Asymmetric patterns in the cranial skeleton of zebrash (Danio rerio)

exposed to sodium pentachlorophenate at different embryonic

developmental stages
Faviel Lo pez-Romero
a
, Gerardo Zu niga
b
, Fernando Martnez-Jero nimo
a,n
a
Laboratorio de Hidrobiolog a Experimental, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Prol. Carpio esq. Plan de Ayala S/N,
Col. Santo Tomas, Mexico, D.F., 11340. Mexico
b
Laboratorio de Variacion y Evolucion Biologica. Escuela Nacional de Ciencias Biologicas, Instituto Polite cnico Nacional, Prol. Carpio esq. Plan de Ayala S/N,
Col. Santo Tomas, Mexico, D.F., 11340. Mexico
a r t i c l e i n f o
Article history:
Received 16 March 2012
Received in revised form
8 June 2012
Accepted 8 June 2012
Available online 19 July 2012
Keywords:
Fluctuating asymmetry
Geometric morphometrics
Pesticides
Teratogenesis
a b s t r a c t
Bilaterally symmetric organisms display mirror copies of their structures on both sides of the body, and
the development of both sides is regulated by the same set of genes. Environmental variations can
directly affect phenotype, and exposure to chemical contaminants at certain stages may modify
embryonic development. The pesticide sodium pentachlorophenate (NaPCP) was used at the no-
observable-effect concentration (NOEC) to determine the degree of susceptibility of zebrash (Danio
rerio) embryos in different developmentally susceptible windows (zygote, blastula, gastrula, segmenta-
tion, pharyngula and larva). Shape variation in the zebrash viscerocranium and uctuating asymmetry
(FA), which increases in direct proportion to environmental stress, induced by exposure to NaPCP were
measured with geometric morphometrics. Procrustes ANOVA was performed to estimate the shape
variation around a symmetric consensus that accounted for the following factors: shape variation in
individuals (I), variation by sides (S), the Individuals Sides interaction (I S), and the stages of
exposure to the toxicant (Stages). Factors I, S and IxS accounted for most of the morphological variation
(po0.0001). Extensive deformities throughout the viscerocranium occurred during the window of
exposure from gastrula to larva. Embryonic mortality occurred and was dependent on the stage of
exposure. The NOEC concentration of NaPCP affected embryonic development in D. rerio and also
induced lethal effects in embryos. FA was determined in both unexposed and NaPCP-exposed embryos
and was greater in the control than in some exposure windows; besides, no correlation was found
between FA and developmental stages, so our results do not support FA as a bioindicator of chemical
stress but conrm its value in the study of morphological effects of toxicants.
& 2012 Elsevier Inc. All rights reserved.
1. Introduction
Bilaterally symmetric organisms use the same set of genes to
develop structures on both sides of the body, and environmental
factors directly inuence this process, resulting in the production
of asymmetric phenotypes under certain conditions (Dongen,
2006). The absolute value of the difference between the left and
right sides (9LR9) of a symmetric character should have a mean
tending to zero; small random variations around the mean value
are termed uctuating asymmetry (FA) (Palmer and Strobeck,
2003). FA has been used as a measure of developmental instability
in environmentally stressed populations, and it has been deter-
mined that FA increases in direct proportion to environmental
stress (Mller and Swaddle, 1997; Allenbach et al., 1999; Estes
et al., 2006; Palmer et al., 2010). Nevertheless, other studies do not
support FA as a measure of environmental stress caused by
temperature (Longson et al., 2007) or chemically-induced stress
(Floate and Coghlin, 2010); according to Fowler and Whitlock
(1994), FA was not related to inbreeding in population. Because
the sensitivity and response to environment stress vary depending
on the developmental stage at which organisms are exposed, it is
also necessary to test for sensitive exposure windows (Gilbert and
Epel, 2009).
FA has been evaluated using geometric morphometric techni-
ques and multivariate analysis (Klingenberg and McIntyre, 1998;
Allen and Leamy, 2001; Klingenberg et al., 2002; Palmer et al.,
2010). Geometric morphometrics are useful tools to study the
shape because they eliminate differences in size, location and
orientation, unlike traditional morphometrics (Zelditch et al., 2004).
Geometric morphometrics use landmarks (biologically homologous
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Ecotoxicology and Environmental Safety
0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ecoenv.2012.06.008
n
Corresponding author. Fax: 55 5396 3503.
E-mail address: fjeroni@ipn.mx (F. Martnez-Jero nimo).
Ecotoxicology and Environmental Safety 84 (2012) 2531
anatomical loci), which permit greater accuracy in the determina-
tion of shape variation and minimise measurement error (Leamy
and Klingenberg, 2005). FA studies using geometric morpho-
metrics should consider the type of symmetry that was to be
evaluated. For example, matching symmetry is present when a
mirror image of the structure is found on either side of the body,
while in object symmetry the structure is divided into equal
halves by a plane running through it (Klingenberg et al., 2002).
The cranium is a symmetric structure, the development of
which involves several processes including the induction of the
cranial neural crest (CNC) and the specication, migration and
differentiation of neural crest cells (NCCs) (Knight and Schilling,
2006). The neurocranium is derived from the CNC and the
mesoderm, and the viscerocranium is derived from the CNC,
mesoderm and endoderm. NCCs arise from the dorsal and lateral
regions of the neural ectoderm, which migrate during segmenta-
tion stages (Kimmel et al., 1995) in separate directions to
populate the seven pharyngeal arches that are separated by
endodermal pouches (Yelick and Schilling, 2002). The specica-
tion of NCCs is related to the location of the rhombomeres
(hindbrain); the more anterior cells form the mandibular arch
and hyoid (Schilling and Kimmel, 1994). Each arch gives rise to
different structures: the rst arch forms Meckels cartilage and
the dorsal palatoquadrate; the second arch gives rise to the
ceratohyal, basihyal, interhyal and hyosimplectic; and the third
to seventh arches form the pharyngobranchial, epibranchial,
ceratobranchial, hypobranchial and basibranchial (Yelick and
Schilling, 2002).
Sodium pentachlorophenate (NaPCP) is a chlorinated hydro-
carbon that is used as a herbicide, insecticide, fungicide and
bactericide, although it is primarily used as a wood preserver
(Wall and Stratton, 1994). NaPCP has toxicological properties: it is
probably an endocrine disruptor, mutagen and carcinogen (Chang
et al., 2009). In sh exposed to NaPCP, this compound causes
negative effects on development, hatching, growth and larval
survival (Nguyen and Janssen, 2001, 2002; Nguyen et al., 1999). In
aquatic toxicology guidelines, this toxicant is proposed as a
reference toxicant (Schirmer et al., 2008).
The zebrash is a model organism for diverse studies, espe-
cially those related to developmental biology (Schilling et al.,
1996; Neuhauss et al., 1996; Heisenberg et al., 1996). It has been
proposed as a test organism for toxicological studies (Hill et al.,
2005) due to its rapid development (somitogenesis is completed
at 24 h post-fertilisation), and its transparent embryos make it
ideal for toxicological and teratogenic studies (Yang et al., 2009).
NaPCP is a potential teratogen and a reference toxicant that can
have effects at different developmental stages. The aim of this
study was to determine the developmental windows at which
zebrash embryos are more susceptible to NaPCP by evaluating
uctuating asymmetry as well as variation in cranium shape and
size using geometric morphometric techniques.
2. Material and methods
2.1. Fish maintenance
Wild-type AB
*
line zebrash adults obtained from the Zebrash International
Resource Center in Oregon (USA) were maintained in 40 L aquariums containing
reconstituted water (de-ionised water with 60 mg L
1
Instant Ocean
s
marine
salts), at pH 77.5 and 2570.5 1C, with a 14 h:10 h (light:dark) photoperiod. The
water quality and cleanliness of aquariums were periodically monitored according
to Trevarrow (2004). Males and females were maintained separately in different
aquariums prior to mating and were fed with nutritionally balanced food pellets
twice daily supplementing this diet with live neonates and juveniles of the
cladoceran Daphnia magna. Zebrash eggs were collected with a siphon and
examined under a stereoscopic microscope to eliminate unfertilised eggs, while
the fertilised eggs were maintained up to the moment of exposure to the toxicant
in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl
2
, 0.33 mM MgSO
4
, 10
5
%
methylene blue) at room temperature (ca. 25 1C).
2.2. Exposure to NaPCP
The concentration we used (0.056 mg L
1
) was the one reported by Nguyen
and Janssen (2001), which, according to these authors, does not have lethal effects
(i.e., NOEC); this was dissolved in E3 medium. Our experimental model was
designed to establish susceptibility windows. The rst exposure treatment (ZB)
was applied between the zygote stage, i.e., 0 h post-fertilisation (hpf), (to ensure
that specimens were at this stage; eggs having 2 4 cells were selected) and the
blastula stage (2.25 hpf). Other treatments were applied between the blastula and
gastrula stages (2.255.5 hpf; BG), the gastrula and segmentation stages 5.5
10 hpf; GS), the segmentation and pharyngula stages (1024 hpf, SP), and the
pharyngula and larva stages (2472 hpf; PL); these treatments were staged as
described by Kimmel et al. (1995). For each described treatment, 24 embryos were
individually exposed to 2 mL of the test solution per well containing the NOEC of
NaPCP, in 24-well plates; individual plates were used for each treatment group. In
the control series, 24 embryos were also individually distributed in 24-well plates
containing 2 mL E3 medium.
After exposure, each set of eggs was washed with E3 medium, placed in a new
multi-well plate with fresh medium (E3), and incubated at 28 1C until preserva-
tion at 120 hpf in 3.7% PBS-buffered formalin. Although two higher concentrations
of NaPCP (0.1 and 0.076 mg L
1
) were also assayed, embryos exposed at these
concentrations were not used since the toxicant induced the loss of branchial
elements, which did not allow for geometric morphometric analysis.
2.3. Staining of larvae with alcian blue
Cranial abnormalities were visualised by staining the cartilage with the alcian
blue technique, as described by Javidan and Schilling (2004): 0.5% alcian blue in
ethanol:acetic acid solution (80:20) and staining for 6 h. The stained larvae were
maintained in a PBSglycerol solution (30:70) until microscopic examination.
2.4. Image processing
The stained larvae were examined under a Nikon Alphaphot-z YS2-H micro-
scope with a Canon PowerShot SD550 photographic camera attached. The eyes
and vitelline sac were removed in most cases to facilitate observation of the
cranial cartilage. At least three images were taken of each specimen in the ventral
view (viscerocranium) at different depths of the eld for subsequent focus
stacking with Photoshop CS4; two sets of images were independently obtained
for each individual as a way to determine the error in taking images.
2.5. Geometric morphometrics
Symmetrically distributed landmarks in the viscerocranium were established
based on the images taken in the ventral view. The initial conguration comprised
41 landmarks, but because of the poor precision of digitisation at the end limits for
the hyomandibular and the basibranchials, seven landmarks corresponding to
these structures were excluded from the analysis (Fig. 1). Landmarks were
digitised with the program ImageJ ver.1.42q (http://rsb. info. nih. gov/ij/); the
conguration of each individual was captured twice to estimate measurement
error; error in taking images was not considered relevant for the analysis, because
Fig. 1. Landmark conguration in viscerocranium of zebrash; 4 median and
30 paired landmarks are shown.
F. Lopez-Romero et al. / Ecotoxicology and Environmental Safety 84 (2012) 2531 26
each set of images for stacking was not uniform for the same individual, so each
group of measures was independent.
A Procrustes analysis was performed to eliminate the differences in the size,
location and orientation of the digitised images. This analysis uses the centroid of
the landmark conguration as the starting point from which rotation and scaling
occur in order to eliminate differences due to discrepancies in size. The Procrustes
analysis was applied based on the symmetric component of variation (Klingenberg
et al., 2002). Principal components analysis (PCA) was carried out on a covariance
matrix to estimate shape variation among individuals. To determine FA values, a
Procrustes ANOVA was performed on a symmetric consensus (Klingenberg and
McIntyre, 1998) considering the following factors: Individual (I), which estimates
shape variation among individuals; Side (S), which estimates the effects related to
directional asymmetry; the Individual Side interaction (I S), which estimates
FA; and the factor associated with developmental windows of exposure to NaPCP
(Stages). The used model was ShapeI SS I Stageerror. According to this
model, directional asymmetry (DA) is supposed to be the same in all treatments;
in order to discard the effect of S, in the present study DA was individually
determined. PCA was performed on the covariance matrix of IxS interactions to
estimate FA-related deformation, and visualised with relative warps to estimate
shape changes; these analyses were made with the full dataset.
To determine the stage at which exposed specimens had increased FA, a
Procrustes ANOVA was performed for the dataset subdivided by each stage, in this
way the processes affected during different developmental stages (e.g., cell
division, specication, migration, and differentiation) could be inferred. Patterns
of shape variation and FA among individuals were visualised on a thin-plate spline
deformation with outliers with respect to symmetric consensus.
All analyses were conducted with MorphoJ 1.2d (Klingenberg, 2011). Although
differences in size were eliminated in the Procrustes analysis, size-related data
were retained in the form of the size of the centroid. The logarithm for centroid
size (logarithm of the squared root of the summed squared distance of each
landmark to the centroid, represented as LogCentroid) was used to determine the
existence of differences in cranium size dependent on the exposure stage to NaPCP
with Tukeys honestly signicant difference (HSD) test; the effect of stage on
individual FA values (distance between the left and right of each individual), from
the covariance matrix of Procrustes ANOVA, was determined with an ANOVA test.
All the analyses were done using R software.
3. Results
3.1. Effects of NaPCP on larval size and survival
NaPCP at the Non-Observed Effect Concentration (NOEC) induced
unexpected mortality in some of the exposed specimens (evident as
coagulated embryos). Observed mortality was 25% in exposure
window ZB, 21% in the exposure window GS, 17% in windows B
G and SP, and 8% mortality in PL exposure window. No mortalities
occurred in the control group through the end of the assay.
The effect of NaPCP on cranium size (Fig. 2) shows that crania
were larger in exposed embryos during the two earliest develop-
mental stages (ZB and BG) compared to all other treatment
groups; the smallest size was found in the SP stage. Signicant
differences in the cranial size among exposure stages (po0.0001)
were observed with ANOVA, and Tukeys HSD test which showed
that the rst two stages did not differ from the control group. The
last three stages were all signicantly different from the control
but not signicantly different from each other (po0.05).
3.2. Cranial morphology and anomalous elements
Larvae showed different modications depending on the stage
at which they were exposed. For example, the protrusion of
Meckels cartilage and the lengthening of the branchial arches
were observed in stages ZB and BG, while in the GS, SP and
PL stages, Meckels cartilage was observed to be retracted. These
last stages showed thickening in all cartilaginous elements and a
wider cranium exposure; in addition, the distance between the
branchial arches in the last three stages was shorter than in the
control group and the rst two exposure stages.
Anomalous cartilaginous elements were found at several exposure
stages. In the GS stage, the midline of Meckels cartilage projected
posteriorly (Fig. 3A). In the SP stage, ectopic cartilage was located
between Meckels cartilage and the ceratohyals (Fig. 3B). Finally, in
the PL stage, a smaller right eye (Fig. 3C) and a thickened cartilage in
the specimens were observed. The described abnormalities occurred
in only one of the 24 specimens in each treatment group. No other
alterations were found, except in those specimens exposed to higher
concentrations of NaPCP (images not shown).
3.3. Procrustes ANOVA
Procrustes ANOVA of viscerocranium shape variation results are
shown in Table 1. The factor Individual explained the largest propor-
tion of the total variation around the symmetric consensus. The
second factor that contributed to the highest variation was Stages,
indicating that shape variation was strongly dependent on the stage
at which individuals were exposed, and there was a signicant
contribution of FA as indicated by the IxS interaction, which was
the third highest factor, denoting an effect of increased developmen-
tal instability caused by NaPCP exposure. Lastly, the Side factor,
related to directional asymmetry (DA), also had an effect on shape
variation, but it was lower than the effect of the I S factor; for this
reason, DA did not mask the effect of the other factor and, according
to Klingenberg and Monteiro (2005), DA could be more relevant in
quantitative genetics studies, since genetics could contribute more to
a deviation of individual symmetry in a population. All factors were
signicant (po0.0001), and measurement error did not exceed FA
values and, because measurement error did not exceed FA values,
results are considered to be valid. When the entire dataset was
considered, changes in symmetry with respect to consensus, which
was used to estimate directional asymmetry, showed that the
ceratobranchial and the articulation between the palatoquadrate
and hyosymplectic are structures where shape variation predomi-
nantly occurs.
The Procrustes ANOVA applied to the subdivided dataset by
stage showed the different contributions of the factors to shape
variation. In the control group, the largest proportion of the total
variation was explained by factors I and I S, which suggests that
FA is present in unexposed specimens, furthermore the effect of
Fig. 2. Effect of sodium pentachlorophenate (NaPCP) on cranium size of zebrash
embryos. Logarithm of centroid size in each developmental stage: zygoteblastula
(ZB), blastulagastrula (BG), gastrulasegmentation (GS), segmentationphar-
yngula (SP), and pharyngulalarva stages (PL); CT is for the control. Standard
error limits are shown; letters indicate signicant differences (Tukeys HSD test,
po0.05). Sample size per treatment n24.
F. Lopez-Romero et al. / Ecotoxicology and Environmental Safety 84 (2012) 2531 27
Side (DA) was not signicant. FA explained most of the variation in
the subset BG stage followed by the subsets SP, GS and PL
stages, which suggests that specimens in these stages were most
susceptible to NaPCP exposure, i.e., they displayed more asymmetric
phenotypes. In all the subsets, FA values were signicantly different
from zero (po0.0001), and exceeded measurement errors.
3.4. Shape variation
The PCA used to display deformation patterns in the sym-
metric component showed that the variation in individual shape
is located at the distal ends of the ceratobranquiales (PC1), which
is expressed as elongated, posteriorly extended branchial arches
that narrow towards the midline (Fig. 4). In addition, shape
changes in the anterior region of Meckels cartilage were also
observed, giving rise to a more protruding mandible. Ceratobran-
chials showed shape variations (PC2), which were different from
those observed in PC1; these structures were shortened, the
basihyal extended anteriorly, and the pharyngeal teeth varied in
location. The articulation between the interhyal and ceratohyal
showed shape variation (PC3): elongation of the ceratohyals
was observed. Finally, the anterior region of Meckels cartilage
showed variation (PC4), which was similar to that observed
in PC1. Changes also occurred in the second ceratobranchial
hypobranchial joint and palatoquadrateMeckels cartilage joint.
The FA displayed different shape variation patterns (Fig. 5A);
changes centred on the ceratohyal joint and especially on the
second ceratobranchialhypobranchial joint (PC1). The distal ends
of the ceratobranchials showed deformation (PC2) and, conse-
quently, the branchial arch length showed greater asymmetry in
this principal component. A similar pattern of these structures
was observed in PC3, although in this case, an asymmetric change
occurred in the palatoquadrateMeckels cartilage joint. Finally,
the ceratobranchial length and the symplectic position displayed
shape variation (PC4). Although deformation patterns were clear,
when comparing the individual FA values among exposure stages
(windows) (Fig. 5B), no relation to FA was found (ANOVA,
p0.901). The magnitude of FA was quite uniform in all the
exposed windows, because of that, a separated Procrustes ANOVA
was performed for each stage (as described above, see Table 1),
and it was observed that the interaction I S had different
signicance values, depending on the exposure window. There-
fore, although no relation in FA dependent on exposure stage was
found with the individual FA values, each stage exhibited a
signicant I S interaction that accounts for its shape variation.
4. Discussion
The results of the present study show that the NOEC value for
NaPCP induced cranial deformities and mortality in zebrash
embryos. Changes were present mainly in Meckels cartilage,
the ceratohyals, the ceratobranchials and, to a lesser extent, in
other cranial cartilages. The anatomical deformities observed in
the larvae were subtle, contrary to what has been reported by
Nguyen and Janssen (2001), who found no changes in zebrash
embryos exposed to NaPCP but did nd pericardial edema
development in Clarias gariepinus larvae exposed to this com-
pound. Likewise, Duan et al. (2008) reported tail malformations in
zebrash embryos exposed to pentachlorophenol (0.08 mg/L), a
compound similar to NaPCP. Expression of anatomical changes in
the various stages of exposure suggests the need to carry out
more complete anatomical and physiological analyses (e.g. on
Fig. 3. Abnormalities in larvae of Danio rerio exposed to NaPCP at different developmental stages, GS (A), SP (B) and PL (C).
Table 1
Procrustes ANOVA for partitioning of the variation around the symmetric
consensus in embryos of Danio rerio exposed to NaPCP at different developmental
stages: control (CT), zygoteblastula (ZB), blastulagastrula (BG), gastrula
segmentation (GS), segmentationpharyngula (SP), and pharyngulalarva
stages (PL).
Factor SS MS df F p
Stage 0.486393 0.003040 160 21.51 o0.0001
Individual 0.515465 0.000141 3648 1.90 o0.0001
Side 0.019687 0.000615 32 8.28 o0.0001
IndSide 0.283076 0.000074 3808 23.70 o0.0001
Error 0.024093 0.000003 7680
CT
Individual 0.135603 0.000202 672 2.72 o0.0001
Side 0.003254 0.000102 32 1.37 0.0864
IndSide 0.049918 0.000074 672 126.79 o0.0001
Error 0.000825 0.000001 1408
ZB
Individual 0.071121 0.000131 544 2.48 o0.0001
Side 0.002981 0.000093 32 1.77 0.0065
IndSide 0.028654 0.000053 544 64.61 o0.0001
Error 0.000939 0.000001 1152
BG
Individual 0.107258 0.000176 608 1.20 0.0134
Side 0.004328 0.000135 32 0.92 0.6
IndSide 0.089614 0.000147 608 213.18 o0.0001
Error 0.000885 0.000001 1280
GS
Individual 0.076935 0.000134 576 1.87 o0.0001
Side 0.005492 0.000172 32 2.40 o0.0001
IndSide 0.041238 0.000072 576 4.48 o0.0001
Error 0.019429 0.000016 1216
SP
Individual 0.070085 0.000115 608 1.37 o0.0001
Side 0.013214 0.000413 32 4.92 o0.0001
IndSide 0.051057 0.000084 608 109.89 o0.0001
Error 0.000978 0.000001 1280
PL
Individual 0.059041 0.000092 640 2.34 o0.0001
Side 0.004912 0.000153 32 3.89 o0.0001
IndSide 0.025265 0.000039 640 40.47 o0.0001
Error 0.001311 0.000001 1344
F. Lopez-Romero et al. / Ecotoxicology and Environmental Safety 84 (2012) 2531 28
cardiac function, hepatic function, etc.) to nd and determine the
range of alterations that a toxic compound can induce during
embryonic development.
While the Organisation for Economic Co-operation and Devel-
opment (OECD) recommends evaluating the toxic effects of a
chemical compound during early developmental stages (OECD,
2006), toxicological studies focus mostly on evaluating exposure
times and toxic concentrations. Our results using zebrash sup-
port the idea of analysing the various windows of exposure
during development (Barata et al., 2002; Gonza lez-Doncel et al.,
2004; Oxendine et al., 2006) to determine the risk level and
probable mechanism of action and to assess better the potential
toxic effect of a given compound.
Geometric morphometrics analysis showed clear and signi-
cant differences in zebrash cranial size in the various stages of
embryonic development. Differences in viscerocranium size may
be the result of changes in the pattern of NCC migration,
alterations during the differentiation, maturation and prolifera-
tion of chondrogenic progenitor cells, or delayed development
(van Boxtel et al., 2010). However, the higher mortality at the
earliest stages, along with higher FA in the BG window, indicates
a possible relation to stress-induced polyphenism. Sandstr om and
Neuman (2003) found that sh exposed for a long term to paper
mill efuents (containing endocrine disruptors) grew faster but
showed a delay in sexual maturation; on the other hand, stressed
sh in the wild that grow at faster rates, possibly as part of an
ontogenic change to cope with predators and starvation during
early stages, were negatively selected, indicating that smaller size
could be an advantageous trait (Gagliano et al., 2007). Likewise,
most studies in toxicology indicate that growth is negatively
affected, and the direction of the modication in individual
growth rate (increase or decrease) could depend on the stressor
type. Furthermore, it has been documented that the cladoceran
Daphnia. schoedleri exposed to toxicants produced broods with
fewer but larger individuals than non- stressed females (Arzate-
Ca rdenas and Martnez-Jero nimo, 2012).
Fluctuating asymmetry (FA) has been considered a bioindica-
tor of population response to environmental stress (Allenbach,
2011). For instance, Gambusia afnis populations that occur in the
vicinity of paper mill efuent have been found to develop a
greater degree of FA than similar populations in undisturbed
rivers (Estes et al., 2006). Likewise, individuals from wild popula-
tions of the species G. afnis and Notropis ludibundus with a
smaller degree of FA exposed to the LC
70
of parathion or lindane
under laboratory conditions have been found to survive longer
than those with greater FA (Allenbach et al., 1999). However, our
results show that FA is present in both unexposed and NaPCP-
exposed zebrash and is greater in unexposed specimens than in
those exposed in stages ZB, GS and PL. Furthermore, in
exposed specimens, no correlation was observed between FA
and developmental stages, i.e., FA did not increase in magnitude
in the course of successive exposure windows. Our results
conrm, as has also been suggested by other studies (review by
Klingenberg, 2003), that the processes generating asymmetry in
organisms are not uniform in the various exposure windows or
stages of embryonic development of this and other species, and
we therefore suggest caution implied be exercised in handling FA
as an indicator of environmental stress.
On the other hand, while our results show that NaPCP affects
cranial morphogenesis in the various exposure windows, its
mechanism of action is poorly understood. It has been suggested
that chlorinated organic compounds interact with retinoic acid
(RA) receptors (Inoue et al., 2010); a possible explanation may be
that NaPCP modies natural ligand signalling in the RA pathway,
similar to the effects of PCP on retinoic acid response elements
(Dorsey et al., 2002). RA is essential for various developmental
processes since it acts on certain genes (e.g. Hox genes) required
for NCC specication (Marshall et al., 1996). This explains why
zebrash embryos exposed to high concentrations of NaPCP (data
not shown) display phenotypes with alterations (e.g., enlarge-
ment and narrowing of Meckels cartilage, defects along the
midline of the ethmoid cartilage and shortening of the distance
Fig. 4. Shape variation around the symmetric component. The diagrams show the principal components of variation among individuals, with respect to the average of the
original and reected landmark congurations. Shape change patterns are shown in each diagram, the symmetric consensus is represented by the black line, and the
variation around it is represented by the grey line. The percentages of the shared shape variation in the symmetric component are as follows: PC141.772%;
PC216.106%; PC38.991%; PC46.921%.
F. Lopez-Romero et al. / Ecotoxicology and Environmental Safety 84 (2012) 2531 29
between the basibranchials) similar to those expressed by speci-
mens treated with inhibitors of the enzyme CYP26, which con-
trols RA levels (Reijntjes et al., 2007). Furthermore, the absence of
Cyp26b1a in the zebrash dolphin mutant (Laue et al., 2008)
produces phenotypes with the same characteristics found in the
present study, suggesting that NaPCP does in fact interact with
the RA metabolic pathway.
In conclusion, our results indicate that morphologic changes
induced at NOEC concentration in apparently normal phenotypes
involve primarily cranial symmetry and shape. Observed effects
may be associated with disruption in RA signalling. The model of
exposure in specic windows allowed us to establish that the
stage most susceptible to NaPCP exposure was BG, and that the
use of geometric morphometrics increases the discriminative
capacity of subtle morphologic changes or abnormalities induced
by toxicants.
Acknowledgments
F. Lo pez-Romero is grateful for support from CONACYT
(323558) as well as the Programa Institucional de Formacio n de
Investigadores (PIFI) of the Comisio n de Operacio n y Fomento de
Actividades Acade micas (COFAA). F. Martnez-Jero nimo and G.
Zu niga are thankful for nancial support provided by the Sistema
de Estmulo al Desempeno de los Investigadores (EDI) and COFAA
of the Instituto Polite cnico Nacional. Also thanks to two anon-
ymous reviewers for the detailed evaluation of this manuscript,
and for many insightful suggestions and comments that helped
improving this nal version.
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