Biological effects of the frequent application of a copper-containing fungicide on the fruit fly Drosophila melanogaster

The increased consumption of pesticides has an environmental impact due to the dispersion of minerals. Bordasul® is a commonly used fungicide composed of 20% Cu, 10% sulfur, and 3.0% calcium to correct its deficiency in plants. The evaluation of fungicide effects in vivo models is designed to assess their impact on the environment more broadly. Drosophila melanogaster offers a unique model due to its ease of handling and maintenance. Here, the effects of Bordasul® were investigated, addressing the development, survival, and behavior of flies. Our findings showed that exposure to Bordasul® prevented the development of flies (p < 0.01). In addition to causing a significant reduction in memory retention (p < 0.05) and locomotion capacity (p < 0.001). Although fungicides are necessary to satisfy the world’s food demand, we conclude that Bordasul® is highly toxic, and that safer media, such as biofertilizers, must be developed as effective alternatives.


Introduction
Continuous growth in the human population worldwide has increased the demand for food production. The projection from the United Nations of 9 billion humans by 2050 translates to a doubling of the current food requirement (World population prospects, 2019). To meet these nutritional needs, the use of fertilizers and pesticides has become an indispensable tool in agriculture, contributing to huge improvements in food production. The evaluation of fungicide effects using in vivo models is a critical requirement to elucidate the broad impact of these inputs on the environment. Drosophila melanogaster, commonly known as the fruit y, is an alternative model providing a bridge between in vitro and mammals and has been extensively used in research for more than 100 years. Its ease of manipulation and maintenance makes it an optimal model to study the environmental impact of fungicides on living organisms.
Herein, the effects of Bordasul powder were investigated in the fruit y, focusing on its development, survival, and behavior.
2.2 D. melanogaster stock and culture D. melanogaster (Harwich strain) used in the present investigation was obtained from the National Species Stock Center (Bowling Green, OH, USA). Diet was prepared as indicated by Adedara and collaborators (Adedara et al., 2015) with some adaptations. The ies were maintained and reared in corn meal medium (1% corn our, 2% w/v brewer's yeast, 2% w/v sucrose, 1% w/v powdered milk, 1% w/v agar, 0.08% w/v nipagin and 93.92% distilled water) at constant temperature (22-24ºC) and relative humidity (60-70%) under 12-h dark/light cycle conditions.
For the experimental assays, adult ies (13 days-old) were anesthetized with ice between 5 to 10 minutes.
Males and females were separated using a paintbrush and 10 females and 10 males were mated for 24 hours in 1% agar media (containing 0,5% defatted milk powder, 2% sucrose; 0,1% nipagin®, 1% yeast and different concentrations of Bordasul® from 0; 0.1, 0.5 or 1 g/L of diet. The ies were removed, and the offspring used for the assays.

Exposure to Bordasul®
The offspring were randomly exposed to 3 different concentrations of the fungicide and the vehicle (water) as a control group. The highest exposure concentration was based on instructions provided in the product's packaging (1 g/L). This was further diluted 1:2 representing the middle concentration, and 1:10 for the low concentration. Thus, the 3 concentrations used in ascending order were 0.1, 0.5, and 1.0 g/L low, medium, and high, respectively).
Given that in preliminary studies these concentrations affected y development, we subsequently used concentrations 10 times lower for the behavioral assays, as follows: 0.01, 0.05, and 0.1 g/L.
The solutions were diluted in distilled autoclaved water, prepared at the time of use and mixed in a warm medium, and allowed to solidify and cool. All experiments were repeated 3 times and the number of ies used in each trial is contained in the description of the experiments.

Development assay
Fly development was measured by counting the survival rate at 5 days (larvae stage), 9 days (pupae stage), and 13 days (adult stage) after exposure to Bordasul or control (Zamberlan et al 2020a).

Lifespan assay
Lifespan assays were conducted as described above (Linford et al 2013), with some adaptations. After mating for 24 hours of the 10 pairs of ies, the offspring exposed to Bordasul® concentrations from their oval phase was used for the lifespan assay. During the experimental period, ies were transferred to new vials containing fresh food 3 times a week in the absence of anesthesia to ensure that the feeding environment of young females was not interrupted by the presence of larvae. The longevity of the ies was counted every 2 days from the beginning of the adult phase.
Lifespan assays were conducted according to the previously described, with some adaptations. During the experimental period, ies were transferred onto new vials containing fresh food 3 times per week in the absence of anesthesia to ensure that the feeding environment for young females was not disrupted by the presence of larvae. The ies' longevity was counted every 2 days from the onset of the adult stage.

Climbing assay
One the day before the climbing assay determination, ies were transferred to clean rearing asks to avoid the interference of humidity in the quanti cation of ies' climbing ability. For climbing assay, a vial (10 cm high x 1.5 cm of diameter) with a dotted line marked 6 cm away from the bottom was used. Groups of 10 ies were placed in the vial. The ies were tapped down to the bottom and allowed to climb.
The number of ies that crossed the line marked in the vial within 6 seconds was recorded. Data were expressed as the percentage of ies that crossed the line within 6 seconds. The assay was repeated 3 times and the mean was calculated. At least 80 ies were tested per group (n equal or greater than 8 per group).

Memory retention assay
The effect of pairing an odorant to electrical shock was evaluated according to protocols previously described (Tully and Quinn, 1985) with adaptations described by Zamberlan et al., 2020. In the training session, the young (4 days-old) or aged (11 days-old) ies (30-50 ies from a given rearing ask) were exposed to a T maze (18 cm x 3 cm x 3 cm) in the presence of the odorant 3-octanol (OCT-1:100).
Immediately after placing the ies in the maze, they received an electrical shock (75 V for 2 min, aversive pairing of OCT with shock). Then, the ies were exposed to methylcyclohexane (MCH-1:25) for 2 min without applying the shock. In the test sessions (which were performed 1, 30 or 60 minutes after the training session), the ies were again transferred to the T maze and allowed to choose for one of the arms of the maze (containing either the odor of MHC or OCT). But in these 3 test trials no shock was applied. The performance index was scored by recording the number of ies which chose the correct nonaversive arm (MCH) minus the number of ies that chose the wrong OCT arm divided by the total number of tested ies and presented as percentage.

Statistical analysis
All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed with GraphPad Prism7 Software. Survival and behavioral signi cance were assessed by one or two-way analysis of variance (ANOVA), followed by Newman-Keuls's post hoc test. Survival curves were assessed by log-rank test for trend. Differences were considered statistically signi cant among groups at p < 0.05.

Bordasul® affects ies' development
Exposure to Bordasul® at 0.5 and 1.0 g/L led to an arrest in ies development (Fig. 1). A signi cant decrease in the number of larvae was observed in both Bordasul® treatment groups when compared to the control group (Fig. 1. A -p < 0.05). Furthermore, ies treated with Bordasul® failed to reach adult ( Fig. 1. C), pupae ( Fig. 1. B), and even the L3 stage ( Fig. 1. A). Accordingly, in the following experiments, we used lower concentrations of Bordasul® (as described in the methods) that did not affect their development.

Bordasul® decreases locomotor activity
In the locomotor activity assay, ies exposed to Bordasul® at 0.05 and 0.1 g/L showed a signi cant decrease in climbing at the young adult stage (p < 0.05), with a more pronounced effect at the higher Bordasul® exposure (Fig. 3.A -p < 0.01). This effect was corroborated at an old-adult stage with an even greater effect (Fig. 3. B -p < 0.001).

Bordasul® disrupts memory in ies
Memory retention test analysis indicated that the test was e cient in short-term memory acquisition since all ies avoided the odor paired with shock for 60 min after the shock, at both young (CI > 85% - Fig. 3. A) and old-adult (CI > 65% - Fig. 3. B) stages. No signi cant difference was observed between the groups in memory acquisition (time zero), except when comparing the young-to old-stage (data not shown). The analysis also demonstrated that ies exposed to 0.1 g/L of Bordasul® had signi cantly lower memory retention at 60 min after memory acquisition at the young-adult stage when compared to control ies (Fig. 3. A -p < 0,05). This effect of treatment was more evident in old-adult ies, demonstrating de cient memory retention as early as 30 min after shock when compared to the control ies at the same age (Fig. 3. B -p < 0,05).

Discussion
Research has increasingly directed its focus toward safer and more e cacious agricultural products. The continuous increase in the consumption of fungicides and fertilizers in farming is necessary to meet global food demand, and as the population has grown, so has the utilization of these products. Concomitantly, their increased application has raised concerns about their environmental impacts. In this report, the in vivo toxicological effects of a chemical fungicide rich in copper (Cu) were demonstrated. We used the fruit y D. melanogaster to investigate the biological effects of exposure to a fungicide commonly used in agriculture, namely, Bordasul®. It was used at environmentally-relevant concentrations per the manufacturer's recommendations to evaluate its impact on the environment, herein, the fruit y.
D. melanogaster is a well-established alternative and complementary model organism extensively studied for toxicity testing due to its inherent advantages over other more complex animal models (Yamaguchi & Yoshida 2018). D. melanogaster has a rapid life cycle and short lifespan, and large numbers of ies can be obtained in laboratory conditions (Reaume & Sokolowski 2006). It has been used as a model for the assessment of toxicological, developmental, as well as cognitive, memory, and learning de cit effects In this study, concentrations equal to or less than those recommended for use on the product's label was tested in an attempt to mimic real-life exposure scenarios. Bordasul® exposure in the larval period disrupted ies' development at 0.5 mg/L, a concentration equivalent to half of what has been recommended for usage by the manufacturer. Furthermore, even lower concentrations, as low as 0.05 and 0.1 mg/L, led to a shorter lifespan, disrupted motor activity, and decreased memory retention at the adult stage when compared to the control. The effect on memory retention was more persistent in old ies compared to young ies, indicating the toxic effect on memory worsens over time. These results are of fundamental importance in view of the constantly increasing demand for fungicides and fertilizers in the world. Agriculture is sustained with the help of chemical fungicides. Without them, it would not be possible to produce the great demand for food necessary to supply the world population. But along with these bene ts comes environmental toxicity. The use of safer means, such as biofertilizers, can be an effective alternative to enable the production of necessary food in the world without affecting the environment.

Conclusion
The use of chemicals in agriculture contributes to environmental pollution, raising major concerns regarding ecological and ultimately human health effects. Tests to evaluate the biological effects of products used in the environment, mainly in food are of fundamental importance for the safety of both the environment and humans. Here, we demonstrated the toxic effects of the fungicide, Bordasul®, commonly used in farms, even in concentrations ten times lower than those indicated on its label. Overall, more extensive and systematic research efforts are required to investigate the effects of fertilizers on living organisms with a major focus on novel products.

Declarations
Funding Figure 1 Effect of the fungicide Bordasul® on D. melanogaster development. Bordasul in the highest concentrations signi cantly avoids ies development at (A) larvae (6 days after mating), (B) pulpae (12 days after mating), and (C) adult (18 days after mating) stage. *p<0.05 and **p<0.001 (compared to Ctrl group) one-way ANOVA followed by Bonferroni post-hoc test.