Comparative study of Insecticide potential of Methyl parathion and diazinon in Drosophila melanogaster

Suman Sahoo², Mausumi Ari Acharyya³_,Rajiniraja Muniyan¹*

¹Department of Biotechnology, School of Bio Sciences and Technology,

Vellore Institute of Technology, Vellore – 632014.

²Department of Biomedical Genetics, School of Bio Sciences and Technology,

Vellore of Technology, Vellore – 632014.

³Institute of Genetic Engineering, Kolkata - 700128.

*Corresponding Author E-mail: rajiniraja.m@vit.ac.in

ABSTRACT:

Several reports indicate that many chemical pollutants which are widely spread in the environment, such as insecticide, pesticide and drugs are mutagenic in various test system. These findings reflect an urgent need to draw more attention to the possible genetic hazards of such pollutants to public health. The present investigation of working hypothesis deals with the effects of two insecticides viz. Methyl Parathion (MeP) and Diazinon (DZ) on non-target organism Drosophila melanogaster. We have carried out the chromosomal aberration test with various concentration for insecticides (MeP and DZ) which infer properties like ectopic pairing, inversion loop, puffing, fusion, and asynapsis. Chromosomal aberration result shows significant effects with DZ even in less concentration (0.02ppm) when compared with MeP (0.2ppm). The present study proposes that diazinon is more cytotoxic than methyl parathion in Drosophila melanogaster.

KEYWORDS: Chromosomal aberration test, Methyl Parathion, Diazinon.

INTRODUCTION:

Pesticide is a biological or chemical agent which has the tremendous use in agriculture and other human activities. Recent study highlights its continuous contamination has badly affects environment, human health and animals. A wide classification of pesticides are herbicide, insecticides, bactericide, antimicrobial, fungicide and others on the basis of their function. Among them, insecticide has a more consumption pattern in India and have a negative feedback like effect on human, animal, birds and mostly on non-target organisms by the way of inhalation, skin, consume foods [1,2]. After expose into the body, different reaction like skin rash, skin disease, breathing problems, heart problem, and brain disease occur.

Organophosphorus pesticides which are still present in the market such as methyl parathion (MeP) (C₈H₁₀NO₅PS) and diazinon (DZ) (C₁₂H₂₁N₂O₃PS) and used for horticultural crops, vegetables like beans, onions and many other fruits. United states use methyl parathion since discover in 1952 for agriculture [3]. Pham LT group have reported that over 500 ongoing products in world market manufacture with diazinon compound [4]. These two organophosphates by their toxicity inhibit many metabolic and physiological protein like acetyl cholinesterase (AchE), glutathione S-transferases D1 (GstD1), protein kinase C (PKC), causing phenotypical abnormalities which may lead to complicated nervous diseases like Alzheimer, Parkinson’s disease etc [5,6,7,8,9]. Sequence analysis revealed that 75% of human diseases are homolog with Drosophila melanogaster [10,11]. This leads us to perform human health related experiments using Drosophila as a model organism.

The aim of our research is to focus on in vivo analysis using polytene chromosome of Drosophila melanogaster larvae treated with pesticides (MeP and DZ) and check the effectiveness of two pesticides.

MATERIALS AND METHOD:

Chemicals:

We used two different commercial pesticides: a) Methyl parathion powder (obtained from local shop of Bardhaman, WB, India), b) Diazinon as liquid state (from local shop of Hoogly district, WB, India). Agar, dried yeast, glacial acetic acid (CH₃COOH), methyl paraben (C₈H₈O_3,Loba Chemicals Pvt. Ltd, India), propionic acid (C₃H₆O₂, Institute of Genetic Engineering, Kolkata), ringer solution, Lacto orcein (for staining) were used in this study.

Food preparation and fly culturing:

Food was prepared in 50ml culture vials with 15ml of standard food which contains dry agar (10gm/L), maize powder (100gm/L), brown sugar (100gm/L), yeast (30gm/L), propionic acid and antifungal agent methyl paraben (each 1 pinch) with 1ml ethanol [12]. Wild type Drosophila melanogaster were collected from Genetics lab, Institute of Genetic Engineering, Kolkata-128, India. Drosophila were cultured at 22°±1°C and 70% humidity maintained in BOD incubator on this standard food. Collect third instar larvae from the culture vial and placed them in each petri plate in a ringer solution and mixed with insecticide methyl parathion at different concentration (0.2ppm, 0.3ppm, 0.4ppm) and other insecticide diazinon at different concentration (0.02ppm, 0.03ppm, 0.04ppm) for 30 minutes.

Dissection and polytene chromosome preparation:

Third instar larvae were transfer into groove slide filled with ringer solution after 30 mins exposure of insecticides and dissected one pair of salivary glands from Drosophila melanogaster under a binocular microscope. Aceto - alcohol (ethanol (3): glacial acetic acid (1)) was added drop wise till three minutes continuously to the salivary gland which was kept in the slide, for fixation. After that process glands were stain with Aceto orcein and covered it with Petri plate for 15 minutes, then slides with salivary glands were washed with 50% acetic acid and added 2-3 drops of lacto orcein stain into the salivary gland. After staining mount with a coverslip over the salivary gland on a slide. Thumb pressure was given on it so, that chromosome can be spread uniformly [13]. Then slides were visualised under microscope and statistically analyzed.

RESULTS:

Chromosomal aberration:

Polytene chromosome analysis showed different chromosomal aberration such as, ectopic pairing, inversion loop, puffing, fusion, asynapsis when wild type Drosophila exposed with pesticides: methyl parathion or diazinon. The average frequency of different types of chromosomal aberration (Fig. 1) in different concentration (i.e. 0.2ppm, 0.3ppm, 0.4ppm) of methyl parathion treated larvae shown in Table 1.

Table 1: Different types of chromosomal aberration frequency when Drosophila larvae treated with methyl parathion (in different conc.) for exposure of 30 mins. This assay was carried out in triplicate.

S. No	Types of aberration	Number of nuclei studied	Number of individual types of aberration (mean ± SD)	Percentage of frequency
1.	Asynapsis	300	15 ±1	4.90%
2.	Inversion loop	300	19±2	6.08%
3.	Ectopic pairing	300	30±1	10.00%
4.	Puffing	300	16±1	6.18%
5.	Deletion	300	5±0	1.69%

Table 2: Different types of chromosomal aberration frequency when Drosophila larvae treated with diazinon (in different conc.) for exposure of 30 mins. This assay was carried out in triplicate.

S. No.	Types of aberration	Total no. of nuclei studied (30 nuclei for each slide)	No. of individual types of aberration	Percentage frequency
1.	Asynapsis	300	15±2	9.21%
2.	Inversion loop	300	19±1	9.77%
3.	Ectopic pairing	300	30±1	18.33%
4.	Puffing	300	16±2	15.9%
5.	Deletion	300	5±0	2.58%

Figure 2: Drosophila polytene chromosome showing aberration after treated with Diazinon at different concentration for 30minutes exposure time. A) Shows chromosome aberration after treated with 0.02ppm Diazinon for 30 minutes; B) Shows chromosome aberration after treated with 0.03ppm Diazinon for 30 minutes; C & D) Shows chromosome aberration after treated with 0.04ppm Diazinon for 30 minutes.

Similarly, the average frequency of different types of chromosomal aberration (Fig. 2) in different concentration (i.e. 0.02ppm, 0.03ppm, 0.04ppm) of diazinon treated larvae shown in Table 2.

DISCUSSION:

Boussabbeh M et al reported that diazinon has cytotoxic effect and causes oxidative damage as well, by using cell line of large intestine [14]. Another group also reported genotoxicity of diazinon through conducting different assays such as MN, FISH on human peripheral blood lymphocytes [15]. Jhon G et al have performed sister chromatid exchange analysis as well as other chromosome aberrations on Etrpolus suratensis by exposing different pesticides. Their experiment stated that commertially available pesticide methyl parathion showed more chromosomal aberration than other pesticide phosphamidon [16]. The wing spot test on Drosophila melanogaster has shown genotoxicity against different pesticides. The study have reported that diazinon showing more genotoxicity than methyl parathion [17]. As regards to the chromosome aberration, our study also showed diazinon in lower concentration was more genotoxic than comparatively higher concentration of methyl parathion using model organism Drosophila melanogaster. During the study on different fish experiments many scientists such as Rao et al.,1994 and Sharbidre et al., 2011 have identified that AchE activity was reduced mainly in brain when exposed with different organopesticides such as methyl parathion and diazinon [18,19].

CONCLUSION:

The results clarify the effects of methyl parathion and diazinon on the chromosome, thereby forwarding the idea of impact of chemicals used as pesticides or insecticides on organism like Drosophila melanogaster. Furthermore, analysis of mutagenic effects of methyl parathion and diazinon suggested at intensive level, that alternatives of insecticides should be implemented, and use of natural predators of target pests.

CONFLICT OF INTEREST:

The authors declares that there is no conflict of interest

ACKNOWLEDGEMENT:

Authors thanks the VIT for the infrastructure and instrumentation facilities. Also, thanks to Institute of Genetic Engineering, Kolkata for carry out the in vitro analysis.

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Received on 15.05.2020 Modified on 30.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2649-2652.

DOI: 10.52711/0974-360X.2021.00467