CRYOLITE INDUCED MORPHOLOGICAL CHANGE IN THE …€¦ · Additionally, sodium fluoride (NaF) and...

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].




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.




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.




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.




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.




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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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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