos exposed to 1.1lM fipronil in 8-h
intervals prior to 24 hpf showed normal spontaneous movement
at 24 hpf and a normal touch response at 48 and 72 hpf. By
contrast, embryos exposed to the same concentration of fipronil
at 24–32, 32–40, or 40–48 hpf were unresponsive to touch.
Fipronil-induced locomotor defects were reversible. After
return to clean water, all animals exposed from 24 to 32 (n ¼
42) and 32 to 40 hpf (n ¼ 44) and most of the fish exposed from
40 to 48 hpf (93%, n ¼ 45) showed normal touch responses
when tested subsequently at 72 hpf. This indicates that
sensorimotor function generally recovers within 24 h.
Reduced Body Length, Notochord Degeneration, and
Abnormal Axial Muscle Morphology
There were no indications of anatomical defects in zebrafish
embryos exposed continuously to fipronil ( 0.23lM) until
~ 30 h of development. At this point, embryos began to show
reduced body length (Fig. 3F), notochord degeneration (Fig.
3G and 3H), and abnormal axial muscle morphology (Fig. 3H–
3J). Histology confirmed that notochord morphology was
normal at 24 hpf and then degenerative at 30 (data not shown)
and 48 hpf (Fig. 3H). Damage to the notochord was evident as
a fragmentation of the notochord vacuoles and the presence of
fibrous material (Fig. 3H). Myotomes in fipronil-exposed
embryos were shorter (Fig. 3I and 3J), and muscle fibers were
abnormally sinusoidal in shape (Fig. 3I). Immunofluorescent
labeling with an antibody against myosin heavy chain (F59)
revealed a disorganization of the superficial slow muscle fibers
in fipronil-exposed embryos at 48 hpf (Fig. 3J).
Whereas the effects of fipronil on the embryonic touch
response were reversible, degeneration of the notochord was
irreversible. For both effects, however, the developmental
window of sensitivity was similar. All embryos (n ¼ 134)
exposed to an 8-h pulse of fipronil (1.1lM) prior to 24 hpf were
normal in their morphology when examined at 24 hpf. The same
SPINAL DEFECTS IN FIPRONIL-EXPOSED ZEBRA FISH
273
FIG. 1. Abnormal response to a mechanical stimulus (touch) following fipronil exposure. (A) Sequential frames from a digital video recording show a 48 hpf
larvae exposed to solvent only (vehicle control) responding to touch with two alternating flicks of the tail. Elapsed time (ms) is indicated in the lower left corner. (B)
The characteristic swimming response was absent in a larva exposed to 1.1lM fipronil from 2–48 hpf. (C) Higher magnification frames of a fipronil-exposed larva
reveal accordion-like, anterior-posterior contractions. Elapsed time (ms) is indicated at the bottom. Vertical white lines mark the rostral-caudal extent of the yolk
extension, and the arrow indicates the caudal end. Simultaneous bilateral contractions result in shortening along the body axis rather than effective alternating tail
flicks. Scale bars are 1 mm (A, B) and 0.5 mm (C).
animals were unaffected at 48 hpf with the exception of
a few fish that had small areas where one or two notochord
vacuoles appeared abnormal. In contrast, all embryos exposed
to a fipronil pulse from 24 to 32 hpf (n ¼ 42) and > 75% of the
animals exposed at 32–40 hpf (n ¼ 44) or 40–48 hpf (n ¼ 45)
showed extensive notochord degeneration, shortened body
length, and disrupted muscle morphology when examined at
48 hpf. When animals were transferred to clean water for 24 h
and examined again at 72 hpf, notochord degeneration and
shortened body length were still present. Shortened body
length was dose dependent (Fig. 2), with an EC50 of 0.37 ±
0.037 (SE) lM. The longitudinal body axis of 5 dpf larvae
continuously exposed to 0.23lM fipronil was significantly
reduced relative to the controls (p < 0.001, Kruskal-Wallis rank
sum). Logistic regression indicated a sigmoidal relationship
between fipronil dose and body length (Fig. 2, r2 ¼ 0.996).
Finally, significant lethality was only observed at the highest
concentration of fipronil (11lM). For this exposure, viability
was 7% (n ¼ 44) at 5 dpf (p < 0.001, Kruskal-Wallis rank sum),
with most fish dying between 3 and 5 dpf. Survival at all other
concentrations ranged from 73 to 100% and were not
significantly different from controls (p > 0.05).
Fipronil-Induced Morphological Defects Are Secondary to
Abnormal Muscle Contraction
The notochord plays an important structural role that
supports locomotion in larval fish. Moreover, during embryonic
development, the notochord is the origin of signals involved in
patterning axial structures such as the somites and overlying
neural tube. Several genes involved in notochord formation and
function are often altered by identified mutations in zebrafish
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STEHR ET AL.
FIG. 2. Dose-dependent effects of fipronil on body length and touch
response. Five-day-old zebrafish were continuously exposed to a range of
nominal fipronil concentrations beginning at 1–2 hpf. The logistic regression
curve for mean length (solid line) is illustrated. The percentage (%) of fish
swimming away from touch (dashed line) is also plotted as a function of fipronil
concentration. Exposure concentrations are plotted on a log scale. Solid
symbols indicate mean length and open symbols indicate percentage of fish
swimming away from touch. Squares are solvent controls and triangles are
untreated controls. Numbers in parentheses, number of fish, and SC, solvent
control; error bars indicate standard deviation. *Significantly different from
controls at p < 0.0001 (ANOVA with Tukey’s HSD post hoc for length and
Kruskal-Wallis for touch response).
(Odenthal et al., 1996; Parsons et al., 2002; Stemple et al.,
1996). To explore potential mechanisms underlying notochord
defects due to fipronil exposure, we monitored the in situ
expression patterns of several genes. Markers of notochord
differentiation, including mRNAs for collagen 2a1 (col2a1), no
tail (ntl), tiggy winkle hedgehog (twhh), floating head (flh),
one-eyed pinhead (oep), and laminin protein (data not shown)
were unchanged in fish exposed to 1.1lM fipronil at 24 and
30 hpf relative to controls. Similarly, fipronil-exposed embryos
showed no changes in the number of engrailed-immunopositive
muscle pioneer cells in the somites (data not shown), providing additional evidence for normal notochord development
(Devoto et al., 1996). These data collectively indicate that
fipronil-induced notochord lesions are unlikely to be related
to the developmental processes that typically guide normal
notochord differentiation.
To determine if notochord lesions and myotome shortening
were related to abnormal muscle contraction, we evaluated the
effects of pharmacologically inhibiting muscle contractions
during fipronil exposure. The anesthetic MS-222, a voltagegated Naþ channel blocker, has previously been used at low
concentrations to chronically produce a flaccid paralysis in
zebrafish embryos and larvae without affecting morphological
FIG. 3. Notochord and muscle morphology of fipronil-exposed zebrafish.
Architecture of the notochord in control zebrafish embryos (A–E) and embryos
continuously exposed to 1.1lM fipronil (F–J) are imaged at 48 hpf. The
fipronil-exposed phenotype is characterized by shortened body length (F),
notochord degeneration (G and H; live specimen and histological sections,
respectively), and disrupted muscle morphology (H–J; histology, Nomarksi,
and confocal imaging of muscle fibers stained with F59 antibody, respectively)
resulting in shortened myotomes (I and J). nc ¼ notochord and m ¼ muscle;
double ended arrows indicate myotome length. Scale bars ¼ 0.5 mm (F),
0.1 mm (G), 50 lm (H), and 25 lm (I and J).
development (Hirata et al., 2004; Teraoka et al., 2006).
Notochord degeneration (Fig. 4C) and reduction of longitudinal body axis (data not shown) were prevented when embryos
were coexposed to fipronil (1.1lM; continuous exposure
beginning at 1–2 hpf) and MS-222 (0.6mM; 24–48 hpf).
Combined with our other results, this phenotypic rescue
indicates that fipronil-induced notochord lesions are likely a
mechanical consequence of muscle hypercontractility and
not due to a defect in notochord differentiation or intrinsic
maintenance.
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SPINAL DEFECTS IN FIPRONIL-EXPOSED ZEBRA FISH
Fipronil Exposure Mimics the Effects of a Vertebrate Glycine
Receptor Antagonist but Not a Potent GABAA Antagonist
To determine if the effects of fipronil on embryonic locomotor activity were due to blockade of GABA receptors, we tested
the effects of a well-characterized vertebrate GABA antagonist. Of the vertebrate GABA receptor classes most likely to be
antagonized by fipronil (GABAA and GABAC), GABAA receptors are known to be involved in spinal locomotor circuits
(Grillner, 2003). Therefore, we exposed zebrafish embryos to
the classical GABAA antagonist gabazine (Heaulme et al.,
1986; Krogsgaard-Larsen et al., 2002; Mienville and Vicini,
1987). As observed with other pharmacologic agents (Milan
et al., 2003) and consistent with the hydrophilicity of gabazine
and presumed poor uptake by fish embryos, waterborne
gabazine exposure had no apparent effect on zebrafish development (data not shown). However, microinjection of
gabazine over doses ranging from ~ 21 to 334 nmol/g wet
weight into fertilized eggs (n 34 embryos per dose) produced
a marked change in locomotor behavior (the motor activity of
embryos injected with water alone was indistinguishable from
controls). Moreover, the effects of gabazine on zebrafish
embryos were markedly different from the effects of fipronil
exposure across the entire dosage range. Rather than showing
a reduced touch response and ‘‘accordion’’-like muscle contractions, gabazine-exposed larvae responded to mechanical
stimulus with prolonged and rapid swimming activity when
examined at 36–48 hpf. Video microscopy was used to monitor
this behavioral response among embryos still within the
chorion (Supplemental Movie 2). Notably, this hyperexcitation
is consistent with known, seizure-inducing effects of gabazine
(Heaulme et al., 1986). At doses of 334 and 167 nmol/g, 100%
of exposed larvae exhibited seizures, 84% of larvae showed
seizures at 84 and 42 nmol/g, and 70.6% showed seizures at
21 nmol/g. None of the morphological defects associated with
fipronil exposure, including notochord degeneration (Fig. 4D),
were observed in gabazine-injected embryos. In contrast,
exposure to the GlyR antagonist strychnine (70lM; 1–2 hpf
through 48 hpf) resulted in the same suite of defects as fipronil,
including impaired touch response, shortened body length,
disorganization of muscle fibers (data not shown), and notochord degeneration (Fig. 4E).
DISCUSSION
The phenotypic effects of fipronil on zebrafish embryos
include impaired motor response, notochord degeneration,
FIG. 4. Notochord degeneration is dependent on muscle contraction and
a strychnine-like activity of fipronil. All panels show a portion of the notochord
just posterior to the yolk sac in embryos at 48 hpf. (A) Normal notochord in
a control zebrafish embryo. (B) Notochord degeneration in an embryo
continuously exposed to 1.1lM fipronil. (C) Normal notochord in an embryo
exposed to fipronil and then coexposed to the Naþ channel blocker MS-222 to
prevent muscle contraction. (D) Normal notochord in a gabazine-injected
embryo. (E) Notochord degeneration in an embryo exposed to the GlyR
antagonist strychnine. nc ¼ notochord. Scale bar ¼ 0.05 mm.
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abnormal muscle fiber morphology, and shortened body length.
These effects first appear at approximately 30 hpf and coincide
with the normal development of both the touch-mediated escape
response and the capacity for sustained swimming movements
(Saint-Amant and Drapeau, 1998). The mode of action for
fipronil appears to be primarily physiological and is characterized by reversible effects on sensorimotor function. This is in
contrast to notochord distortions resulting from exposure to
dithiocarbamate pesticides, which are dependent on normal
muscle contractions, but are most likely due to a pesticideinduced structural defect in the notochord itself (Haendel et al.,
2004; Teraoka et al., 2006). The reduced body length and
abnormal muscle fiber morphology in fipronil-exposed larvae
reflect sustained bilateral contractions of the axial muscles, and
this in turn leads to notochord degeneration. The mechanical
damage to the notochord is effectively blocked by a pharmacological paralytic (MS-222). While sensorimotor function recovered after presumptive depuration of fipronil, damage to the
notochord was irreversible throughout the duration of the pulse
experiment (3 dpf) and was still evident at the end of the continuous exposure (5 dpf). Although our analysis indicates that
initial differentiation of the notochord and its early signaling
functions are unaffected by fipronil, it is possible that fipronilinduced notochord lesions could affect the subsequent motor
behavior through structural impact on notochord function.
Although the developmental defects caused by fipronil are
consistent with effects on neuromuscular physiology, the precise targets are unclear. Fipronil is by design a selective
antagonist of insect GABA receptors. However, treatment with
the potent vertebrate GABAA receptor antagonist gabazine did
not mimic the effects of fipronil in zebrafish embryos, but rather
produced a seizurelike activity in response to touch, consistent
with the previous observations in mammalian systems
(Heaulme et al., 1986). Instead, the suite of fipronil-induced
defects was almost identical to the pharmacological effects of
the GlyR antagonist strychnine (Granato et al., 1996; Hirata
et al., 2005). Moreover, exposure to fipronil or strychnine
phenocopied zebrafish accordion class mutants, so named for
the abnormal bilateral axial muscle contractions in response to
touch (Granato et al., 1996). As with fipronil exposure,
accordion class mutants have disorganized axial muscle fibers
and notochord degeneration that are prevented by paralysis with
MS-222 treatment. The glycinergic reciprocal inhibitory pathway in the hindbrain and spinal cord is thought to be involved in
these mutants, and the affected loci are therefore potential
targets for fipronil. Of the seven originally identified accordion
class mutants, three have been characterized at the molecular
level. Consistent with the effects of strychnine, a mutation in the
GlyR [beta] subunit underlies the defects in the bandoneon
mutant (Hirata et al., 2005). However, the zieharmonika and
accordion mutants were found to affect acetylcholinesterase
(Downes and Granato, 2004) and the sarco(endo)plasmic
reticulum Ca2þ-ATPase 1 (SERCA1) (Gleason et al., 2004;
Hirata et al., 2004), respectively. While the bandoneon mutants
are unable to relax axial muscle due to loss of central
glycinergic inhibition of motor neurons, the zieharmonika and
accordion mutants display sustained muscle contraction due to
elevated acetylcholine levels at the neuromuscular junction and
impaired uptake of intracellular Ca2þ in skeletal myofibers,
respectively (Gleason et al., 2004; Hirata et al., 2004).
At the level of analysis performed here, we cannot distinguish between effects on spinal or other CNS circuitry and
peripheral effects on the neuromuscular junction or axial
musculature. However, the most likely explanation for our
results is that fipronil acts as a GlyR antagonist in developing
zebrafish. Sequence identity and structural similarity are high
among all members of the ligand-gated ion channel superfamily (including GABAA and GlyRs), and cross-selectivity
among both agonists and antagonists is common (Bueno and
Leidenheimer, 1998; Machu, 1998; Sun and Machu, 2000; Yan
et al., 1998). It should be noted, however, that the developing
spinal and hindbrain circuitry involved in sensorimotor integration and swimming behavior in zebrafish have not been
completely characterized, and the effects of fipronil could
reflect a novel role for GABAergic signaling. This is unlikely,
in part, because of the marked differences between the effects
of fipronil and gabazine. Finally, fipronil could act on unidentified targets that are unrelated to neurotransmission.
Zebrafish are proving increasingly useful as a model system
to identify novel mechanisms of developmental toxicity in
vertebrates (Hill et al., 2005) and fish in particular (Hinton et al.,
2005). This is due in part to the fact that patterning, organogenesis, and other developmental processes are very well understood in zebrafish relative to most other fish species. Moreover,
as we have shown here, a comparison of the fipronil-induced
phenotype to the zebrafish mutant bandoneon is an example of
how a chemical-genetic approach can be used to reveal potential
cellular targets for poorly characterized toxicants. These and
other tools in zebrafish (i.e., reverse genetics) are making it
increasingly possible to explore long-standing and difficult
challenges in aquatic toxicology such as the mechanistic
underpinnings of mixture toxicity (Incardona et al., 2005).
Finally, the aim of this study was to explore potential
mechanisms of fipronil-induced developmental toxicity using
the zebrafish model. Accordingly, more work is needed to
establish the environmental relevance of these results for
human health as well as for the health of wild fish that spawn
in fipronil-contaminated habitats. Fipronil concentrations as
high as 5.3 lg/l (Cary et al., 2004), 8 lg/l (Wirth et al., 2004),
and 9 lg/l (Schlenk et al., 2001) have been detected in surface
waters downstream of rice fields planted with fipronil-treated
rice seed, and the insecticide has been previously been shown
to be toxic to invertebrates at these levels. For example, caged
crayfish placed in a pond receiving effluent from a field with
treated rice containing 9 lg/l fipronil showed significantly
higher mortality compared to crayfish similarly exposed to
untreated rice field effluent (Schlenk et al., 2001). In the
present study, we observed developmental defects in zebrafish
SPINAL DEFECTS IN FIPRONIL-EXPOSED ZEBRA FISH
at nominal concentrations beginning at 333 lg/l, which is well
above levels that have previously been measured in the aquatic
environment. However, we did not analytically determine
dissolved-phase fipronil in our exposure solutions, and the
actual concentrations of fipronil (vs. nominal) may have been
much lower due to adsorption to the glass exposure chambers
and the partitioning of the pesticide to other matrices (i.e., air).
Also, native species may be more sensitive than zebrafish to the
effects of fipronil, as has been previously shown for dioxins
and salmonids (Henry et al., 1997). Irrespective, our findings
indicate that fipronil is a developmental neurotoxicant in
vertebrates and that future investigations should focus on axial
muscle hyperexcitation as a toxicological end point for fish in
aquatic habitats contaminated with this insecticide.
SUPPLEMENTAL DATA
Supplemental data includes two .mov (Quicktime) movie
clips. Movie 1 (Fipronil touch response) illustrates the normal
touch response behavior of untreated 48 hpf zebrafish embryos
(dechorionated) compared to embryos exposed from 2 to 48
hpf to 1.1lM fipronil. Movie 2 (Gabazine touch response)
illustrates the touch response behavior of 48 hpf embryos in
the chorion injected with sterile distilled water compared to
embryos injected with ~ 240lM gabazine. Supplemental data
are available online at www.toxsci.oxfordjournals.org.
ACKNOWLEDGMENTS
This study was supported by the National Oceanic and Atmospheric
Administration’s (NOAA) Coastal Storms Program and the NOAA Oceans
and Human Health Program. We thank U. Pyati and D. Kimelman for
generously providing probes for in situ hybridization and Jamie Colman,
Heather Day, Jana Labenia, and Tracy Collier for their critical review of the
manuscript.
REFERENCES
277
Gant, D. B., Chalmers, A. E., Wolff, M. A., Hoffman, H. B., and Bushey, D. F.
(1998). Fipronil: Action at the GABA receptor. Rev. Toxicol. 2, 147–156.
Gleason, M. R., Armisen, R., Verdecia, M. A., Sirotkin, H., Brehm, P., and
Mandel, G. (2004). A mutation in SERCA underlies motility dysfunction in
accordion zebrafish. Dev. Biol. 276, 441–451.
Granato, M., van Eeden, F. J., Schach, U., Trowe, T., Brand, M., FurutaniSeiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C. P., Jiang, Y. J.,
et al. (1996). Genes controlling and mediating locomotion behavior of the
zebrafish embryo and larva. Development 123, 399–413.
Grillner, S. (2003). The motor infrastructure: From ion channels to neuronal
networks. Nat. Rev. Neurosci. 4, 573–586.
Haendel, M. A., Tilton, F., Bailey, G. S., and Tanguay, R. L. (2004).
Developmental toxicity of the dithiocarbamate pesticide sodium metam in
zebrafish. Toxicol. Sci. 81, 390–400.
Hagedorn, M., Kleinhans, F. W., Freitas, R., Liu, J., Hsu, E. W., Wildt, D. E.,
and Rall, W. F. (1997). Water distribution and permeability of zebrafish
embryos, Brachydanio rerio. J. Exp. Zool. 278, 356–713.
Hainzl, D., Cole, L. M., and Casida, J. E. (1998). Mechanisms for selective
toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl
photoproduct. Chem. Res. Toxicol. 11, 1529–1535.
Heaulme, M., Chambon, J. P., Leyris, R., Molimard, J. C., Wermuth, C. G., and
Biziere, K. (1986). Biochemical characterization of the interaction of three
pyridazinyl-GABA derivatives with the GABAA receptor site. Brain Res.
384, 224–231.
Henry, T. R., Spitsbergen, J. M., Hornung, M. W., Abnet, C. C., and Peterson,
R. E. (1997). Early life stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin
in zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 142, 56–68.
Higashijima, S., Schaefer, M., and Fetcho, J. R. (2004). Neurotransmitter
properties of spinal interneurons in embryonic and larval zebrafish. J. Comp.
Neurol. 480, 19–37.
Hill, A. J., Teraoka, H., Heideman, W., and Peterson, R. E. (2005). Zebrafish
as a model vertebrate for investigating chemical toxicity. Toxicol. Sci. 86,
6–19.
Hinton, D. E., Kullman, S. W., Hardman, R. C., Volz, D. C., Chen, P. J.,
Carney, M., and Bencic, D. C. (2005). Resolving mechanisms of toxicity
while pursuing ecotoxicological relevance? Mar. Pollut. Bull. 51, 635–648.
Hirata, H., Saint-Amant, L., Downes, G. B., Cui, W. W., Zhou, W., Granato, M.,
and Kuwada, J. Y. (2005). Zebrafish bandoneon mutants display behavioral
defects due to a mutation in the glycine receptor beta-subunit. Proc. Natl.
Acad. Sci. U.S.A. 102, 8345–8350.
Hirata, H., Saint-Amant, L., Waterbury, J., Cui, W., Zhou, W., Li, Q., Goldman, D.,
Granato, M., and Kuwada, J. Y. (2004). Accordion, a zebrafish behavioral
mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2þ
pump SERCA1. Development 131, 5457–5468.
Bueno, O. F., and Leidenheimer, N. J. (1998). Colchicine inhibits GABAA
receptors independently of microtubule depolymerization. Neuropharmacology 37, 383–390.
Hosie, A., Sattelle, D., Aronstein, K., and French-Constant, R. (1997).
Molecular biology of insect neuronal GABA receptors. Trends Neurosci.
20, 578–583.
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. Environ. Sci. Technol. 38,
522–528.
Incardona, J. P., Carls, M. G., Teraoka, H., Sloan, C. A., Collier, T. K., and
Scholz, N. L. (2005). Aryl hydrocarbon receptor-independent toxicity of
weathered crude oil during fish development. Environ. Health Perspect. 113,
1755–1762.
Chandler, G. T., Cary, T. L., Volz, D. C., Walse, S. S., Ferry, J. L., and
Klosterhaus, S. L. (2004). Fipronil effects on estuarine copepod (Amphiascus
tenuiremis) development, fertility, and reproduction: A rapid life-cycle assay
in 96-well microplate format. Environ. Toxicol. Chem. 23, 117–124.
Incardona, J. P., Collier, T. K., and Scholz, N. L. (2004). Defects in cardiac
function precede morphological abnormalities in fish embryos exposed to
polycyclic aromatic hydrocarbons. Toxicol. Appl. Pharmacol. 196, 191–205.
Devoto, S. H., Melancon, E., Eisen, J. S., and Westerfield, M. (1996).
Identification of separate slow and fast muscle precursor cells in vivo, prior
to somite formation. Development 122, 3371–3380.
Downes, G. B., and Granato, M. (2004). Acetylcholinesterase function is
dispensable for sensory neurite growth but is critical for neuromuscular
synapse stability. Dev. Biol. 270, 232–245.
Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002). Molecular
structure and physiological function of chloride channels. Physiol. Rev. 82,
503–568.
Key, P. B., Chung, K. W., Opatkiewicz, A. D., Wirth, E. F., and Fulton, M. H.
(2003). Toxicity of the insecticides fipronil and endosulfan to selected life
stages of the grass shrimp (Palaemonetes pugio). Bull. Environ. Contam.
Toxicol. 70, 533–540.
278
STEHR ET AL.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling, T. F.
(1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,
253–310.
Krogsgaard-Larsen, P., Frølund, B., and Liljefors, T. (2002). Specific GABAA
agonists and partial agonists. Chem. Rec. 2, 419–430.
Kullander, K. (2005). Genetics moving to neuronal networks. Trends Neurosci.
28, 239–247.
Lee, R. K., Finger, T. E., and Eaton, R. C. (1993). GABAergic innervation of
the Mauthner cell and other reticulospinal neurons in the goldfish. J. Comp.
Neurol. 338, 601–611.
Machu, T. K. (1998). Colchicine competitively antagonizes glycine receptors
expressed in Xenopus oocytes. Neuropharmacology 37, 391–396.
Mienville, J. M., and Vicini, S. (1987). A pyridazinyl derivative of gammaaminobutyric acid (GABA), SR 95531, is a potent antagonist of Cl
channel opening regulated by GABAA receptors. Neuropharmacology 26,
779–783.
Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., and MacRae, C. A.
(2003). Drugs that induce repolarization abnormalities cause bradycardia in
zebrafish. Circulation 107, 1355–1358.
Odenthal, J., Haffter, P., Vogelsang, E., Brand, M., van Eeden, F. J., FurutaniSeiki, M., Granato, M., Hammerschmidt, M., Heisenberg, C. P., Jiang, Y. J.,
et al. (1996). Mutations affecting the formation of the notochord in the
zebrafish, Danio rerio. Development 123, 103–115.
Parsons, M. J., Pollard, S. M., Saude, L., Feldman, B., Coutinho, P., Hirst,
E. M., and Stemple, D. L. (2002). Zebrafish mutants identify an essential
role for laminins in notochord formation. Development 129, 3137–3146.
Petersen, G. I., and Kristensen, P. (1998). Bioaccumulation of lipophilic
substances in fish early life stages. Environ. Toxicol. Chem. 17, 1385–1395.
Peterson, R. T., Link, B. A., Dowling, J. E., and Schreiber, S. L. (2000). Small
molecule developmental screens reveal the logic and timing of vertebrate
development. Proc. Natl. Acad. Sci. U.S.A. 97, 12965–12969.
Ratra, G. S., Erkkila, B. E., Weiss, D. S., and Casida, J. E. (2002). Unique
insecticide specificity of human homomeric [rho]1 GABAC receptor.
Toxicol. Lett. 129, 47–53.
Ratra, G. S., Kamita, S. G., and Casida, J. E. (2001). Role of human GABA(A)
receptor beta3 subunit in insecticide toxicity. Toxicol. Appl. Pharmacol. 172,
233–240.
Saint-Amant, L., and Drapeau, P. (1998). Time course of the development of
motor behaviors in the zebrafish embryo. J. Neurobiol. 37, 622–632.
Saint-Amant, L., and Drapeau, P. (2000). Motoneuron activity patterns related
to the earliest behavior of the zebrafish embryo. J. Neurosci. 20, 3964–3972.
Schlenk, D., Huggett, D. B., Allgood, J., Bennett, E., Rimoldi, J., Beeler, A. B.,
Block, D., Holder, A. W., Hovinga, R., and Bedient, P. (2001). Toxicity of
fipronil and its degradation products to Procambarus sp.: Field and
laboratory studies. Arch. Environ. Contam. Toxicol. 41, 325–332.
Stemple, D. L., Solnica-Krezel, L., Zwartkruis, F., Neuhauss, S. C., Schier,
A. F., Malicki, J., Stainier, D. Y., Abdelilah, S., Rangini, Z., MountcastleShah, E., et al. (1996). Mutations affecting development of the notochord
in zebrafish. Development 123, 117–128.
Sun, H., and Machu, T. K. (2000). Bicuculline antagonizes 5-HT3A and
[alpha]2 glycine receptors expressed in Xenopus oocytes. Eur. J. Pharmacol.
391, 243–249.
Tegner, J., Matsushima, T., el Manira, A., and Grillner, S. (1993). The spinal
GABA system modulates burst frequency and intersegmental coordination in
the lamprey: Differential effects of GABAA and GABAB receptors. J.
Neurophysiol. 69, 647–657.
Teraoka, H., Urakawa, S., Nanba, S., Nagai, Y., Dong, W., Imagawa, T.,
Tanguay, R. L., Svoboda, K., Handley-Goldstone, H. M., Stegeman, J. J.,
et al. (2006). Muscular contractions in the zebrafish embryo are necessary to
reveal thiuram-induced notochord distortions. Toxicol. Appl. Pharmacol.
212, 24–43.
Thisse, C., Thisse, B., Schilling, T. F., and Postlethwait, J. H. (1993). Structure
of the zebrafish snail1 gene and its expression in wild-type, spadetail and no
tail mutant embryos. Development 119, 1203–1215.
Tingle, C. C., Rother, J. A., Dewhurst, C. F., Lauer, S., and King, W. J. (2003).
Fipronil: Environmental fate, ecotoxicology, and human health concerns.
Rev. Environ. Contam. Toxicol. 176, 1–66.
Triller, A., Rostaing, P., Korn, H., and Legendre, P. (1997). Morphofunctional
evidence for mature synaptic contacts on the Mauthner cell of 52-hour-old
zebrafish larvae. Neuroscience 80, 133–145.
Westerfield, M. (2000). The Zebrafish Book. University of Oregon Press,
Eugene, OR.
Wirth, E. F., Pennington, P. L., Lawton, J. C., DeLorenzo, M. E., Bearden, D.,
Shaddrix, B., Sivertsen, S., and Fulton, M. H. (2004). The effects of the
contemporary-use insecticide (fipronil) in an estuarine mesocosm. Environ.
Pollut. 131, 365–371.
Yan, D., Pedersen, S. E., and White, M. M. (1998). Interaction of D-turbocurarine
analogs with the 5HT3 receptor. Neuropharmacology 37, 251–257.