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Potential risk of organophosphate exposure in male reproductive system of a non-target insect model Drosophila melanogaster
Journal Pre-proof Potential risk of organophosphate exposure in male reproductive system of a non-target insect model Drosophila melanogaster Moutushi MandiExperimental methodologies) (Investigation)Data collection)Data analysis) (Validation) (Visualization)Preparation of original draft), Salma KhatunExperimental investigation) (Methodology)data collection) (Visualization), Prem Rajak (Formal analysis) (Software) (Writing - review and editing), Abhijit Mazumdar (Supervision)Data Interpretation)Suggestion)Review), Sumedha Roy (Conceptualization) (Data curation)Interpretation) (Supervision) (Writing review and editing) (Project administration)
PII:
S1382-6689(19)30183-8
DOI:
https://doi.org/10.1016/j.etap.2019.103308
Reference:
ENVTOX 103308
To appear in:
Environmental Toxicology and Pharmacology
Received Date:
11 June 2019
Revised Date:
25 November 2019
Accepted Date:
26 November 2019
Please cite this article as: Mandi M, Khatun S, Rajak P, Mazumdar A, Roy S, Potential risk of organophosphate exposure in male reproductive system of a non-target insect model Drosophila melanogaster, Environmental Toxicology and Pharmacology (2019), doi: https://doi.org/10.1016/j.etap.2019.103308
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Potential risk of organophosphate exposure in male reproductive system of a non-target insect model Drosophila melanogaster
Moutushi Mandia, Salma Khatuna, Prem Rajakb, Abhijit Mazumdarc and Sumedha Roya,*
Toxicology Research Unit, Department of Zoology, The University of Burdwan, West Bengal,
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Department of Animal Science, Kazi Nazrul University, Asansol, West Bengal, India
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India
Entomology Research Lab, Department of Zoology, The University of Burdwan, West Bengal,
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India
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Corresponding author: Email: [email protected]
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Graphical abstract
The plate represents the findings of the entire study. Study demonstrates that chronic sub-lethal
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acephate exposure causes significant alteration in male reproductive structures, physiology and behavior which have been reflected through the alterations in the studied parameters. The results
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confirm that the altered expressions of the selected parameters might be the reason for the compromised male reproductive efficiency in the non-target model organism, Drosophila
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melanogaster following chronic acephate exposure.
HIGHLIGHTS
Chronic sub-lethal Acephate exposure reduces Drosophila male germ cell viability Acephate exposure leads to altered adult male body weight and increased apoptosis Acephate(1-6µg/mL) exposure increases CAT and LPO activities in male adults Acephate alters testis architecture and Mitoferrin and vitellogenin content 2
Acephate exposure reduces number of mating pairs
Abstract Based on several adverse reports of pesticides on reproductive efficiency of various organisms, studies on “reproductive toxicity” have gained importance. Fecundity, reflecting reproductive
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success of any organism, is governed by several factors from female and male reproductive
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systems. This present study explored morphological and biochemical alterations in the male reproductive system of a non-target model organism, Drosophila melanogaster following
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chronic sub-lethal exposure (1st instar larvae differentially exposed to 1-6µg/mL until adulthood) to the organophosphate (OP) pesticide, acephate (LC50 8.71µg/mL). This study demonstrates
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altered testis structure, decreased germ cell viability and gross body weight, increased activities
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of oxidative stress markers lipid peroxidase (LPO), and the endogenous antioxidant enzyme catalase (CAT), and altered expression of reproductive marker proteins like vitellogenin and
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mitoferrin in acephate-exposed flies when compared to control counterparts. Altered reproductive behavior, indicated by a significant decline in the number of mating pairs, validates the adverse effect of chronic acephate exposure on male reproduction in the non-target insect model D. melanogaster.
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Keywords: D. melanogaster, Male reproduction, Mitoferrin, Organophosphate, Vitellogenin.
1. Introduction:
Pesticides are potent chemicals specifically designed for repelling, preventing or destroying pests (Landrigan, 2018). In recent decades, significant human as well as non-target toxicity from pesticide exposure have been documented (Frazier et al., 2008, Dutta et al., 2014, Dutta et al., 3
2016, Rajak et al., 2017). People, through dietary and other routes, get exposed to several pesticides that undergo accumulation in measurable concentration inside the body (Frazier et al., 2008). Therefore, it is essential to investigate deleterious impacts, if any of these biocides on non-target organisms. Pesticide poisoning has been increased by several folds in recent decades. According to a report (Yang et al., 2013), around 250000 people die throughout the world each year due to pesticide
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poisoning. Unfortunately, 750000-3000000 people are unintentionally poisoned by
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organophosphate (OP) chemicals every year, with an estimated 300000 deaths (Balali-Mood and Saber, 2012).
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OP pesticides are esters of phosphoric and thiophosphoric acids and their toxicity mainly rely on their ability to inhibit acetylcholine esterase (AChE) activity. In addition, OPs may lead
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to oxidative stress, axonal transport deficits, neuroinflammation, and autoimmune conditions
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(Xu et al., 2015; Naughton and Terry, 2018). Acephate (O,S-dimethyl acetylphosphoramidothioate), a popular OP in most of the developing countries is used as foliar spray (Thapar et al., 2002, Trevizan et al., 2005) to control wide range of biting and sucking
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insects, especially aphids, leaf miners, lepidopteran larvae, sawflies and thrips (Douglas and Hamish, 1983; Dhingra and Dhingra, 2009). Acephate toxicity is attributed to its bio-activation and metabolic conversion into more toxic
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intermediate, methamidophos, which is also a potent AChE inhibitor. Des-o-methyl acephate is a metabolized form of acephate produced as a byproduct in mammals (Farag et al., 2000, Mahajna et al., 1997). Acephate and methamidophos residues are higher in protected cultivation conditions in leaves and soil, than in the open-field (Cabras et al., 1985, Frank et al., 1987, Von Stryk, and Jarvis, 1978, Meloni et al., 1984). Insects metabolize acephate into des-omethyl acephate accounting for acephate’s relatively high selectivity against insects (Roberts 4
and Hutson, 1999). More recently, another plausible mechanism of action of acephate toxicity through ROS mediated pathways was confirmed by our laboratory (Rajak et al., 2018) and simultaneously protective potential of co-administrated L-ascorbic acid validated the idea of acephate induced oxidative stress in Drosophila. Acephate is toxic to non-targets like pollinators (bees), fishes, birds and mammals. Recent study conducted by Yao et al., 2018 concluded that, honey bees exposed to 6.97 mg/mL killed 50
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percent of the population. In addition, individuals who survived after 48h of exposure,
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manifested reduced body weight and suppressed esterase activity. Some studies documented histo-pathological impacts of acephate on fish population. Kavita and Shakila, 2015 were
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documented remarkable alterations in histological architecture of vital organs like liver, intestine, muscle and skin of Poecilia sphenops. Acute and chronic exposure to acephate decreases
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amylase activity in Labeo rohita (Bhilave and Kulkarni, 2016). Hematological parameters like
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RBC count, WBC count and packed cell volume (PCV) were significantly declined in Channa punctata following sub-lethal exposure to acephate (Satish et al., 2018). Significant reduction in hatching rates of embryos of zebra fish has been reported following exposure to OPs like
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chlorpyrifos and diazinon (Cao et al., 2018). Decreased growth rate, survivability and higher morphological abnormalities were evident in acephate treated larvae of salamander, Ambystoma gracile (Geen et al., 1984). Acephate and other OPs may affect breeding success, suppression
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of egg formation, eggshell thinning, impaired incubation, behavior and migratory orientation in avian population (Brigs, 1992; Mitra and Mitra, 2018). Furthermore, immunotoxic response was known to be induced by acephate in White Leghorn cockerel chicks (Tripathi et al., 2012). Some toxic effects of organophosphates and acephate have also been demonstrated on humans. Upon inhalation, organophosphates are known to cause dizziness, headache, nausea and vomiting (Reregistration Elegibility Decision, 2006; Reigart et al., 1999). Moreover, acute 5
dermal exposure results in redness, swelling and rashes in the skin. Chronic exposure to acephate generates intermediate compound called methamidophos which leads to muscular weakness in neck, limb and even causes death (Christiansen et al., 2011). In a study, tobacco field workers exposed to acephate manifested decreased SOD activity in their erythrocytes (Panemangalore et al., 1999). Acephate is also known to affect human sperm motility, vitality and functional integrity of sperm plasma membrane (Dhanushka and Peiris, 2017).
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The no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) of acephate have been established for some mammalian species. For rat, rabbit and
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human, NOAEL was documented as 2.5, 3 and 1.2mg/kg body weight respectively (Fiedler,
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1987, Gavit and Patil, 2016, Zinkl et al., 1980, Behera and Bhunya, 1989). Subsequently, LOAEL has also been established for rat and rabbit as 5 and 10mg/kg body weight but the same
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is still unexplored for human beings (Gupta and Moretto, 2005).
Many epidemiological studies have recognized the connection between OP exposure and
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reproductive disorders such as infertility, birth defect, adverse pregnancy outcomes, and perinatal deaths (Peiris-Jhon and Wickremasinghe, 2008). Interestingly, Perry et al. (2007) have
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suggested that, the reproductive toxicity from OP exposure results due to reduced brain acetylcholinesterase activity which in turn interferes with pituitary gonadotropin thereby causing infertility. Male infertility has become a major manifestation of reproductive toxicity. In the last two decades, sperm counts in human males have reduced below the threshold concentration of
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55 million/mL. In addition, significant decline in semen quality has also been reported (Carlsen et al., 1992). Other documentation regarding impaired male reproductive function in human and wildlife populations (Phillips and Tanphaichitr, 2008) have increased interest in studies exploring toxicological effects of OPs on male reproductive system (Oakes et al., 2002, Sharma et al., 2015) Additional concerns were raised because sperm counts are found to decline at an
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alarming rate, 2% per annum for the past 20 years (Skakkebaek et al., 2001, Toppari et al., 2001, Fisher, 2004). In another study, modern lifestyle was reported to compromise sperm quantity and quality in human (Jayachandra and Srinivasa, 2011). Furthermore, it has been reported that, pyriproxifen impairs the spermatogenesis in B. germanica, (Fathpour et al., 2007). Farag et al., (2000) confirmed about acephate’s potential to cause reproductive toxicity in male mice (e.g., decreased sperm motility and count) from high dose oral administration.
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Joshi and Sharma (2011) reported that, acephate exposure reduced male fertility through altered serum testosterone, LH, and FSH levels in albino rats. Another chemical atrazine has
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been found to affect expression of stress genes (Le Goff et al., 2006) and proteins (Thornton et
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al., 2010) in D. melanogaster, egg production in Orchesella cincta (Badejo and Van Straalen, 1992) and mating choices in Tenebrio molitor (Mc Callum et al., 2013). Mukhopadhyay et al.,
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2003, documented reduced fertility and reproductive performance in both male and female fruit flies following differential exposure to argemone oil. Therefore, these studies indicated that,
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males are at risk of becoming infertile due to environmental exposure to innumerable chemicals and pollutants. Thus, it is essential to investigate xenobiotic induced hazardous effects on male
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fertility. D. melanogaster is a well-established model showing almost 75% functional homology with the disease-causing genes of human beings (Pandey and Nichols, 2011, Demir et al., 2013). Developmental, cellular and molecular mechanisms in Drosophila are well established. Hence, it has emerged as a perfect model organism for researchers from several fields, like
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genetics, medicine, developmental biology, immunology and toxicology to elucidate problems of human interest (Koh et al., 2006, Rajak et al., 2017). From past several decades, fruit fly Drosophila melanogaster is in use as non-target organism for toxicity assessment of various chemicals. Hence, a new discipline in research known as “Drosophotoxicology” is gaining popularity in recent days. Furthermore, Drosophila, with its well characterized developmental
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and reproductive phenomena, offers itself as a potent alternative and a promising model for the male reproductive toxicity assessment. Thus, in the present study, our major goal was to explore variation in reproductive fitness of Drosophila males following chronic sub-lethal acephate exposure through analysis of selected relevant parameters. 2. Materials and methods:
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2.1 Experimental organism
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D. melanogaster (Oregon R strain) was used for the present study. Drosophila were maintained
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at 23±1°C temperature, 50-60 % humidity within environmental test chamber in the laboratory. 2.2 Selection and preparation of experimental concentrations in food media
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In order to investigate the effects of chronic acephate exposure in Drosophila, six sub-lethal
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concentrations (1-6 µg/mL) below the evaluated chronic LC50 (Rajak et al., 2017) were selected and mixed with SDM (standard Drosophila medium) for use in the present study.
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2.3 Treatment schedule
Freshly hatched 1stinstar larvae were reared on food containing selected concentrations of acephate until their emergence as adults. These adult males were considered for experiments in
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the current study.
2.4 Parameters for assessment of toxic effects: 2.4.1 Body weight measurement 30 adult male flies were taken from each treatment cluster for measuring their body weight using electronic weight machine (Afcoset, Sr No. 0413050). Equal number of male flies from control 8
group was used for comparison. Assessments were carried out in triplicate sets hence number of individuals considered for each experimental group was n= 90 (3*30). 2.4.2 Acridine orange (AO) staining for localization of apoptotic lesions AO staining was performed following the protocol of Alone et al., 2005 with slight modifications (Khatun et al., 2017). Testes from adult flies were isolated in PBS and stained with AO (10µg/mL) for 2min. Tissues were washed with PBS and images were taken under
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fluorescent microscope (Leica DMI6000B).
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2.4.3 Germ cell viability study through MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-
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diphenyltetrazolium bromide] assay
Testes were dissected and were processed for determination of germ cell viability through MTT
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assay (Babot et al., 2005). Testes from adults were taken out in PBS and processed with
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collagenase (0.5mg/mL) to enzymatically individualize the cells. MTT (5mg/mL) solution was added to cell suspensions and incubated overnight. After incubation, DMSO was added to
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solubilize the formazan crystals. Absorbance was recorded at 578nm using ELISA plate-reader (Erba-Mannheim, LisaScan EM).
2.5 Biochemical assays for OS markers
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The activity of oxidative stress marker enzymes such as lipid peroxidase (LPO) and catalase (CAT) were quantified in adult male fly to detect alterations from their normal status under chronic sub-lethal acephate exposure. 2.5.1 Preparation of tissue homogenates
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10% tissue homogenates of control and treated flies were prepared from pooled samples of each experimental set using 0.1M phosphate buffer containing 0.15M KCl (pH 7.4) and centrifuged at 10,000g for 20min. Supernatant containing the crude cytosol was collected for protein estimation and enzyme assays. Protein estimation was done following the method of Lowry et al., 1951. 2.5.1.1 CAT assay CAT assay was performed following the procedure of Sinha, 1972 with minor modifications.
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The reaction mixture contained tissue sample, distilled water, 0.01M sodium pyrophosphate
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buffer (0.01 M, pH 7.0) and H2O2 (0.2M). Reaction was allowed for 2min and was stopped by adding dichromate-acetic acid reagent. Mixture was heated for 10min and the absorbance was
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recorded at 570nm.
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2.5.1.2 LPO Assay
Malonyldialdehyde (MDA), a major product of ROS-induced lipid peroxidation, was quantified
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following the method of Ohkawa et al., 1979 with minor modifications. Briefly, tissue homogenate (10%), SDS (8.1%) and acetic acid (20%) were taken in microfuge tubes and the pH
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adjusted to 3.5. Later, the reaction mixture was treated with thiobarbituric acid (0.8%) and heated at 95 °C for 1 h. The solution was cooled and mixed with a butanol–pyridine mixture. The absorbance of the final pink coloured organic layer was recorded at 532 nm against n-butanol as
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the blank.
2.6 Biochemical assays for reproductive protein marker 2.6.1 Vitellogenin assay: Vitellogenin assay was carried out following the method of Hallgren et al., 2010 with some modifications. Tissue homogenate prepared from adult male D. melanogaster was mixed with 10
acetone (35%) and centrifuged at 10000g for 5 min. The pellet obtained was washed with ethanol. Following that, the medium was made alkaline with addition of NaOH, and kept at 70oC, later cooled down to room temperature. Thereafter, trichloroacetic acid was added and centrifuged at 20,000g for 5 min. The resulting supernatant was transferred to a new tube followed by dilution with distilled water and 1-butanol in 1:1 ratio. The mixture was recentrifuged and from lower aqueous phase 145μL sample was carefully transferred in duplicate
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to the wells of tarson polystyrene plates. The plates containing the reaction mixtures were placed in oven at 50oC for 120 min to remove traces of butanol. Finally, 65μL of acid malachite green
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was added to each well and kept at 50oC to increase the reaction rate. The absorbance was
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recorded at 630nm. 2.6.2 Mitofferin assay:
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Iron is an essential micronutrient for almost all organisms and acts as co-factor for many
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enzymes which have redox-reaction potentiality (Metzendorf and Lind, 2010).12 adult males were homogenized in 125 µL of lysis buffer. They then were centrifuged twice at 16,000 g in a bench-top cold centrifuge at 4°C, and 80 µL was recovered. It was then heated for 20 min at
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95°C and centrifuged at 16,000g for 2 min. We placed 66 µL of supernatant in a fresh tube and re-centrifuged at 16,000 g for 2 min. We added 50 µL of supernatant to 20 µL of 75 mM ascorbate, and it was then vortexed and spun down. Next, 20 µL of 10 mM ferrozine was added,
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vortexed, and spun down. Last, 40 µl of saturated ammonium acetate was added to each tube and vortexed, and absorbance was measured at 562 nm. 2.6.3 Mating pair counting: We quantified total number of mating in the control and treated groups following the method of Sing et al., (2015) with minor modifications. 20 virgin flies (10 male and 10 female flies) were
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transferred to a petri plate and observed for half an hour and the total number of mating pairs were recorded. Such observation was carried out 4 times for control and for each of the treatment categories. The cumulative number of mating pairs across all the observations for a given petri plate provided an estimate of total number of mating pair for a particular category. 3. Results: 3.1 Body weight of adult male D. melanogaster: Body weight is seen to be significantly
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(p<0.05) decreased (Fig. 1) in a dose dependent manner after chronic exposure to acephate. A
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maximum of 2.723-fold decrease in weight was found after 6 μg/mL acephate treatment. There is a negative correlation (Correlation coefficient= -0.992) between the exposure concentrations and
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the body weight.
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3.2 Apoptotic bodies in testis: Testis from different treatment categories of acephate showed (Fig. 2) appearance of apoptotic lesions when compared to the control group. The control testis
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presented no detectable apoptotic body whereas testis from individuals exposed to 1, 2, 3, 4, 5 and 6µg/mL acephate showed appearance of apoptotic bodies. In addition to that, the
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morphological structure of the testes from exposed categories manifested irregularities in comparison to the control ones.
3.3 Germ cell viability (MTT) of testis: The result of MTT assay (Fig. 3) confirmed that a
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significant (p<0.01) decrease in number of available viable germ cells in testis after chronic exposure to acephate when compared to control group. The decrease in cell viability is found to be dose dependent. In comparison to control, 9.035-folddecrease in number of viable germ cells was noticed after exposure to 6 µg/mL acephate concentration. Interestingly the number of viable germ cells were found to be negatively correlated (Coefficient of correlation =-0.99) to the treatment concentrations. 12
3.4 Catalase activity in male body: The result showed (Fig.4 [A] Left side) significantly increased (p<0.05) catalase activity in acephate treated flies in comparison to control group. The treatment groups receiving 1µg/mL- 4 µg/mL acephate exposure, demonstrated significantly higher catalase activity when compared with the control group whereas treatment concentration 5 µg/mL and 6 µg/mL significantly (p<0.05) lowered the catalase activity. 3.5 Lipid peroxidation in male body: Graph represented (Fig. 4 [B] Right side) here showed
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significantly altered lipid peroxidation when compared to control set. The MDA
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(malondialdehyde) content represents the lipid peroxidation in adult male body. Treatment
categories from1- 5 µg/mL showed an increasing trend of MDA content in the male body in
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comparison to control group which reflects the increasing lipid peroxidation in the body. But
3,4 and 5 µg/mL treatment categories.
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after 6 µg/mL acephate treatment, the MDA content is found to be lowered in comparison with
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3.6 Mitoferrin activity: Iron content of the body is measured to demonstrate the mitoferrin activity in this study. Graphical representation (Fig. 5) of the result showed decrease in iron
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content in male flies with increasing acephate exposure (1, 2, 3, 4, 5, and 6 µg/mL) when compared to the control group. Iron content decreased by1.684 fold at maximum exposure concentration (6µg/mL) of acephate.
3.7 Vitellogenin content in male flies: The vitellogenin content is found to get decreased after
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chronic acephate exposure. After 6 µg/mL acephate exposure, the vitellogenin content showed (Fig. 6) 2.754-fold decrease in comparison to control group. The result manifests a negative correlation between exposure concentration and vitellogenin content (coefficient of correlation 0.949). 3.8 Mating pair observed: Distinct alteration in the number of available mating pairs was observed after chronic acephate exposure. With increase in treatment concentrations, number of 13
mating pair observed [Fig. 7] presented a decreasing trend in comparison to control group. The result demonstrates a negative correlation between increasing treatment concentrations and subsequent decrease in number of mating pairs. 4. Discussion: Successful reproduction is controlled by both male and female individuals of a population. The role of body size and body mass of an individual is crucial since they influence insect
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physiology, ecological performance and behavior (Dial et al., 2008, Whitman, 2008). Adult
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male flies exposed to differential concentrations of acephate through food demonstrated
significant reduction in wet weight in a concentration-dependent manner. This confirmed the
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adverse effect of acephate on the body weight of a non-target organism, D. melanogaster.
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Works of Jemenez- Perez and Wang, 2004 suggested that, reproductive behaviors of insects are influenced by body weight, body size and fitness. Their study on lepidopteran insect Cnephasia
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jactatana confirmed that, male reproductive potentiality follows the “larger-the-better theory”. Another study with flesh fly Neobellieria bullata revealed that, larger bodied males produced
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more sperms than small bodied ones (Berrigan and Locke, 1991). In this light it can be assumed that, depletion in body weight of Drosophila males as observed in this study (Fig. 1), might be an indirect indicator of reduced reproductive potential.
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The acridine orange staining of testes following acephate exposure demonstrated presence of apoptotic lesions in the form of fluorescent green spots. On comparison with untreated controls, testes of treated flies revealed greater number of apoptotic lesions. Increased frequency of acridine orange positive lesions after treatment (Fig. 2) suggests augmented DNA damage and cellular disintegration. Previously, similar apoptotic lesions were visualized in Drosophila ovary following chronic fluoride insult (Khatun et al., 2017). 14
Furthermore, germ cell viability assay (MTT assay) revealed gradual decrease in number of viable cells in testes with increasing acepahte onslaught (Fig. 3). Pesticide-induced oxidative stress is a well-known factor responsible for triggering cellular apoptosis via extrinsic and intrinsic pathways. Other organophosphates such as basudin, cidial and fenix (Trielli et al., 2006) were also reported to affect cell viability significantly. Rajak et al., 2018 has suggested that besides traditional cholinesterase inhibition, acephate triggers ROS-mediated toxicosis in
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various tissues resulting in DNA, protein and cell damage. Interestingly, co-administration of vitamin C along with acephate rescued from adverse effects (Rajak et al., 2017). In this line,
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acephate having cytotoxic potential is expected to reduce germ cell viability as detected in the
compromised reproductive fitness of treated males.
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present study. Reduced availability of mature germ cells might be a decisive factor behind the
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To have a better insight, this study was extended towards monitoring the activities of stress
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marker enzymes like catalase (CAT) and lipid peroxidase (LPO). Results have demonstrated an increased activity of catalase enzyme at 1- 4 µg/mL concentrations of acephate (Fig.4 [A]). This endogenous antioxidant constitutes the first line of defense against free-radical mediated injury
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(Lobo et al., 2010). Lipid peroxidation is a potent marker of oxidative stress and in this study; organisms manifested higher lipid peroxidation in testes following chronic sub-lethal acephate treatment. Therefore, increased activities of both antioxidant and oxidative stress marker
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enzymes suggest acephate-induced oxidative stress in adult males of Drosophila, as also documented by Rajak et al., 2017, in Drosophila larvae. Interestingly exposures to higher concentrations of acephate (5µg/mL and 6µg/mL) resulted in reduced enzyme activities due to unavailability of viable cells expressing the said enzyme in reproductive organs and tissues. Like mammals, Drosophila male reproductive system consists of a pair of testes, seminal vesicles and seminal proteins (Bairati, 1968). Drosophila spermatogenesis shares similarities 15
with that of mammals (Castrillon et al., 1993). Studies have suggested several conserved genes involved in development of reproductive organs in both Drosophila and mammals. For example, SOX [Sry (sex determining region Y)-related HMGbox] genes in mammals are known to play critical role in testes development (Sekido et al., 2004, Nanda et al., 2009). Mechanism of spermatogenesis in Drosophila and human indicates conservation of genetic regulators throughout the evolution (King et al., 1968). In addition, ecdysone in Drosophila has
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been projected as a primitive counterpart of the mammalian sex hormones, testosterone and
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estrogens (De Loof and Huybrechts, 1998). In the present study, reproductive enzymes
manifested compromised activity following chronic acephate exposure. Vitellogenin, a major
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yolk precursor protein in female body is also found in trace amount in male insects (e.g., Bombyx mori) (Lamy, 1984). Lamy, 1984 reported that, vitellogenesis in males is common for all
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lepidopteran species along with few species of Diptera, Dictyoptera and Orthopteran insects.
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Production of vitellogenin (Vg) in the fat body has been described as a feature of normal ontogenesis in males of various species. An increase in the level of vitellogenin transcript has been shown to correlate with the high level of vitellogenin in the haemolymph of larvae and
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adult drones of Apis mellifera (Colonello-Frattini et al., 2010, Guidugli et al., 2005). Interestingly, study by Majewska et al., 2014 has shown a strong linkage between yolk proteins and spermatogenesis in D. melanogaster.
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Furthermore, it is known that vitellogenin acts as an antioxidant, and elevated vitellogenin content leads to prolonged life span in honeybee (Havukainen et al., 2013). Vitellogenin is one of the largest lipid transfer proteins (LLTPs) which manifest multifaceted roles in animals like lipid transporters, inflammation suppressors, immune modulators, and blood coagulators (Havukainen et al., 2013). Though the specific role of yolk proteins are not yet confirmed, it is assumed that, yolk proteins have a definite role to play in sperm maturation in the male fly. In 16
the present study, decline in vitellogenin level was observed in male flies following chronic sublethal exposure to Acephate (Fig.6) Thus, decreased vitellogenin level might impede with the normal maturation of sperms thereby causing a significant alteration in the male reproductive system. Ecdysis-triggering hormone (ETH) has been reported to remain functional in adult D. melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction (Meiselman et al., 2017). Further, Meiselman et al., 2017 opined
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that, ETH might contribute towards increased plasticity to the stress response system ranging from few hours to even several days. Thus, in the present study, ETH signaling could have been
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upregulated after acephate exposure. Upregulation of apoptosis, excessive oxidative stress and
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compromised reproductive potential suggest that, ETH signaling might be affected in case of acephate- exposed flies to deficit JH availability. It is important to note that, ETH shortage leads
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to reduced JH level which consequently reduces ovary size, egg production, and yolk deposition in mature oocytes of female Drosophila (Meiselmanet al., 2017) A balance between JH and 20
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hydroxyecdysone determines whether oogenesis will progress beyond mid-oogenesis checkpoint stage or not (Gruntenko et al., 2010, Liu et al., 2008). On the other hand, JH deficiency has
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been reported to induce caspase mediated cell death (Soller et al., 1999, Pritchett et al., 2009). In the present study, significant decline in viable cell number in reproductive tissues were noted. Furthermore, in case of JH deficient males, partner fecundity impairment has been reported to occur because of a reduced rate of accessory gland protein synthesis (Meiselmanet al., 2017). It
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has further been demonstrated that, reduced female fecundity as well as impaired male reproductive potential in RNAi knock down of EcR in Inka cells could be rescued through administration of JH analog. Thus, acephate exposure might have followed the above discussed pathway to hinder optimum sperm maturation and thereby male fertility in Drosophila.
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The present study has further shown the decrease in iron content in adult male flies following chronic acephate treatment (Fig. 5). The iron content is considered as a function of mitoferrin enzyme activity. Mitoferrin is a mitochondrial iron carrier protein that helps in iron metabolism during spermatogenesis (Metzendorf and Lind, 2010). Earlier studies have confirmed that, D. melanogaster and other invertebrates (i.e., Sea urchin, Caenorhabditis elegans, bee, wasp, mosquito and flour beetle) have only one mitoferrin gene, which has been suggested to be a
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functional homolog of vertebrate mitoferrin 2 (Metzendorf et al., 2009). Findings of Metzendorf and Lind, 2010 imply that, Drosophila mitoferrin and the mitochondrial iron
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metabolism are essential for regulation of spermatogenesis. It is also an established fact that, iron
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is needed during sperm maturation (Metzendorf and Lind, 2010, Detmer and Chan, 2007). Genes involved in mitochondrial iron metabolism are expressed in testis of both vertebrates and
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Drosophila. It has further been reported that, inadequate mitoferrin activity can result in defective energy metabolism in mitochondria, which during spermatogenesis causes
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morphological alterations like decreased testis length (Metzendorf and Lind, 2010). Earlier studies on mammalian spermatogenesis indicated a nutritional function of iron during
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spermatogenesis. Orlando et al., 1985 has suggested that, levels of transferrin, a ubiquitous iron transfer protein in human seminal plasma, can be correlated with sperm abundance. Thus, in the present study, significant reduction in mitoferrin activity indicates altered iron metabolism which in turn might have impeded the process of spermatogenesis and thereby male fertility in treated
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flies.
Several studies across the globe have been performed on Drosophila mating patterns to demonstrate the basic factors that are crucial for reproductive success. Interestingly, mating speed has been categorized as one of the main components depicting male fitness in Drosophila (Parsons, 1974, Cade, 1984). Hence, the frequency or the number of available mating pairs 18
might be considered as a relevant index demonstrating the male reproductive fitness. Hoffmann 1994 suggested that, aggressive behaviour is also an important criterion for determining male mating success in some species of fruit flies. Generally larger males of D. pseudoobscura and D. melanogaster are better selected in the field (Partridge et al., 1987) and in laboratory respectively (Partridge and Farquhar, 1983). But results of this study have shown a significant decline in wet weight of the male body after
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acephate exposure. This implies that, flies exposed to higher concentrations of acephate might
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automatically have taken a back seat in reproductive selection.
Traditional concept of sexual selection suggests that, in most animal species, males are less
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discriminating in their choice of mating partners than females; because their investment in
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offspring is much lower (Bateman, 1948, Trivers, 1972). However, in case of many species, the males demonstrate a high energy investment in reproduction (mating) because of energetically
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expensive courtship displays (Judge and Brooks, 2001) and the production of ejaculates (Dewsbury, 1982, Galvani and Johnstone, 1998).
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It has been demonstrated that, lesser male mate discrimination is predicted when mating requires less time and/or resources whereas greater mate discrimination by males is obvious when investment in time and energy is greater (Engqvist and Sauer 2001, Kokko and Johnstone 2002). This substantially affects future mating opportunities of males (Simmons, 1990, Clutton-
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Brock, and Langley, 1997, Bonduriansky, 2001). In the present study, the males inhabiting an environment with graded concentrations of acephate are expected to spend considerable energy currency for combating the stress. Hence, males would be left with lower energy thereby resulting in either greater mate discrimination or reduction in the frequency of mating. The
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results validate this fact, since observed numbers of mating pairs in treated groups are found to decrease with increase in treatment concentrations. Sing et al., 2015 demonstrated that, due to environmental stress like cold shock, there happens to be a shift in reproductive behavior. Similarly, in the present study we have observed a change in the frequency of mating ability in males upon chemical stress. Sing et al., 2015 also reported that, females in D. melanogaster when held with males post cold shock, the number of mating
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observed is significantly higher in comparison to control. In contrary to that, the present finding
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depicts decrease in mating frequency after acephate mediated stress. This might be due to tradeoff between survival and reproductive potential. Since greater energy is required for
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survival in stressed environment, hence less energy remains at disposal for reproduction and /
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mating activities. 5. Conclusions:
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In the present study, morphological, biochemical and behavioural parameters were studied to explore the toxic effects of chronic sub-lethal organophosphate exposure in male reproductive
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system of a non-target model organism D. melanogaster. It was observed that after chronic sublethal exposure to organophosphate chemical acephate, physical, biochemical and reproductive fitness of the exposed individuals demonstrated a compromised state in comparison with their control counterparts. Individual variation in male reproductive success in a lifetime is controlled
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by the expression of many different traits relevant for reproduction. It has been seen that natural selection favors phenotypes that give the males an advantage of fathering more offspring, though the expression of these characters might be influenced by trade-offs. Several studies have established the fact that environmental factors are instrumental in mediating these trade-offs (Filice and Long, 2018, Roff and Fairbairn, 2007, Joseph et al., 2017). Thus, in case of males
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having a fixed energy budget, increased investment into a specific reproductive phenotype might result in reduced expression of another. In the present study the male Drosophilids might have expressed reduced reproductive fitness as a result of altered resource allocation supporting survival in the acephate-contaminated (stressed) environment. Additionally, the results of this exposure analysis in Drosophila can be extrapolated to other non-target organisms as well as human beings because of the developmental and physiological similarities. Furthermore, this
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study yet again validates the use of Drosophila melanogaster as a successful model organism in
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toxicological assessment of chemicals.
Author contribution
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To Whom it may concern
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This is to mention the Credit roles of the different authors involved in the conceptualization, shaping of experiments, execution of the experimental methodologies, data collection, data analysis, interpretation of results and writing of the manuscript
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MOUTUSHI MANDI: Experimental methodologies, Investigation, Data collection, Data analysis, Validation; Visualization; Preparation of original draft.
SALMA KHATUN: Experimental investigation; Methodology, data collection, Visualization PREM RAJAK: Formal analysis of data, Software, Review and Editing ABHIJIT MAZUMDAR: Supervision, Data Interpretation, Suggestion, Review
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SUMEDHA ROY: Conceptualization, Data curation, Interpretation, Supervision, Review and editing, Project administration
Conflict of interest: There is no known conflict of interest.
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Acknowledgments: Authors are grateful to the Head, Department of Zoology (UGC DRS, DST-FIST and DST PURSE sponsored), The University of Burdwan for providing necessary infrastructural facilities to carry out the present work. Authors are thankful to Prof. Niladri Hazra (BU) for providing environmental chambers to maintain Drosophila melanogaster stock and extending the
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microscopic facilities. The authors are indebted to Dr. Sanjib Ray (BU) and Dr. Koushik Ghosh (BU) for providing chemicals and analytical instruments time to time through the course of this
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work.
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Thapar, A., Sandhir, R., SivaKiran, R. R., 2002. Acephate induced oxidative damage in erythrocytes. Indian J. Exp. Biol.40 (8), 963-966. Thornton, B.J., Elthon, T.E., Cerny, R.L., Siegfried, B.D., 2010. Proteomic analysis of atrazine exposure in Drosophila melanogaster (Diptera: Drosophilidae). Chemosphere 81, 235-241.
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Toppari, J., Kaleva, M.,Virtanen, H.E., 2001. Trends in the incidence of cryptorchidism and hypospadias, and methodological limitations of registry-based data. Hum.Reprod. Update. 7(3), 282-286. Trevizan, L. R. P., De Baptista. G. C., Papa, G., 2005. Acephate and methamidophos residues in greenhouse and in field grown tomatoes. Hortic. Bras. 23 (1), 38-43. 34
Trielli, F., Chessa, M.G., Amaroli, A., Ognibene, M., Delmonte Corrado, M.U., 2006. Effects of organophosphate compounds on a soil protist, Colpoda inflata(Ciliophora, Colpodidae).Chemosphere 65(10), 1731-1737. Tripathia, S.M., Thakera, A.M., Joshi C.G., Sankhalaa, L.N., 2012. Acephate immunotoxicity in White Leghorn cockerel chicks upon experimental exposure. Environ. Toxicol. Pharmacol. 34, 192-199
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Trivers, R., 1972. Parental investment and sexual selection, In B. Campbell (Ed.), Sexual
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selection and the descent of man New York: Aldine DeGruyter, 136-179.
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Von Stryk, F. G., Jarvis, W. R., 1978. Residues of mancozeb, maneb and ethylene thiourea in fungicide-treated field and greenhouse tomatoes, Canadian Journal of Plant Science 58(3),
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623-628.
17, 117-134.
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Whitman, D.E., 2008. The significance of body size in the Orthoptera: a review. J. Orthopt. Res.
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Xu, M.Y., Wang, P., Sun, Y.J., Wang, H.P., Liang, Y.J., Zhu, L., et al. 2015. Redox status in liver of rats following subchronic exposure to the combination of low dose dichlorvos and deltamethrin. Pestic Biochem Physiol. 124, 60-65.
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Yang,Y., Hou, L., Li, Y., Ni, J., Liu, L., 2013. Neuronal necrosis and spreading death in a Drosophila genetic model. Cell Death Dis. 4, e723.
Yao, J., Zhu, Y.C., Adamczyk, J., Luttrell, R., 2018. Influences of Acephate and mixtures with other commonly used pesticides on honey bee (Apis mellifera) survival and detoxification enzyme activities. Comp. Biochem. Physiol. .C Toxicol. Pharmacol. 209, 9-17. doi: 10.1016/j.cbpc.2018.03.005. 35
Zinkl, J.G., Roberts, R.B., Henry, C.J, Lenhart, D.J., 1980. Inhibition of brain cholinesterase activity in forest birds and squirrels exposed to aerially applied Acephate. Bull. Environ.
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Contam. Toxicol. 24, 676-683.
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Legends to Figures: Fig.1. Alterations in the wet weight of adult body in Drosophila melanogaster after chronic exposure to graded concentrations of acephate (1-6µg/mL). Treated groups were compared with untreated control group for assessment of significant change. The data represents mean ± S.E. of three pooled determinations. Each pool consisted of 30 individuals. “*” denoted significant
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change in comparison to control. *p < 0.05 were ascribed as statistically significant.
Fig.2.Effect of acephate exposure on testis: Figure shows AO staining of testis from (3-5) days old adult D. melanogaster facing chronic insult to six different acephate concentrations(b-1, c-2,
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d-3, e-4, f-5, g-6 µg/mL) along with control (a) group. Bright green fluorescent bodies (indicated by circle) correspond to apoptotic masses in the stained tissues. Apoptosis was negligible in case of control category (a) but prominently visible in case of (b, c, d, e, f and g). The structure of the
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testes in case of treated flies (b-g) is found to be altered in comparison to the control (a).
Fig.3.Figure represents results of MTT assay of germ cell from adult male D. melanogaster exposed to six different treatment categories (1-6 µg/mL) along with control counterparts. Data
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specify mean±S.E. Data represents numerical abundance of viable germ cells reflected as absorbance at 578nm wavelength. *p <0.05 were considered as statistically significant when
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compared to control category.
Fig.4. Changes in the endogenous detoxification enzyme (Catalase) and oxidative stress marker enzyme (Lipid peroxidase) activities. (A) Catalase (CAT) activity in control and differentially exposed flies. (B) Lipid peroxidase (LPO) activity in control and treated groups. The data 39
represents mean ± S.E. of three pooled determinations. “*” denoted significant changes in treated
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group in comparison to control.*p <0.05are ascribed as statistically significant.
Fig.5.The figure represents mitoferrin activity represented through the iron content in male adults of D. melanogaster after chronic exposure to six different concentrations of acephate (1-6
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µg/mL). The vertical bars specify standard error. Data represents mean ± S.E. (*) representing p<0.05 are considered as statistically significant when compared between treated and control category.
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Fig.6.The figure represents vitellogenin content in adult male of D. melanogaster after chronic exposure to six different concentrations of acephate (1-6 µg/mL). The vertical bars specify standard error. Data represents mean ± S.E. (*) representing p<0.05 are considered as
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statistically significant when compared among treated and control category.
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Fig.7. The scattered plot represents number of mating pair in control and acephate treated D. melanogaster. Data represents four separate observations for control and each of the treatment categories. Each observation was carried out for duration of 30 min, during which number of
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observed mating pair was recorded.
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PII:
S1382-6689(19)30183-8
DOI:
https://doi.org/10.1016/j.etap.2019.103308
Reference:
ENVTOX 103308
To appear in:
Environmental Toxicology and Pharmacology
Received Date:
11 June 2019
Revised Date:
25 November 2019
Accepted Date:
26 November 2019
Please cite this article as: Mandi M, Khatun S, Rajak P, Mazumdar A, Roy S, Potential risk of organophosphate exposure in male reproductive system of a non-target insect model Drosophila melanogaster, Environmental Toxicology and Pharmacology (2019), doi: https://doi.org/10.1016/j.etap.2019.103308
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Potential risk of organophosphate exposure in male reproductive system of a non-target insect model Drosophila melanogaster
Moutushi Mandia, Salma Khatuna, Prem Rajakb, Abhijit Mazumdarc and Sumedha Roya,*
Toxicology Research Unit, Department of Zoology, The University of Burdwan, West Bengal,
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Department of Animal Science, Kazi Nazrul University, Asansol, West Bengal, India
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India
Entomology Research Lab, Department of Zoology, The University of Burdwan, West Bengal,
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India
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Corresponding author: Email: [email protected]
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Graphical abstract
The plate represents the findings of the entire study. Study demonstrates that chronic sub-lethal
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acephate exposure causes significant alteration in male reproductive structures, physiology and behavior which have been reflected through the alterations in the studied parameters. The results
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confirm that the altered expressions of the selected parameters might be the reason for the compromised male reproductive efficiency in the non-target model organism, Drosophila
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melanogaster following chronic acephate exposure.
HIGHLIGHTS
Chronic sub-lethal Acephate exposure reduces Drosophila male germ cell viability Acephate exposure leads to altered adult male body weight and increased apoptosis Acephate(1-6µg/mL) exposure increases CAT and LPO activities in male adults Acephate alters testis architecture and Mitoferrin and vitellogenin content 2
Acephate exposure reduces number of mating pairs
Abstract Based on several adverse reports of pesticides on reproductive efficiency of various organisms, studies on “reproductive toxicity” have gained importance. Fecundity, reflecting reproductive
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success of any organism, is governed by several factors from female and male reproductive
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systems. This present study explored morphological and biochemical alterations in the male reproductive system of a non-target model organism, Drosophila melanogaster following
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chronic sub-lethal exposure (1st instar larvae differentially exposed to 1-6µg/mL until adulthood) to the organophosphate (OP) pesticide, acephate (LC50 8.71µg/mL). This study demonstrates
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altered testis structure, decreased germ cell viability and gross body weight, increased activities
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of oxidative stress markers lipid peroxidase (LPO), and the endogenous antioxidant enzyme catalase (CAT), and altered expression of reproductive marker proteins like vitellogenin and
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mitoferrin in acephate-exposed flies when compared to control counterparts. Altered reproductive behavior, indicated by a significant decline in the number of mating pairs, validates the adverse effect of chronic acephate exposure on male reproduction in the non-target insect model D. melanogaster.
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Keywords: D. melanogaster, Male reproduction, Mitoferrin, Organophosphate, Vitellogenin.
1. Introduction:
Pesticides are potent chemicals specifically designed for repelling, preventing or destroying pests (Landrigan, 2018). In recent decades, significant human as well as non-target toxicity from pesticide exposure have been documented (Frazier et al., 2008, Dutta et al., 2014, Dutta et al., 3
2016, Rajak et al., 2017). People, through dietary and other routes, get exposed to several pesticides that undergo accumulation in measurable concentration inside the body (Frazier et al., 2008). Therefore, it is essential to investigate deleterious impacts, if any of these biocides on non-target organisms. Pesticide poisoning has been increased by several folds in recent decades. According to a report (Yang et al., 2013), around 250000 people die throughout the world each year due to pesticide
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poisoning. Unfortunately, 750000-3000000 people are unintentionally poisoned by
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organophosphate (OP) chemicals every year, with an estimated 300000 deaths (Balali-Mood and Saber, 2012).
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OP pesticides are esters of phosphoric and thiophosphoric acids and their toxicity mainly rely on their ability to inhibit acetylcholine esterase (AChE) activity. In addition, OPs may lead
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to oxidative stress, axonal transport deficits, neuroinflammation, and autoimmune conditions
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(Xu et al., 2015; Naughton and Terry, 2018). Acephate (O,S-dimethyl acetylphosphoramidothioate), a popular OP in most of the developing countries is used as foliar spray (Thapar et al., 2002, Trevizan et al., 2005) to control wide range of biting and sucking
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insects, especially aphids, leaf miners, lepidopteran larvae, sawflies and thrips (Douglas and Hamish, 1983; Dhingra and Dhingra, 2009). Acephate toxicity is attributed to its bio-activation and metabolic conversion into more toxic
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intermediate, methamidophos, which is also a potent AChE inhibitor. Des-o-methyl acephate is a metabolized form of acephate produced as a byproduct in mammals (Farag et al., 2000, Mahajna et al., 1997). Acephate and methamidophos residues are higher in protected cultivation conditions in leaves and soil, than in the open-field (Cabras et al., 1985, Frank et al., 1987, Von Stryk, and Jarvis, 1978, Meloni et al., 1984). Insects metabolize acephate into des-omethyl acephate accounting for acephate’s relatively high selectivity against insects (Roberts 4
and Hutson, 1999). More recently, another plausible mechanism of action of acephate toxicity through ROS mediated pathways was confirmed by our laboratory (Rajak et al., 2018) and simultaneously protective potential of co-administrated L-ascorbic acid validated the idea of acephate induced oxidative stress in Drosophila. Acephate is toxic to non-targets like pollinators (bees), fishes, birds and mammals. Recent study conducted by Yao et al., 2018 concluded that, honey bees exposed to 6.97 mg/mL killed 50
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percent of the population. In addition, individuals who survived after 48h of exposure,
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manifested reduced body weight and suppressed esterase activity. Some studies documented histo-pathological impacts of acephate on fish population. Kavita and Shakila, 2015 were
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documented remarkable alterations in histological architecture of vital organs like liver, intestine, muscle and skin of Poecilia sphenops. Acute and chronic exposure to acephate decreases
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amylase activity in Labeo rohita (Bhilave and Kulkarni, 2016). Hematological parameters like
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RBC count, WBC count and packed cell volume (PCV) were significantly declined in Channa punctata following sub-lethal exposure to acephate (Satish et al., 2018). Significant reduction in hatching rates of embryos of zebra fish has been reported following exposure to OPs like
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chlorpyrifos and diazinon (Cao et al., 2018). Decreased growth rate, survivability and higher morphological abnormalities were evident in acephate treated larvae of salamander, Ambystoma gracile (Geen et al., 1984). Acephate and other OPs may affect breeding success, suppression
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of egg formation, eggshell thinning, impaired incubation, behavior and migratory orientation in avian population (Brigs, 1992; Mitra and Mitra, 2018). Furthermore, immunotoxic response was known to be induced by acephate in White Leghorn cockerel chicks (Tripathi et al., 2012). Some toxic effects of organophosphates and acephate have also been demonstrated on humans. Upon inhalation, organophosphates are known to cause dizziness, headache, nausea and vomiting (Reregistration Elegibility Decision, 2006; Reigart et al., 1999). Moreover, acute 5
dermal exposure results in redness, swelling and rashes in the skin. Chronic exposure to acephate generates intermediate compound called methamidophos which leads to muscular weakness in neck, limb and even causes death (Christiansen et al., 2011). In a study, tobacco field workers exposed to acephate manifested decreased SOD activity in their erythrocytes (Panemangalore et al., 1999). Acephate is also known to affect human sperm motility, vitality and functional integrity of sperm plasma membrane (Dhanushka and Peiris, 2017).
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The no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) of acephate have been established for some mammalian species. For rat, rabbit and
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human, NOAEL was documented as 2.5, 3 and 1.2mg/kg body weight respectively (Fiedler,
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1987, Gavit and Patil, 2016, Zinkl et al., 1980, Behera and Bhunya, 1989). Subsequently, LOAEL has also been established for rat and rabbit as 5 and 10mg/kg body weight but the same
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is still unexplored for human beings (Gupta and Moretto, 2005).
Many epidemiological studies have recognized the connection between OP exposure and
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reproductive disorders such as infertility, birth defect, adverse pregnancy outcomes, and perinatal deaths (Peiris-Jhon and Wickremasinghe, 2008). Interestingly, Perry et al. (2007) have
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suggested that, the reproductive toxicity from OP exposure results due to reduced brain acetylcholinesterase activity which in turn interferes with pituitary gonadotropin thereby causing infertility. Male infertility has become a major manifestation of reproductive toxicity. In the last two decades, sperm counts in human males have reduced below the threshold concentration of
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55 million/mL. In addition, significant decline in semen quality has also been reported (Carlsen et al., 1992). Other documentation regarding impaired male reproductive function in human and wildlife populations (Phillips and Tanphaichitr, 2008) have increased interest in studies exploring toxicological effects of OPs on male reproductive system (Oakes et al., 2002, Sharma et al., 2015) Additional concerns were raised because sperm counts are found to decline at an
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alarming rate, 2% per annum for the past 20 years (Skakkebaek et al., 2001, Toppari et al., 2001, Fisher, 2004). In another study, modern lifestyle was reported to compromise sperm quantity and quality in human (Jayachandra and Srinivasa, 2011). Furthermore, it has been reported that, pyriproxifen impairs the spermatogenesis in B. germanica, (Fathpour et al., 2007). Farag et al., (2000) confirmed about acephate’s potential to cause reproductive toxicity in male mice (e.g., decreased sperm motility and count) from high dose oral administration.
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Joshi and Sharma (2011) reported that, acephate exposure reduced male fertility through altered serum testosterone, LH, and FSH levels in albino rats. Another chemical atrazine has
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been found to affect expression of stress genes (Le Goff et al., 2006) and proteins (Thornton et
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al., 2010) in D. melanogaster, egg production in Orchesella cincta (Badejo and Van Straalen, 1992) and mating choices in Tenebrio molitor (Mc Callum et al., 2013). Mukhopadhyay et al.,
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2003, documented reduced fertility and reproductive performance in both male and female fruit flies following differential exposure to argemone oil. Therefore, these studies indicated that,
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males are at risk of becoming infertile due to environmental exposure to innumerable chemicals and pollutants. Thus, it is essential to investigate xenobiotic induced hazardous effects on male
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fertility. D. melanogaster is a well-established model showing almost 75% functional homology with the disease-causing genes of human beings (Pandey and Nichols, 2011, Demir et al., 2013). Developmental, cellular and molecular mechanisms in Drosophila are well established. Hence, it has emerged as a perfect model organism for researchers from several fields, like
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genetics, medicine, developmental biology, immunology and toxicology to elucidate problems of human interest (Koh et al., 2006, Rajak et al., 2017). From past several decades, fruit fly Drosophila melanogaster is in use as non-target organism for toxicity assessment of various chemicals. Hence, a new discipline in research known as “Drosophotoxicology” is gaining popularity in recent days. Furthermore, Drosophila, with its well characterized developmental
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and reproductive phenomena, offers itself as a potent alternative and a promising model for the male reproductive toxicity assessment. Thus, in the present study, our major goal was to explore variation in reproductive fitness of Drosophila males following chronic sub-lethal acephate exposure through analysis of selected relevant parameters. 2. Materials and methods:
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2.1 Experimental organism
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D. melanogaster (Oregon R strain) was used for the present study. Drosophila were maintained
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at 23±1°C temperature, 50-60 % humidity within environmental test chamber in the laboratory. 2.2 Selection and preparation of experimental concentrations in food media
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In order to investigate the effects of chronic acephate exposure in Drosophila, six sub-lethal
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concentrations (1-6 µg/mL) below the evaluated chronic LC50 (Rajak et al., 2017) were selected and mixed with SDM (standard Drosophila medium) for use in the present study.
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2.3 Treatment schedule
Freshly hatched 1stinstar larvae were reared on food containing selected concentrations of acephate until their emergence as adults. These adult males were considered for experiments in
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the current study.
2.4 Parameters for assessment of toxic effects: 2.4.1 Body weight measurement 30 adult male flies were taken from each treatment cluster for measuring their body weight using electronic weight machine (Afcoset, Sr No. 0413050). Equal number of male flies from control 8
group was used for comparison. Assessments were carried out in triplicate sets hence number of individuals considered for each experimental group was n= 90 (3*30). 2.4.2 Acridine orange (AO) staining for localization of apoptotic lesions AO staining was performed following the protocol of Alone et al., 2005 with slight modifications (Khatun et al., 2017). Testes from adult flies were isolated in PBS and stained with AO (10µg/mL) for 2min. Tissues were washed with PBS and images were taken under
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fluorescent microscope (Leica DMI6000B).
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2.4.3 Germ cell viability study through MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-
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diphenyltetrazolium bromide] assay
Testes were dissected and were processed for determination of germ cell viability through MTT
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assay (Babot et al., 2005). Testes from adults were taken out in PBS and processed with
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collagenase (0.5mg/mL) to enzymatically individualize the cells. MTT (5mg/mL) solution was added to cell suspensions and incubated overnight. After incubation, DMSO was added to
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solubilize the formazan crystals. Absorbance was recorded at 578nm using ELISA plate-reader (Erba-Mannheim, LisaScan EM).
2.5 Biochemical assays for OS markers
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The activity of oxidative stress marker enzymes such as lipid peroxidase (LPO) and catalase (CAT) were quantified in adult male fly to detect alterations from their normal status under chronic sub-lethal acephate exposure. 2.5.1 Preparation of tissue homogenates
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10% tissue homogenates of control and treated flies were prepared from pooled samples of each experimental set using 0.1M phosphate buffer containing 0.15M KCl (pH 7.4) and centrifuged at 10,000g for 20min. Supernatant containing the crude cytosol was collected for protein estimation and enzyme assays. Protein estimation was done following the method of Lowry et al., 1951. 2.5.1.1 CAT assay CAT assay was performed following the procedure of Sinha, 1972 with minor modifications.
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The reaction mixture contained tissue sample, distilled water, 0.01M sodium pyrophosphate
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buffer (0.01 M, pH 7.0) and H2O2 (0.2M). Reaction was allowed for 2min and was stopped by adding dichromate-acetic acid reagent. Mixture was heated for 10min and the absorbance was
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recorded at 570nm.
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2.5.1.2 LPO Assay
Malonyldialdehyde (MDA), a major product of ROS-induced lipid peroxidation, was quantified
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following the method of Ohkawa et al., 1979 with minor modifications. Briefly, tissue homogenate (10%), SDS (8.1%) and acetic acid (20%) were taken in microfuge tubes and the pH
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adjusted to 3.5. Later, the reaction mixture was treated with thiobarbituric acid (0.8%) and heated at 95 °C for 1 h. The solution was cooled and mixed with a butanol–pyridine mixture. The absorbance of the final pink coloured organic layer was recorded at 532 nm against n-butanol as
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the blank.
2.6 Biochemical assays for reproductive protein marker 2.6.1 Vitellogenin assay: Vitellogenin assay was carried out following the method of Hallgren et al., 2010 with some modifications. Tissue homogenate prepared from adult male D. melanogaster was mixed with 10
acetone (35%) and centrifuged at 10000g for 5 min. The pellet obtained was washed with ethanol. Following that, the medium was made alkaline with addition of NaOH, and kept at 70oC, later cooled down to room temperature. Thereafter, trichloroacetic acid was added and centrifuged at 20,000g for 5 min. The resulting supernatant was transferred to a new tube followed by dilution with distilled water and 1-butanol in 1:1 ratio. The mixture was recentrifuged and from lower aqueous phase 145μL sample was carefully transferred in duplicate
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to the wells of tarson polystyrene plates. The plates containing the reaction mixtures were placed in oven at 50oC for 120 min to remove traces of butanol. Finally, 65μL of acid malachite green
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was added to each well and kept at 50oC to increase the reaction rate. The absorbance was
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recorded at 630nm. 2.6.2 Mitofferin assay:
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Iron is an essential micronutrient for almost all organisms and acts as co-factor for many
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enzymes which have redox-reaction potentiality (Metzendorf and Lind, 2010).12 adult males were homogenized in 125 µL of lysis buffer. They then were centrifuged twice at 16,000 g in a bench-top cold centrifuge at 4°C, and 80 µL was recovered. It was then heated for 20 min at
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95°C and centrifuged at 16,000g for 2 min. We placed 66 µL of supernatant in a fresh tube and re-centrifuged at 16,000 g for 2 min. We added 50 µL of supernatant to 20 µL of 75 mM ascorbate, and it was then vortexed and spun down. Next, 20 µL of 10 mM ferrozine was added,
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vortexed, and spun down. Last, 40 µl of saturated ammonium acetate was added to each tube and vortexed, and absorbance was measured at 562 nm. 2.6.3 Mating pair counting: We quantified total number of mating in the control and treated groups following the method of Sing et al., (2015) with minor modifications. 20 virgin flies (10 male and 10 female flies) were
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transferred to a petri plate and observed for half an hour and the total number of mating pairs were recorded. Such observation was carried out 4 times for control and for each of the treatment categories. The cumulative number of mating pairs across all the observations for a given petri plate provided an estimate of total number of mating pair for a particular category. 3. Results: 3.1 Body weight of adult male D. melanogaster: Body weight is seen to be significantly
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(p<0.05) decreased (Fig. 1) in a dose dependent manner after chronic exposure to acephate. A
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maximum of 2.723-fold decrease in weight was found after 6 μg/mL acephate treatment. There is a negative correlation (Correlation coefficient= -0.992) between the exposure concentrations and
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the body weight.
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3.2 Apoptotic bodies in testis: Testis from different treatment categories of acephate showed (Fig. 2) appearance of apoptotic lesions when compared to the control group. The control testis
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presented no detectable apoptotic body whereas testis from individuals exposed to 1, 2, 3, 4, 5 and 6µg/mL acephate showed appearance of apoptotic bodies. In addition to that, the
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morphological structure of the testes from exposed categories manifested irregularities in comparison to the control ones.
3.3 Germ cell viability (MTT) of testis: The result of MTT assay (Fig. 3) confirmed that a
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significant (p<0.01) decrease in number of available viable germ cells in testis after chronic exposure to acephate when compared to control group. The decrease in cell viability is found to be dose dependent. In comparison to control, 9.035-folddecrease in number of viable germ cells was noticed after exposure to 6 µg/mL acephate concentration. Interestingly the number of viable germ cells were found to be negatively correlated (Coefficient of correlation =-0.99) to the treatment concentrations. 12
3.4 Catalase activity in male body: The result showed (Fig.4 [A] Left side) significantly increased (p<0.05) catalase activity in acephate treated flies in comparison to control group. The treatment groups receiving 1µg/mL- 4 µg/mL acephate exposure, demonstrated significantly higher catalase activity when compared with the control group whereas treatment concentration 5 µg/mL and 6 µg/mL significantly (p<0.05) lowered the catalase activity. 3.5 Lipid peroxidation in male body: Graph represented (Fig. 4 [B] Right side) here showed
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significantly altered lipid peroxidation when compared to control set. The MDA
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(malondialdehyde) content represents the lipid peroxidation in adult male body. Treatment
categories from1- 5 µg/mL showed an increasing trend of MDA content in the male body in
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comparison to control group which reflects the increasing lipid peroxidation in the body. But
3,4 and 5 µg/mL treatment categories.
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after 6 µg/mL acephate treatment, the MDA content is found to be lowered in comparison with
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3.6 Mitoferrin activity: Iron content of the body is measured to demonstrate the mitoferrin activity in this study. Graphical representation (Fig. 5) of the result showed decrease in iron
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content in male flies with increasing acephate exposure (1, 2, 3, 4, 5, and 6 µg/mL) when compared to the control group. Iron content decreased by1.684 fold at maximum exposure concentration (6µg/mL) of acephate.
3.7 Vitellogenin content in male flies: The vitellogenin content is found to get decreased after
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chronic acephate exposure. After 6 µg/mL acephate exposure, the vitellogenin content showed (Fig. 6) 2.754-fold decrease in comparison to control group. The result manifests a negative correlation between exposure concentration and vitellogenin content (coefficient of correlation 0.949). 3.8 Mating pair observed: Distinct alteration in the number of available mating pairs was observed after chronic acephate exposure. With increase in treatment concentrations, number of 13
mating pair observed [Fig. 7] presented a decreasing trend in comparison to control group. The result demonstrates a negative correlation between increasing treatment concentrations and subsequent decrease in number of mating pairs. 4. Discussion: Successful reproduction is controlled by both male and female individuals of a population. The role of body size and body mass of an individual is crucial since they influence insect
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physiology, ecological performance and behavior (Dial et al., 2008, Whitman, 2008). Adult
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male flies exposed to differential concentrations of acephate through food demonstrated
significant reduction in wet weight in a concentration-dependent manner. This confirmed the
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adverse effect of acephate on the body weight of a non-target organism, D. melanogaster.
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Works of Jemenez- Perez and Wang, 2004 suggested that, reproductive behaviors of insects are influenced by body weight, body size and fitness. Their study on lepidopteran insect Cnephasia
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jactatana confirmed that, male reproductive potentiality follows the “larger-the-better theory”. Another study with flesh fly Neobellieria bullata revealed that, larger bodied males produced
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more sperms than small bodied ones (Berrigan and Locke, 1991). In this light it can be assumed that, depletion in body weight of Drosophila males as observed in this study (Fig. 1), might be an indirect indicator of reduced reproductive potential.
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The acridine orange staining of testes following acephate exposure demonstrated presence of apoptotic lesions in the form of fluorescent green spots. On comparison with untreated controls, testes of treated flies revealed greater number of apoptotic lesions. Increased frequency of acridine orange positive lesions after treatment (Fig. 2) suggests augmented DNA damage and cellular disintegration. Previously, similar apoptotic lesions were visualized in Drosophila ovary following chronic fluoride insult (Khatun et al., 2017). 14
Furthermore, germ cell viability assay (MTT assay) revealed gradual decrease in number of viable cells in testes with increasing acepahte onslaught (Fig. 3). Pesticide-induced oxidative stress is a well-known factor responsible for triggering cellular apoptosis via extrinsic and intrinsic pathways. Other organophosphates such as basudin, cidial and fenix (Trielli et al., 2006) were also reported to affect cell viability significantly. Rajak et al., 2018 has suggested that besides traditional cholinesterase inhibition, acephate triggers ROS-mediated toxicosis in
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various tissues resulting in DNA, protein and cell damage. Interestingly, co-administration of vitamin C along with acephate rescued from adverse effects (Rajak et al., 2017). In this line,
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acephate having cytotoxic potential is expected to reduce germ cell viability as detected in the
compromised reproductive fitness of treated males.
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present study. Reduced availability of mature germ cells might be a decisive factor behind the
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To have a better insight, this study was extended towards monitoring the activities of stress
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marker enzymes like catalase (CAT) and lipid peroxidase (LPO). Results have demonstrated an increased activity of catalase enzyme at 1- 4 µg/mL concentrations of acephate (Fig.4 [A]). This endogenous antioxidant constitutes the first line of defense against free-radical mediated injury
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(Lobo et al., 2010). Lipid peroxidation is a potent marker of oxidative stress and in this study; organisms manifested higher lipid peroxidation in testes following chronic sub-lethal acephate treatment. Therefore, increased activities of both antioxidant and oxidative stress marker
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enzymes suggest acephate-induced oxidative stress in adult males of Drosophila, as also documented by Rajak et al., 2017, in Drosophila larvae. Interestingly exposures to higher concentrations of acephate (5µg/mL and 6µg/mL) resulted in reduced enzyme activities due to unavailability of viable cells expressing the said enzyme in reproductive organs and tissues. Like mammals, Drosophila male reproductive system consists of a pair of testes, seminal vesicles and seminal proteins (Bairati, 1968). Drosophila spermatogenesis shares similarities 15
with that of mammals (Castrillon et al., 1993). Studies have suggested several conserved genes involved in development of reproductive organs in both Drosophila and mammals. For example, SOX [Sry (sex determining region Y)-related HMGbox] genes in mammals are known to play critical role in testes development (Sekido et al., 2004, Nanda et al., 2009). Mechanism of spermatogenesis in Drosophila and human indicates conservation of genetic regulators throughout the evolution (King et al., 1968). In addition, ecdysone in Drosophila has
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been projected as a primitive counterpart of the mammalian sex hormones, testosterone and
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estrogens (De Loof and Huybrechts, 1998). In the present study, reproductive enzymes
manifested compromised activity following chronic acephate exposure. Vitellogenin, a major
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yolk precursor protein in female body is also found in trace amount in male insects (e.g., Bombyx mori) (Lamy, 1984). Lamy, 1984 reported that, vitellogenesis in males is common for all
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lepidopteran species along with few species of Diptera, Dictyoptera and Orthopteran insects.
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Production of vitellogenin (Vg) in the fat body has been described as a feature of normal ontogenesis in males of various species. An increase in the level of vitellogenin transcript has been shown to correlate with the high level of vitellogenin in the haemolymph of larvae and
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adult drones of Apis mellifera (Colonello-Frattini et al., 2010, Guidugli et al., 2005). Interestingly, study by Majewska et al., 2014 has shown a strong linkage between yolk proteins and spermatogenesis in D. melanogaster.
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Furthermore, it is known that vitellogenin acts as an antioxidant, and elevated vitellogenin content leads to prolonged life span in honeybee (Havukainen et al., 2013). Vitellogenin is one of the largest lipid transfer proteins (LLTPs) which manifest multifaceted roles in animals like lipid transporters, inflammation suppressors, immune modulators, and blood coagulators (Havukainen et al., 2013). Though the specific role of yolk proteins are not yet confirmed, it is assumed that, yolk proteins have a definite role to play in sperm maturation in the male fly. In 16
the present study, decline in vitellogenin level was observed in male flies following chronic sublethal exposure to Acephate (Fig.6) Thus, decreased vitellogenin level might impede with the normal maturation of sperms thereby causing a significant alteration in the male reproductive system. Ecdysis-triggering hormone (ETH) has been reported to remain functional in adult D. melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction (Meiselman et al., 2017). Further, Meiselman et al., 2017 opined
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that, ETH might contribute towards increased plasticity to the stress response system ranging from few hours to even several days. Thus, in the present study, ETH signaling could have been
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upregulated after acephate exposure. Upregulation of apoptosis, excessive oxidative stress and
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compromised reproductive potential suggest that, ETH signaling might be affected in case of acephate- exposed flies to deficit JH availability. It is important to note that, ETH shortage leads
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to reduced JH level which consequently reduces ovary size, egg production, and yolk deposition in mature oocytes of female Drosophila (Meiselmanet al., 2017) A balance between JH and 20
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hydroxyecdysone determines whether oogenesis will progress beyond mid-oogenesis checkpoint stage or not (Gruntenko et al., 2010, Liu et al., 2008). On the other hand, JH deficiency has
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been reported to induce caspase mediated cell death (Soller et al., 1999, Pritchett et al., 2009). In the present study, significant decline in viable cell number in reproductive tissues were noted. Furthermore, in case of JH deficient males, partner fecundity impairment has been reported to occur because of a reduced rate of accessory gland protein synthesis (Meiselmanet al., 2017). It
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has further been demonstrated that, reduced female fecundity as well as impaired male reproductive potential in RNAi knock down of EcR in Inka cells could be rescued through administration of JH analog. Thus, acephate exposure might have followed the above discussed pathway to hinder optimum sperm maturation and thereby male fertility in Drosophila.
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The present study has further shown the decrease in iron content in adult male flies following chronic acephate treatment (Fig. 5). The iron content is considered as a function of mitoferrin enzyme activity. Mitoferrin is a mitochondrial iron carrier protein that helps in iron metabolism during spermatogenesis (Metzendorf and Lind, 2010). Earlier studies have confirmed that, D. melanogaster and other invertebrates (i.e., Sea urchin, Caenorhabditis elegans, bee, wasp, mosquito and flour beetle) have only one mitoferrin gene, which has been suggested to be a
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functional homolog of vertebrate mitoferrin 2 (Metzendorf et al., 2009). Findings of Metzendorf and Lind, 2010 imply that, Drosophila mitoferrin and the mitochondrial iron
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metabolism are essential for regulation of spermatogenesis. It is also an established fact that, iron
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is needed during sperm maturation (Metzendorf and Lind, 2010, Detmer and Chan, 2007). Genes involved in mitochondrial iron metabolism are expressed in testis of both vertebrates and
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Drosophila. It has further been reported that, inadequate mitoferrin activity can result in defective energy metabolism in mitochondria, which during spermatogenesis causes
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morphological alterations like decreased testis length (Metzendorf and Lind, 2010). Earlier studies on mammalian spermatogenesis indicated a nutritional function of iron during
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spermatogenesis. Orlando et al., 1985 has suggested that, levels of transferrin, a ubiquitous iron transfer protein in human seminal plasma, can be correlated with sperm abundance. Thus, in the present study, significant reduction in mitoferrin activity indicates altered iron metabolism which in turn might have impeded the process of spermatogenesis and thereby male fertility in treated
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flies.
Several studies across the globe have been performed on Drosophila mating patterns to demonstrate the basic factors that are crucial for reproductive success. Interestingly, mating speed has been categorized as one of the main components depicting male fitness in Drosophila (Parsons, 1974, Cade, 1984). Hence, the frequency or the number of available mating pairs 18
might be considered as a relevant index demonstrating the male reproductive fitness. Hoffmann 1994 suggested that, aggressive behaviour is also an important criterion for determining male mating success in some species of fruit flies. Generally larger males of D. pseudoobscura and D. melanogaster are better selected in the field (Partridge et al., 1987) and in laboratory respectively (Partridge and Farquhar, 1983). But results of this study have shown a significant decline in wet weight of the male body after
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acephate exposure. This implies that, flies exposed to higher concentrations of acephate might
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automatically have taken a back seat in reproductive selection.
Traditional concept of sexual selection suggests that, in most animal species, males are less
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discriminating in their choice of mating partners than females; because their investment in
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offspring is much lower (Bateman, 1948, Trivers, 1972). However, in case of many species, the males demonstrate a high energy investment in reproduction (mating) because of energetically
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expensive courtship displays (Judge and Brooks, 2001) and the production of ejaculates (Dewsbury, 1982, Galvani and Johnstone, 1998).
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It has been demonstrated that, lesser male mate discrimination is predicted when mating requires less time and/or resources whereas greater mate discrimination by males is obvious when investment in time and energy is greater (Engqvist and Sauer 2001, Kokko and Johnstone 2002). This substantially affects future mating opportunities of males (Simmons, 1990, Clutton-
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Brock, and Langley, 1997, Bonduriansky, 2001). In the present study, the males inhabiting an environment with graded concentrations of acephate are expected to spend considerable energy currency for combating the stress. Hence, males would be left with lower energy thereby resulting in either greater mate discrimination or reduction in the frequency of mating. The
19
results validate this fact, since observed numbers of mating pairs in treated groups are found to decrease with increase in treatment concentrations. Sing et al., 2015 demonstrated that, due to environmental stress like cold shock, there happens to be a shift in reproductive behavior. Similarly, in the present study we have observed a change in the frequency of mating ability in males upon chemical stress. Sing et al., 2015 also reported that, females in D. melanogaster when held with males post cold shock, the number of mating
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observed is significantly higher in comparison to control. In contrary to that, the present finding
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depicts decrease in mating frequency after acephate mediated stress. This might be due to tradeoff between survival and reproductive potential. Since greater energy is required for
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survival in stressed environment, hence less energy remains at disposal for reproduction and /
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mating activities. 5. Conclusions:
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In the present study, morphological, biochemical and behavioural parameters were studied to explore the toxic effects of chronic sub-lethal organophosphate exposure in male reproductive
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system of a non-target model organism D. melanogaster. It was observed that after chronic sublethal exposure to organophosphate chemical acephate, physical, biochemical and reproductive fitness of the exposed individuals demonstrated a compromised state in comparison with their control counterparts. Individual variation in male reproductive success in a lifetime is controlled
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by the expression of many different traits relevant for reproduction. It has been seen that natural selection favors phenotypes that give the males an advantage of fathering more offspring, though the expression of these characters might be influenced by trade-offs. Several studies have established the fact that environmental factors are instrumental in mediating these trade-offs (Filice and Long, 2018, Roff and Fairbairn, 2007, Joseph et al., 2017). Thus, in case of males
20
having a fixed energy budget, increased investment into a specific reproductive phenotype might result in reduced expression of another. In the present study the male Drosophilids might have expressed reduced reproductive fitness as a result of altered resource allocation supporting survival in the acephate-contaminated (stressed) environment. Additionally, the results of this exposure analysis in Drosophila can be extrapolated to other non-target organisms as well as human beings because of the developmental and physiological similarities. Furthermore, this
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study yet again validates the use of Drosophila melanogaster as a successful model organism in
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toxicological assessment of chemicals.
Author contribution
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To Whom it may concern
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This is to mention the Credit roles of the different authors involved in the conceptualization, shaping of experiments, execution of the experimental methodologies, data collection, data analysis, interpretation of results and writing of the manuscript
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MOUTUSHI MANDI: Experimental methodologies, Investigation, Data collection, Data analysis, Validation; Visualization; Preparation of original draft.
SALMA KHATUN: Experimental investigation; Methodology, data collection, Visualization PREM RAJAK: Formal analysis of data, Software, Review and Editing ABHIJIT MAZUMDAR: Supervision, Data Interpretation, Suggestion, Review
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SUMEDHA ROY: Conceptualization, Data curation, Interpretation, Supervision, Review and editing, Project administration
Conflict of interest: There is no known conflict of interest.
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Acknowledgments: Authors are grateful to the Head, Department of Zoology (UGC DRS, DST-FIST and DST PURSE sponsored), The University of Burdwan for providing necessary infrastructural facilities to carry out the present work. Authors are thankful to Prof. Niladri Hazra (BU) for providing environmental chambers to maintain Drosophila melanogaster stock and extending the
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microscopic facilities. The authors are indebted to Dr. Sanjib Ray (BU) and Dr. Koushik Ghosh (BU) for providing chemicals and analytical instruments time to time through the course of this
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work.
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Legends to Figures: Fig.1. Alterations in the wet weight of adult body in Drosophila melanogaster after chronic exposure to graded concentrations of acephate (1-6µg/mL). Treated groups were compared with untreated control group for assessment of significant change. The data represents mean ± S.E. of three pooled determinations. Each pool consisted of 30 individuals. “*” denoted significant
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change in comparison to control. *p < 0.05 were ascribed as statistically significant.
Fig.2.Effect of acephate exposure on testis: Figure shows AO staining of testis from (3-5) days old adult D. melanogaster facing chronic insult to six different acephate concentrations(b-1, c-2,
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d-3, e-4, f-5, g-6 µg/mL) along with control (a) group. Bright green fluorescent bodies (indicated by circle) correspond to apoptotic masses in the stained tissues. Apoptosis was negligible in case of control category (a) but prominently visible in case of (b, c, d, e, f and g). The structure of the
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testes in case of treated flies (b-g) is found to be altered in comparison to the control (a).
Fig.3.Figure represents results of MTT assay of germ cell from adult male D. melanogaster exposed to six different treatment categories (1-6 µg/mL) along with control counterparts. Data
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specify mean±S.E. Data represents numerical abundance of viable germ cells reflected as absorbance at 578nm wavelength. *p <0.05 were considered as statistically significant when
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compared to control category.
Fig.4. Changes in the endogenous detoxification enzyme (Catalase) and oxidative stress marker enzyme (Lipid peroxidase) activities. (A) Catalase (CAT) activity in control and differentially exposed flies. (B) Lipid peroxidase (LPO) activity in control and treated groups. The data 39
represents mean ± S.E. of three pooled determinations. “*” denoted significant changes in treated
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group in comparison to control.*p <0.05are ascribed as statistically significant.
Fig.5.The figure represents mitoferrin activity represented through the iron content in male adults of D. melanogaster after chronic exposure to six different concentrations of acephate (1-6
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µg/mL). The vertical bars specify standard error. Data represents mean ± S.E. (*) representing p<0.05 are considered as statistically significant when compared between treated and control category.
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Fig.6.The figure represents vitellogenin content in adult male of D. melanogaster after chronic exposure to six different concentrations of acephate (1-6 µg/mL). The vertical bars specify standard error. Data represents mean ± S.E. (*) representing p<0.05 are considered as
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statistically significant when compared among treated and control category.
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Fig.7. The scattered plot represents number of mating pair in control and acephate treated D. melanogaster. Data represents four separate observations for control and each of the treatment categories. Each observation was carried out for duration of 30 min, during which number of
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observed mating pair was recorded.
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