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Research report Fluoride 45(1)58–64January-March 2012
Cryolite induced morphological change in the compound eyeof
Drosophila melanogaster
Podder, Akbari, Roy5858
CRYOLITE INDUCED MORPHOLOGICAL CHANGE IN THE COMPOUND EYE OF
DROSOPHILA MELANOGASTER
Sayanti Podder,a Shabana Akbari,a Sumedha Roya
Burdwan, India
SUMMARY: Treatment of the larvae of the common fruit fly
Drosophila melanogasterwith 10, 20, and 40 ppm of the F-containing
insecticide cryolite (Na3AlF6) through itsnormal food resulted in
abnormal morphology of its compound eye. A ridge-likeappearance of
the mechanosensory bristles of the ommatidia was apparent
byscanning electron microscopy (SEM). Interestingly, ommatidial
disorganization wasgreater with 10 ppm cryolite than with 20 and 40
ppm. These results indicate that theuse of cryolite as an
insecticide in fruit orchards may cause morphologicalalterations in
a non-target organism.Keywords: Cryolite; Drosophila melanogaster;
Fruit fly eyes; Ommatidia; Scanning electron microscopy (SEM).
INTRODUCTION
Among agricultural pesticides, certain inorganic fluorides are
noted for havingundesirable side effects, e.g., on the reproductive
cycle of the silkworm.1Additionally, sodium fluoride (NaF) and
stannous fluoride (SnF2) have been foundto have mutagenic effects
on the common fruit fly Drosophila melanogaster.2Atmospheric HF
also increases the frequency of sex-linked recessive lethality
andsterility in Drosophila melanogaster.3 Another F compound,
cryolite (Na3AlF6), isa widely used insecticide used on many
fruits, vegetables, and ornamental cropsfor protection against leaf
eating pests. Several studies with different
insecticides,fungicides, and pesticides have manifested
well-defined effects on the life cycle,hatchability, and emergence
of flies in Drosophila melanogaster.4-6 The presentwork aimed to
explore any detectable change in the external morphology of
thecompound eye of the adult fruit fly as a function of exposure of
the eggs and larvaeto cryolite. Interestingly, this model insect
used in this study is not considered to bean insect pest.
Investigation of non-target organisms from this perspective
wouldtherefore appear to be beneficial for evaluating the risk
under which otherorganisms including the humans are exposed.7
MATERIALS AND METHODS
For this study of the effect of cryolite on the compound eyes of
Drosophilamelanogaster, a Scanning Electron Microscope (S-530
HITACHI) was used toexamine the external morphology and structural
changes in the eyes.
Cryolite exposure: For treatment of the eggs and larvae, three
concentrations (10,20, and 40 ppm) of cryolite (Loba Chemie Pvt.
Ltd, India) were prepared in waterand mixed with Drosophila food
medium containing agar, corn meal, sucrose, andyeast at 22±1ºC.
Fifty Drosophila eggs were introduced into each food
preparationcontaining the three cryolite solutions, along with a
control containing only thenormal food constituents. Triplicate
sets of each treatment as well as the control
aCytogenetics Laboratory, University of Burdwan, Rajbati,
Burdwan, Pin-713104, West Bengal,India. Corresponding author: Dr
Sumedha Roy, Cytogenetics Laboratory, University of
Burdwan,Rajbati, Burdwan, Pin-713104, West Bengal, India. E-mail:
[email protected].
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Research report Fluoride 45(1)58–64January-March 2012
Cryolite induced morphological change in the compound eyeof
Drosophila melanogaster
Podder, Akbari, Roy5959
group were used for the study. The eggs were allowed to hatch
and the larvae wereallowed to grow in the respective food medium
throughout their development.
Adult flies were collected randomly from each treatment group in
2.5%gluteraldehyde for fixation and then dried in graded alcohol.
Critical point dryingwas carried out in the CPD Machine (HCP-2
HITACHI). Finally gold coating ofthe eyes was done using IB-2 Ion
Coater (EIKO ENGINEERING) for study underSEM.
RESULTS
Examination after gold coating of the compound eye of
Drosophilamelanogaster, which consists of a regular,
crystalline-like array of some 800ommatidia (simple eyes), under
scanning electron microscopy (SEM) imaging at300× magnification
showed the even distribution of the mechanosensory bristlesbetween
ommatidia in the control flies (Figure 1).
However, as seen in Figure 2, these bristles became very
disrupted anddisorganized with disordered ommatidia from the
treatment with 10 ppm cryolite.With the 20 ppm cryolite (Figure 3)
the bristles were also disoriented and showed aprominent ridge-like
appearance, but the effect appeared to be less in comparisonto the
effect from the 10 ppm treatment.
Figure 1. SEM imaging at 300× magnification showing the normal,
organized alignment ofmechanosensory bristles along with properly
arranged ommatidia in control adult Drosophilamelanogaster fed a
normal diet from larval hatching.
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Research report Fluoride 45(1)58–64January-March 2012
Cryolite induced morphological change in the compound eyeof
Drosophila melanogaster
Podder, Akbari, Roy6060
Figure 2. Complete disorientation of the mechanosensory bristles
and disorganized ommatidialstructures visible by SEM under 300×
magnification in Drosophila melanogaster maintainedfrom egg
hatching on food containing 10 ppm cryolite.
Figure 3. Disoriented mechanosensory bristles and disorganized
ommatidial structures visible by SEM at 300× magnification in adult
Drosophila melanogaster maintained from egg hatching on food
containing 20 ppm cryolite.
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Research report Fluoride 45(1)58–64January-March 2012
Cryolite induced morphological change in the compound eyeof
Drosophila melanogaster
Podder, Akbari, Roy6161
With the higher 40 ppm concentration, the ommatidial alignment
and bristleorientation appeared to be similarly affected as in the
20 ppm group (Figure 4).
DISCUSSION
The compound eye of Drosophila melanogaster is an effective
model fordetailed investigation of cell signaling, control of cell
proliferation, neuronalconnectivity, vesicular transport, etc.
Undoubtedly vesicular transport has a veryimportant role in eye
development. Rab1 and Rab6 genes have a key role inprocessing
and/or transporting rhodopsins.8-10 The Drosophila eye is also a
modelsystem for analyzing mutations that disrupt trafficking of
molecules to lysosomesand lysosome-related organelles.11 It is also
well documented that the Rab11 geneis one of the key players
associated with Drosophila eye development.12Mammalian genomes
contain more than 60 known Rab genes.13
Pattern formation in the developing Drosophila eye begins in the
eye disc at thethird larval instar and is known to be closely
associated with a morphologicalindentation in the disc called
morphogenetic furrow, which moves across the discepithelium in the
direction from posterior to anterior.14 From there it is
apparentthat the pattern of classical eye development occurs during
the third larval instar.Hence, any change in the developmental
programme of third instar larvae wouldbe expected to manifest
impairment in adult eye development. As seen in thepresent study,
distinct morphological changes in the eye seen in Figures 2, 3, and
4indicate that the eggs and larvae subjected to the different
concentrations ofcryolite during the larval stage caused distinct
phenotypic changes in the adult eye.
Figure 4. Lesser disorientation of the mechanosensory bristles
in the disorganized ommatidial structures visible by SEM at 300×
magnification showing ridge-like alignment of ommatidia in adult
Drosophila melanogaster maintained from egg hatching on food
containing 40 ppm cryolite.
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Research report Fluoride 45(1)58–64January-March 2012
Cryolite induced morphological change in the compound eyeof
Drosophila melanogaster
Podder, Akbari, Roy6262
In Drosophila melanogaster, the fat facets (faf) gene encodes a
deubiquitinatingenzyme essential in differentiating tissues of the
eye and ovary of fly.15,16 Thedevelopment of the eye discs in D.
melanogaster is reported to be impaired due tothe facet-inhibitory
effect of chemicals like mitomycin-C and nitromine.17Similarly, in
the present study, it appears likely that cryolite might have
inducedaberrant deubiquitinisation resulting in the changes
observed here in themorphology of the eye.
Over the years, D. melanogaster has been used as a model to
study the effect ofvarious pesticides and insecticides. In the
present study, the effect of the fluorine-containing insecticide
cryolite on the morphology of Drosophila has beeninvestigated. To
the best of our knowledge, to date there seems to be only
fewreports on the effect of F on the ommatidia of Drosophila. Here,
as noted in theresults, we have found that treatment of the larvae
with cryolite caused distinctchanges in the morphology of ommatidia
as observed through scanning electronmicroscopy (SEM). Owing to its
precisely organized architecture, the Drosophilacompound eye can be
viewed as a very good model for addressing questionsconcerning
several important biological processes including cell
signaling.
Ommatidia are facets that make up the Drosophila eye that are
connected to thenervous system. The adult compound eye consists of
a regular, almost crystalline,array of nearly 800 ommatidia,18 each
containing 8 photoreceptor neurons.Ommatidia develop in a monolayer
epithelium, contained within the eye-antennalimaginal disc in which
cells extend from the apical surface to the basalmembrane.19 During
photoreceptor differentiation, ommatidial clusters rotate 90ºtoward
the poles of the eye disc.20 Ommatidial rotation can be divided
into twophases: the first 45º of quick rotation that is completed
by ommatidial row 6posterior to the morphogenetic furrow, followed
by a second 45º of slowrotation.21 The nemo (nmo) gene has been
identified as the only gene that has beenspecifically found to
affect this rotation process.22 Figures 2, 3, and 4 show adistinct
disorganization and ridge formation in the compound eye from
exposure ofthe larvae to cryolite that may be due to abnormal
functioning of the nemo gene atthe time of differentiation.
Interestingly, maximum noticeable disorganizationoccurred with the
10 ppm treatment. At the higher cryolite concentrations
similarommatidial disorganization occurred, but it decreased
noticeably with increasingconcentration. In agreement with this
finding, it should be noted that other studieshave shown that F in
vivo can often be more genotoxic at lower concentrationsthan at
higher concentrations.23,24.
Finally, the rough surface of the compound eyes with unusual
ridge and furrow-like changes may be due to subtle defects in cone
cells, pigment cells, and/orbristles in the final stages of eye
development.25 The intensely roughened eyesurface seen in Figure 2
may have arisen from these kinds of defects. Thecomparison of the
affected eyes with the normal eye structure (Figure 1) clearlyshows
that the ordered and well-orchestrated nature of normal eye
developmentseems to be completely lost as a result of exposure of
the larvae to the variousconcentrations of cryolite.
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Research report Fluoride 45(1)58–64January-March 2012
Cryolite induced morphological change in the compound eyeof
Drosophila melanogaster
Podder, Akbari, Roy6363
Further study with a wider range of cryolite concentrations
(above 40 ppm andbelow 10 ppm) would no doubt help to determine the
instar stage at which thesechanges occur. In the present study the
concentrations of cryolite were selected inaccordance with those
actually applied in agriculture. Generally, in practice, lowerdoses
are preferable for fruit trees. Since the present study dealt with
the effect ofcryolite on a single generation of Drosophila, it
cannot offer a justified theory forthe impact of repeated exposure
to this insecticide generation after generation. Afurther
investigation with three or more generations of fruit flies can be
expected toprovide a fuller understanding necessary for proposing a
plausible mechanism forthe findings reported here.
ACKNOWLEDGEMENTS
We thank the Head, Department of Zoology, and we gratefully
acknowledge theDepartment of Zoology for providing the
infrastructural facilities. The Drosophilamelanogaster stock was
provided by Professor RN Chatterjee, Department ofZoology, Calcutta
University. We also thank Dr Srikanta Chakraborty, USIC(University
Science Instrumentation Centre), The University of Burdwan, for
hishelp during the Scanning Electron Microscopy.
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Copyright © 2012 The International Society for Fluoride Research
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Editorial Office: 727 Brighton Road, Ocean View, Dunedin 9035,
New Zealand.
SUMMARY: Treatment of the larvae of the common fruit fly
Drosophila melanogaster with 10, 20, and 40 ppm of the F-containing
insecticide cryolite (Na3AlF6) through its normal food resulted in
abnormal morphology of its compound eye. A ridge-l...
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