Abstract
The prerequisite for toxicity assessment of any chemical is the determination of its median lethal concentrations and thus, this study initiates with determination of chronic median lethal concentration of sodium fluoride (NaF) in a non-target organism Drosophila melanogaster, which was found to be 125–130 µg/mL. Further, chronic exposure to NaF at sub-lethal concentrations (10–100 µg/mL) resulted in significant reduction in total hemocyte number in general and plasmatocytes in particular, whereas, crystal cells were significantly increased in number. Since hemocytes are an integral part of innate immune system in Drosophila, their numerical fluctuation confirms fluoride-induced compromise in innate immunity status of Drosophila.
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Introduction
Any chemical substance, biological agent or antimicrobial disinfectant used against pest (like insects, plant pathogen, birds, mammals, fish, and nematodes) is known as pesticide. Use of pesticides is indispensable because physical and biological pest control methods are far less cost effective, thus, chemical control is an obvious choice. But most of these chemical pesticides are persistent in the environment and cause significant problem to non-target organisms. Among various pesticides used in agriculture, fluoride-containing ones form a small group. Sodium fluoride (NaF) being one such chemical, is widely used as fungicide, wood preservative for utility poles and for water fluoridation. Interestingly, it is also an active ingredient of toothpastes and mouth rinses. Drosophila melanogaster (fruit fly) which is genetically best known of all eukaryotic organisms is equally popular as a model organism in evaluating the toxic potential of any chemical (Mishra et al. 2013). Various studies report the effect of chemicals on the life cycle, morphology, fecundity and hatchability of Drosophila (Podder and Roy 2013; Rajak et al. 2013). To evaluate the toxicity of any chemical it is very important to determine its concentrations which can kill 50% of the organisms. The acute and chronic LC50 of another fluoride-containing pesticide, cryolite (sodium aluminum fluoride) have been worked out (Podder and Roy 2013) and the values are seen to have huge differences (acute LC50 is in the range of 150–158 µg/mL and chronic LC50 is 67,000 µg/mL cryolite concentrations). The acute LC50 of NaF in adult D. melanogaster has been reported to be 48,000 µg/mL (Mitchell and Gerdes 1973). Interestingly no report on chronic LC50 of the chemical in Drosophila is yet available. Due to this reason, the authors aim to determine the chronic LC50 of NaF in the larvae and pupae of Drosophila. This finding would essentially help in the process of dose selection for study on sub-lethal toxicity of the chemical.
Like other living organisms, D. melanogaster is known to combat any infection through immune system machineries, particularly the innate immune system, which comprises of humoral and cell mediated immune responses. Hemocytes are responsible for cell-mediated immune response and three different types of hemocytes, namely plasmatocyte, lamellocyte and crystal cells (PC, LC and CC), are reported from the fruit fly’s hemolymph (Shrestha and Gateff 1982) and their structures were reported earlier by three authors (Rajak et al. 2014). The total hemocyte count in D. melanogaster varied to a great extent after acute exposure to acephate, an organophosphate insecticide (Rajak et al. 2014). In another study, Das et al. (2006) noted sodium fluoride (NaF) induced oxidative stress and immunotoxicity following NaF exposure in rats. Further it was shown that NaF treatment lowered cellular immunity in the rats resulting from significant diminution in peripheral blood lymphocyte, monocyte and neutrophil counts in conjunction with a reduction in splenocyte counts. Similarly, chronic fluoride (F) intoxication causes biochemical changes as well as chromosome aberrations in mice brain and bone marrow cells (Bhatnagar et al. 2006; Podder et al. 2008a, b). Thus, in this study, chronic LC50 of NaF in larvae and pupae of D. melanogaster has been determined followed by selection of sub-lethal concentrations of NaF for further experiments. The experimental findings on differential hemocyte abundance in treated insects would prove helpful to confirm the immunotoxic potential of NaF.
Materials and Methods
Model Organism
Oregon-R strain of D. melanogaster cultured in Standard Drosophila Medium (SDM) was used as the model organism. SDM contain agar–agar (0.83%), sucrose (4.16%), corn meal (4.72%), yeast extract (1.66%), nipagin (0.27%), and propionic acid (0.27%). The flies were maintained in environmental test chamber at 22 ± 1 °C temperature, 50–60% humidity and 12L:12D conditions.
Test Chemical Preparation
NaF powder (MERCK, India) was dissolved in distilled water to prepare the stock solution (1000 µg/mL). This stock was further diluted to prepare different concentrations of working NaF solutions.
LC50 Determination
To determine the chronic lethal concentration 50 (LC50) of NaF in 3rd instar larvae of D. melanogaster, five graded concentrations (120,130,140,150 and160 µg/mL) of NaF were prepared in SDM from stock solution of 1000 µg/mL. Since previous study in our laboratory recorded the chronic median lethal concentration of another fluoridated chemical cryolite (Na3AlF6), which was found to be within the range 100–160 µg/mL (Podder and Roy 2013), the present study was designed to the range 120–160 µg/mL. Thirty early 1st instar larvae (freshly hatched, within 6 h) in triplicate sets were maintained for each grade along with the control set and data on LC50 were collected and recorded following the methodologies described by Ashburner et al. (2005).
Hemolymph Smear Preparation
Following determination of median lethal concentration newly hatched 1st instar larvae were exposed to sub-lethal concentrations of NaF (10, 20, 40, 80, and 100 µg/mL) and maintained up to 3rd instar stage. Hemolymph of late 3rd instar larvae was collected and hemocytes were counted and compared according to the method of Rajak et al. (2014). Here 10 larvae (per treatment category) were bled for hemolymph on a slide having 20 µl PBS and mixed properly using micropipette. Thereafter, hemolymph smear was prepared on the slide and was air dried, followed by fixation in methanol and stained using Giemsa stain to visualize the hemocytes. For each treatment category, three replicates of slides were prepared for differential count of hemocytes.
Trypan Blue Dye Exclusion Assay
For differentiating the living from the dead hemocytes, hemolymph collected from treated 3rd instar larvae was diluted in 20 µl of phosphate buffered saline (PBS) (pH 7.4). Then the cells were stained with 20 µl of 0.2% trypan blue staining solution for 10 min. The extra stain was removed by washing with 20 µl of PBS and observed under microscope at (10 × 40) × magnification. Similar type of experiment was carried out for untreated (control) larval hemolymph. Total number of cells (hemocytes) were counted using hemocytometer and the number of dead and live cells were recorded from which the difference between control and treatment categories could easily be suggested. To obtain the total number of cells the following formula was applied:
Statistical Analysis
All data are expressed as mean ± Standard Error (SE). Mean, standard error, linear regression equation and correlation coefficient of data were calculated using MS Excel 2007. Student’s t-test was carried out to determine significant differences between control and treatment groups, where p < 0.05 was considered statistically significant. Chronic median lethal concentration (LC50) of NaF in the third instar larvae and pupae of Drosophila melanogaster was determined by the log probit transformation method as described by Finney (1971). The results of probit calculations as represented in the graphs were obtained with the help of Microsoft Excel 2007.
Results
The result shows that increasing concentrations of NaF significantly (p < 0.05) reduces the percentage of pupae formation (Table 1) when compared to the control data. The percentage of pupae formation declined up to 61.66% at 120 µg/mL of NaF when compared to the control group (in which 95% pupae formation occurred). The percentage of pupae formation was least (6.66%) at 160 µg/mL. Fifty percent of larval mortality was observed between 120–130 µg/mL as represented in the Table 1. Probit analysis of the data reveals that larval LC50 of NaF is 125.314 µg/mL (Fig. 1). Thus 50% of the larvae failed to become pupae at this concentration.
Percentage of emergence that represents pupal mortality determined the LC50 of NaF in case of the pupae. Chronic LC50 of NaF in the pupae has been presented in Fig. 2. As stated above, the results show that pupal mortality was significantly increased (p < 0.05) with increasing concentrations of NaF (Table 1), that is greater number of pupae fail to emerge as adults. Maximum pupal mortality was observed at 160 µg/mL where no pupa successfully emerged as adult. From the probit analysis of the data, it was observed that 50% pupal mortality occurred between 125 and 130 µg/mL of NaF.
Hematological studies reveal mean percentage of PC for control group to be 94.147 ± 1.85% whereas for treatment groups (10, 20, 40, 80 and 100 µg/mL), the mean PC count was found to be 90.382 ± 0.728, 81.759 ± 0.798, 84.966 ± 2.178, 89.160 ± 0.777 and 80.436 ± 1.37% respectively (Fig. 3). Data of plasmatocyte number in treatment categories (20, 40 and 100 µg/mL) are significantly (p < 0.05) reduced when compared to the control set.
On the other hand, the mean percentage of LC for control group was recorded as 0 where as for treatment groups such as 10, 20, 40, 80 and 100 µg/mL, the mean LC count was found to be 0.672 ± 0.001, 0.326 ± 0.002, 0.067 ± 0.028, 0.376 ± 0.011 and 0.483 ± 0.001% respectively (Fig. 3). The number of lamellocyte in different treatment categories of NaF increased significantly (p < 0.05) when compared to the control. Interestingly, the mean percentage of CC for different treatment groups such as 10, 20, 40, 80 and 100 µg/mL was 8.18 ± 0.017, 24.25 ± 0.020, 11.72 ± 0.024, 11.82 ± 0.014, and 9.71 ± 0.020, respectively whereas the control group showed crystal cells percentage as 3.85 ± 0.017 (Fig. 3). The number of crystal cells in different concentrations of NaF increased significantly (p < 0.05) when compared to the control. Since the number of LC is maintained at negligible low number, the focus was confined to variation in the count of PC and CC in the present study.
Through trypan blue dye exclusion assay it was observed that in control larval hemolymph the number of dead cells was 12.6 × 104 ± 1.8 /mL of hemolymph. Interestingly, the number of dead cells increased significantly with the increasing concentrations of NaF i.e., 15.942 × 104 ± 1.56, 15.0 × 104 ± 1.50, 18.0 × 104 ± 1.8, 15.9 × 104 ± 0.3, 25.8 × 104 ± 2.83 cells/mL of hemolymph respectively (Fig. 5).
Discussion
In the context of the above LC50 determination, death caused by slow and delayed poisonous effect of NaF has only been considered. Hence the chronic LC50 is expected to be lower in comparison to the acute LC50. Another fluoride-containing chemical cryolite has been shown to cause 50% of larval mortality in Drosophila between 150–158 µg/mL concentrations (Podder and Roy 2013). The toxicity caused by NaF depends on its easy and free dissociation into elemental fluoride ion (F−). Further, F− of NaF has been suggested to inhibit a variety of vital enzymes causing death (Bhatnagar et al. 2006). Hence, lower concentration might be more effective at prolonged exposure. Since larva of D. melanogaster is a voracious eater, hence, the selection of the route of exposure through food seemed to be quite justified. Thus the relevance of determination of LC50 is unquestionable.
Plasmatocyte cells (PC) are known to be involved in phagocytosis (Tan et al. 2014) and lamellocyte (LC), a variant form of PC (Rizki and Rizki 1980), is involved in encapsulation of large foreign particle which may be too large to be phagocytosed (Crossley 1964; Ware and Whitacre 2004; Tan et al. 2014). In the present study, a significant (p < 0.05) reduction in the number of PC in the hemolymph of D. melanogaster after exposure to sub-lethal concentrations of NaF has been observed. Interestingly, similar type of reduction in number of PC and LC is seen after treatment with the organophosphorous chemical acephate (Rajak et al. 2014), indicating compromise in immunity due to chemical insult. NaF is known to influence actin polymerization and thus is likely to interfere with the process of phagocytosis as has been reported in Venerupis philippinarum (Ballarin et al. 2014). Hence the present observation where significant (p < 0.05) reduction in the number of PC was seen can be correlated with probable compromise in phagocytic machinery.
Due to rise in the level of free radicals resulting from F− exposure, lipid peroxidation is reported to increase in sheep (Guven and Kaya 2005) and Drosophila melanogaster (Dutta et al. 2017). Fluoride induces oxidative stress and apoptosis resulting in elevation of lipid peroxidation (Karube et al. 2009; Anuradha et al. 2001). So from results of the present studies it seems that, there exists a possibility that, NaF might cause death of PC and thereby their number in circulating hemolymph would be low. NaF is also known to reduce mitosis rate which might result in reducing proliferation of hemocyte precursor cells during hematopoiesis thereby decreasing the cell count as suggested by Podder et al. (2008a, b) in mice. Since PCs are involved in phagocytosis and encapsulation of larger pathogen, their reduction might adversely affect the immune response of the insect. Another kind of hemocyte reported in Drosophila hemolymph are crystal cells (CC), which possess dark crystals of prophenoloxidase (proPO) inside their cytoplasm (Meister and Lagueux 2003).Various stress inducing factors like temperature, pathogen attack, exposure to toxic chemicals etc. might activate a cascade of serine protease activity that ultimately cleaves prophenoloxidase into active phenoloxidase to produce melanin. Melanin is reported to help insects to combat different stress conditions (Hamilton and Gomez 2002). In the present study, increased CC after chronic exposure to NaF seems to be associated with detoxification mechanism in the insect body as this might be helpful to protect the organism from excessive F induced damage.
As trypan blue is a vital dye, dead cells with compromised cell membrane are unable to exclude the dye; as a result, those cells appear blue in colour when observed under microscope (Fig. 4). Hence, this assay successfully confirms the decrease in number of PC and it may be assumed that NaF being an inducer of apoptosis might cause the death of the immune cells (Fig. 5).
Conclusion
The study, for the first time reported about larval and pupal chronic LC50 of NaF in the range of 125–130 µg/mL in D. melanogaster. The sub-lethal concentrations of NaF, were found to cause significant variations in differential hemocyte count of the third instar larvae of Drosophila. This variation, in turn, could compromise the immune system function of the insect. Similarly, chronic exposure to NaF through pesticides, tooth paste and water might also adversely affect the immune system of genetically similar higher order organisms including human beings and result in reduced immune response against pathogenic infections and diseases.
References
Anuradha, C.D., S. Kanno, and S. Hirano. 2001. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL- 60 cells. Free Radical Biology and Medicine 31: 367–373.
Ashburner, M., K.G. Golic, and R.S. Hawley. 2005. Drosophila: a laboratory handbook, 2nd ed, 162–164. New York, NY: Cold Spring Harbor Laboratory Press.
Ballarin, L., V. Covre, L. Masiero, and S. Casellato. 2014. Immunotoxic effects of fluoride on the hemocytes of Venerupis philippinarum. Information Systems Journal 11: 22–29.
Bhatnagar, M., P. Rao, A. Saxena, R. Bhatnagar, P. Meena, S. Barbar, A. Chouhan, and S. Vimal. 2006. Biochemical changes in brain and other tissues of young adult female mice from F in their drinking water. Fluoride 39: 280–284.
Crossley, A.C.S. 1964. An experimental analysis of the origins and physiology of hemocytes in the blue bottle blowfly Calliphora erythrocephala (Meig.). Journal of Experimental Zoology 157: 375–397.
Das, S, R Maiti, and D Ghosh. 2006 Fluoride-Induced Immunotoxicity in Adult Male Albino Rat: A Correlative Approach to Oxidative Stress. Journal of Immunotoxicology 3(2): http://www.tandfonline.com/doi/full/10.1080/15476910600631587.
Dutta, M., P. Rajak, S. Khatun, and S. Roy. 2017. Toxicity assessment of sodium fluoride in Drosophila melanogaster after chronic sub-lethal exposure. Chemosphere 166: 255–266.
Finney, D.J. 1971. Probit analysis. Cambridge: Cambridge University Press.
Guven, A., and N. Kaya. 2005. Effect of fluoride intoxication on lipid peroxidation and reduced glutathione in Tuj sheep. Fluoride 38: 139–142.
Hamilton, A.J., and B.L. Gomez. 2002. Melanins in fungal pathogens. Journal of Medical Microbiology 51: 189–191.
Karube, H., G. Nishitai, K. Inageda, H. Kurosu, and M. Matsuoka. 2009. NaF activates MAPKs and induces apoptosis in odontoblast-like cells. Journal of Dental Research 88: 461–465.
Meister, M., and M. Lagueux. 2003. Drosophila blood cells. Cellular Microbiology 5: 573–580.
Mishra, M., A. Sharma, A.K. Shukla, P. Pragya, R.C. Murthy, D. de Pomerai, U.N. Dwivedi, and D.K. Chowdhuri. 2013. Transcriptomic analysis provides insights on hexavalent chromium induced DNA double strand breaks and their possible repair in midgut cells of Drosophila melanogaster larvae. Mutation Research. doi:10.1016/j.mrfmmm.2013.04.005.
Mitchell, B., and R.A. Gerdes. 1973. Mutagenic effects of sodium and stannous fluoride upon Drosophila melanogaster. Fluoride 6: 113–117.
Podder, S., A. Chattopadhyay, and S. Bhattacharya. 2008a. In vivo suppression by fluoride of chromosome aberrations induced by mitomycin-c in mouse bone marrow cells. Fluoride 41: 40–43.
Podder, S., A. Chattopadhyay, S. Bhattacharya, and M.R. Ray. 2008b. Differential in-vivo genotoxic effects of lower and higher concentrations of fluoride in mouse bone marrow cells. Fluoride 41: 301–307.
Podder, S., and S. Roy. 2013. Study of the changes in life cycle parameters of Drosophila melanogaster exposed to fluorinated insecticide, cryolite. Toxicology and Industrial Health 31: 1341–1347.
Rajak, P., M. Dutta, and S. Roy. 2014. Effect of acute exposure of acephate on hemocyte abundance in a non-target victim Drosophila melanogaster. Toxicological and Environmental Chemistry 96: 768–776.
Rajak, P., S. Sahana, and S. Roy. 2013. Acephate-induced shortening of developmental duration and early adult emergence in a non-target insect Drosophila melanogaster. Toxicological and Environmental Chemistry 95: 1369–1379.
Rizki, R.M., and T.M. Rizki. 1980. The direction of evolution in the Drosophila melanogaster species subgroup based on functional analyses of the crystal cells. Journal of Experimental Zoology 212: 323–328.
Shrestha, R., and E. Gateff. 1982. Ultrastructure and cytochemistry of the cell types in the larval hematopoietic organs and hemolymph of Drosophila melanogaster. Development, Growth and Differentiation 24: 65–82.
Tan, K.L., I. Vlisidou, and W. Wood. 2014. Ecdysone mediates the development of immunity in the Drosophila embryo. Current Biology 24: 1145–1152.
Ware, G.W., and D.M. Whitacre. 2004. An introduction to insecticides. Ohio: Willoughby.
Acknowledgements
The authors are grateful to the Head, DST-FIST, DST-PURSE and UGC-DRS sponsored Department of Zoology, the University of Burdwan for providing the infrastructural facilities. Thanks are due to Dr. A. Mazumdar for kindly providing microscopic facilities.
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Dutta, M., Rajak, P. & Roy, S. Determination of Chronic Median Lethal Concentration of Sodium Fluoride in Drosophila melanogaster and Exploring Effect of Sub-lethal Concentrations on Differential Hemocyte Count. Proc Zool Soc 72, 111–117 (2019). https://doi.org/10.1007/s12595-017-0235-x
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DOI: https://doi.org/10.1007/s12595-017-0235-x