Effect of bisphenol A on Drosophila melanogaster behavior – A new model for the studies on neurodevelopmental disorders

Behavioural Brain Research 284 (2015) 77–84

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Effect of bisphenol A on Drosophila melanogaster behavior – A new model for the studies on neurodevelopmental disorders Kulbir Kaur a,b,c , Anne F. Simon d , Ved Chauhan a , Abha Chauhan a,∗ a

Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA Biology/Neuroscience Graduate Program, City University of New York – Graduate Center, 365 5th Avenue, New York, NY 10016, USA c Center for Developmental Neuroscience and Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA d Department of Biology, Faculty of Science, Western Ontario University, Ontario, Canada b

h i g h l i g h t s • • • •

Perinatal exposure to bisphenol A (BPA) leads to behavioral modifications in the Drosophila. BPA exposure changes the exploratory behavior of the Drosophila in the open field assay. In BPA-treated Drosophila, there was an increase in the grooming episodes, which suggests abnormal repetitive behavior. Along with the motor changes, we also observed uncharacteristic social interaction in Drosophila exposed to BPA.

a r t i c l e

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Article history: Received 23 December 2014 Received in revised form 30 January 2015 Accepted 1 February 2015 Available online 7 February 2015 Keywords: Autism Bisphenol A Drosophila melanogaster Grooming Locomotion Social interaction

a b s t r a c t Developmental disorders such as autism and attention deficit hyperactivity disorder (ADHD) appear to have a complex etiology implicating both genetic and environmental factors. Bisphenol A (BPA), a widely used chemical in the plastic containers and in the linings of food and beverage cans, has been suggested to play a possible causative role in some developmental disorders. Here, we report behavioral modifications in Drosophila melanogaster following early exposure to BPA, which may suggest BPA as an environmental risk factor for the behavioral impairments that are the basis of diagnosis of autism and ADHD. In an open field assay with perinatally BPA-exposed and vehicle-treated control Drosophila, different parameters of locomotion (distance traveled, walking speed, spatial movement, mobility, turn angle, angular velocity and meander) were analyzed using the ethovision software. We also examined the repetitive and social interaction behaviors in these flies. In an open field assay, we identified disturbances in the locomotion patterns of BPA-exposed Drosophila that may relate to the decision-making and the motivational state of the animal. An increase in repetitive behavior was observed as an increase in the grooming behavior of Drosophila following BPA exposure. Furthermore, we also observed abnormal social interaction by the BPA-exposed flies in a social setting. These results demonstrate the effect of the environmentally prevalent risk agent BPA on the behavior of Drosophila, and suggest the practicability and the ease of using Drosophila as a model in the studies of neurobehavioral developmental disorders. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The Centers for Disease Control and Prevention (CDC) estimates the prevalence of developmental disorders at 1 in every 6 children [1]. Neurodevelopmental disorders such as intellectual disability, attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and fragile X syndrome have core

∗ Corresponding author. Tel.: +1 718 494 5258; fax: +1 718 698 7916. E-mail address: [email protected] (A. Chauhan). http://dx.doi.org/10.1016/j.bbr.2015.02.001 0166-4328/© 2015 Elsevier B.V. All rights reserved.

abnormal behavioral components that are fundamental to their diagnosis. The core components central to an autism diagnosis consist of abnormal social interactions, impairments in verbal and non-verbal communication as well as repetitive and restricted behavior or interests. In addition, other features are often associated to this triad, such as difficulties with decision-making [2]. A major finding from epidemiologic studies has been the increase in the prevalence of autism in recent years, with about 1 in every 68 children being identified with ASD [3]. With no single identifiable cause linked to autism, the roles of genetic factors as well as oxidative stress, mitochondrial dysfunction, inflammation, and

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immune abnormalities have been reported, leading to a multifactorial model for its etiology [4–12]. Environment is considered an especially strong contributor to the increased prevalence of autism [11–15]. Exposure to maternal infections during critical periods of development, such as prenatal and perinatal periods, has also gained attention in the search for the etiology of autism [16,17]. The increased incidence of autism in certain regions has suggested that there is a link between geography and the genetic predisposition to autism [18–20]. Bisphenol A (4,4 -dihydroxy-2,2-diphenylpropane; BPA) is one of the environmental toxins that has recently received increased attention. BPA is widely utilized in the production of polycarbonate plastics such as drinking bottles, food containers, toys and dental sealants. It is also employed in the production of epoxy resins used in the linings of food and beverage cans. The U.S. Environmental Protection Agency (EPA) recommends the lowest observed adverse effect level (LOAEL) of BPA to be 50 mg/kg body weight/day [21], which is set as the reference dose (RfD) for the maximum acceptable level for daily exposure. BPA and its metabolites are found in the majority of biological fluids, including blood and urine. Analysis by the CDC showed detectable levels of BPA in 92.6% of urine samples in human [22]. BPA is known to cross the placenta [23] and is found in the amniotic fluid, placental tissue, umbilical cord, and fetal serum [24]. Estimated intake of BPA was found to be much higher in bottle-fed infants (1 ␮g–11 ␮g/kg body weight/day) than in breast-fed infants (about 0.2 ␮g–1 ␮g/kg body weight), thus mandating BPA-free baby bottles [25]. BPA acts mainly as an endocrine disrupting chemical (EDC), with prenatal and postnatal exposure leading to neurodevelopmental disturbances [26,27], along with behavioral changes [28–30]. Many studies have indicated a role of EDCs in the development of ASD and ADHD [31]. Drosophila melanogaster is a widely used model for studies of brain disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, fragile X syndrome, and Angelman syndrome [32]. Drosophila is a comparatively simple organism with its genome consisting of four chromosomes encoding approximately 14,000 genes [33,34], which is about 50% of the estimated 25,000–30,000 genes in humans [35,36]. Among 59 of the human neurological genes examined, 38 have orthologs in the Drosophila genome [37]. A single Drosophila gene may serve the same function as multiple related genes of mammals, thus decreasing the redundancy seen in other vertebrate models. Although flies and humans are distinctly different from each other, many molecular processes are conserved between them. The advantage of studying neurobehavioral disorders in Drosophila is the presence of genes that are similar to human genes for normal cognitive functions, as a result of phylogenetic conservation of these genes [38,39]. The Drosophila model exhibits complex behaviors relevant to humans, including courtship [40,41], circadian rhythms [42], learning and memory [43], aggression [44], grooming [45], and open field exploration [46]. The fly is used as a model in studies because of its compact genome, which has been fully sequenced, and the availability of sophisticated genetic approaches. The Drosophila model is also attractive because of its quicker generation time, large number of progeny for better selection, and easy maintenance of the animal model. Nevertheless, Drosophila has been underused in the study of complex disorders with abnormal behavioral components because of lack of reliable tests to assess complex behavioral phenotypes relevant to human [47,48]. We combined several behavioral assays evaluating autismrelated impairments to examine the effects of exposure to BPA in Drosophila melanogaster. Here, we report that exposure to BPA leads to features of repetitive behaviors, abnormal social interaction, and significant impairment in locomotion in an open field

arena, a decision-making process. Most importantly, we present the use of Drosophila as a prospective model for the study of neurobehavioral disorders with complex gene-environment etiology such as autism. 2. Materials and methods 2.1. Drosophila stocks Wild-type Oregon-R Drosophila stocks were maintained at 25 ◦ C on a standard cornmeal diet (Jazz-mix Drosophila food, Fisher Scientific, Pittsburg, PA, USA) under 12 h/12 h light and dark cycle. 2.2. BPA treatment of Drosophila On the basis of a previously used drug dosing protocol [49], we assumed that a 1-mg fly would consume food equaling 5% of its body weight per day. In our study, the highest BPA dose (1 mM) (>99% purity; Sigma–Aldrich, St. Louis, MO, USA) corresponds to the approximate human LOAEL of 50 mg/kg body weight/day. For the oral administration, the BPA dissolved in dimethylsulfoxide (DMSO) (Sigma–Aldrich, St. Louis, MO, USA) was mixed with recently cooked and cooled standard fly food. Different concentrations of BPA (0.001, 0.01, 0.025, 0.05, 0.1, 0.1, and 1 mM) were used for the social interaction assay, while the higher doses (0.5 and 1 mM) were used for all the other experiments. For all the treatments, the amount of DMSO was kept below 0.1% of the volume added. In the controls (no BPA), only 0.1% DMSO (vehicle) was used. Five virgin female and three male flies (3–5 days old) were mated in vials with the BPA-treated food. The flies were allowed to feed and lay eggs in the treatment vials for 3–4 days, after which the flies were discarded and the vials placed in the incubator. Newly enclosed flies (F1) were anesthetized on ice, and separated according to their gender. The F1 progeny were transferred into fresh vials containing their respective BPA-treated or vehicle control food prior to behavioral testing. 2.3. Behavioral assays All behavioral experiments were performed between 12 pm and 3 pm (between Zeitgeber time ZT5 – 5 h after the onset of light and ZT8) and in adequate daylight, to reduce variations in performance due to circadian rhythms. 2.3.1. Social interaction Social interaction was determined in male and female flies by using the social space assay as described by Simon et al. with slight modifications [50]. One day prior to the experiment, flies from each treatment group (n = 30–40) were separated by gender. On the day of the experiment, the flies were allowed to acclimate in the dedicated behavior room (environmentally controlled: 50% humidity, 25 ◦ C) for 1 h before the experiment was initiated. The male and female flies were kept separate for each treatment to avoid interference with courtship behavior. Flies were placed in the same kind of rectangular vertical chambers used by Simon et al. [50]. After the flies spent 20 min exploring and settling in the chamber, a digital image of the chamber with the flies was taken with a camera and exported into iPhoto (Macintosh computer). Image J (NIH software – http://imagej.nih.gov/ij/) was then used to process the image into an 8-bit binary image. The binary image was then imported in a Lispix (NIH image analysis software – http://www.nist.gov/lispix/) program to calculate the distance of the fly to its nearest neighboring fly.

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2.3.2. Open field assay: locomotion Locomotion was studied in the open field assay as described previously, with a few modifications [51]. The chamber for the assay was a circular arena 8 mm in diameter and 0.1 mm in height, open on both sides, placed on a white background and covered with a clear glass slide that could be moved for the placement of the fly and then moved back to prevent the fly from escaping. For this assay, 3–6 day-old male flies were used. On the day of the experiment, the flies were removed from the treatment vials into clean empty vials and placed in the behavior room for 2 h before the experiment. For the experiment, an individual naïve male fly (n = 9–10) was aspirated into the arena and allowed to walk freely. The chamber was placed directly under a mounted digital video camera (Panasonic) for live video recording. The fly was allowed to acclimate to the chamber for 1 min, followed by video recording for a 5-min observation period. The video recordings were converted to mpeg format for analysis of the following parameters using Noldus EthoVision-XT system (Noldus Information Technology, Netherlands). (1) Distance traveled: The distance moved was used as an estimate of the general activity of the fly. Total distance that the fly traveled during the 5-min observation period was measured. (2) Velocity or walking speed: Velocity was also used as a measure of the activity of the subject. The velocity of the movement of the fly was measured as the walking speed with which the fly moved in the arena during the 5-min observation period. (3) Movement: Spatial movement of the fly was used as an indicator of the activity and inactivity period of the fly. The movement of the fly was calculated by applying a threshold to the analysis; this was done to remove small wobbling movements due to the capture rate of the video by the camera. Moving vs. not moving (s) was characterized according to the previously used rates [46,51]. (4) Mobility: Mobility is defined on the basis of changes in the pixels of the sample independent of the spatial displacement of the center of the body point. Mobility was calculated independent of movement of the center point. A threshold of 10–80% was used for calculating duration of mobility of the flies in the arena, while <10% and >80% defined the fly as immobile and highly mobile, respectively. (5) Turn angle: Turn angle is the angle formed by the change in the direction of the movement of the fly, based on the change in the center point between two consecutive samples. (6) Angular velocity: Angular velocity is the change in the moving direction of the fly per unit of time or speed of change of direction. It was computed as the ratio between turn angle and sample interval. (7) Meander: Meander is the change in the direction of movement of a subject relative to the distance moved by the subject, and was predicted using the ratio of the turn angle and the distance moved. The meander gives the level of tortuosity. 2.3.3. Repetitive behavior Repetitive behavior was assessed by the grooming assay, as previously described [45]. For this assay, we used the previously recorded videos of the open field assay and manually analyzed the number of grooming episodes during the 5 min observation period. 2.4. Statistical analysis For the social interaction assay, non-parametric Kruskal–Wallis analysis was performed to test for significance between the groups. For the locomotor and the repetitive behavior assay, one-way ANOVA was used to measure the significance between the groups. To evaluate the correlation between BPA concentration and various test parameters, linear regression analysis of the data was done, and Spearman correlation coefficient (r) was calculated.

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Fig. 1. Exposure to BPA decreases the distance maintained between a Drosophila fly and its closest neighbor in a social setting. The data are shown as box and whisker plots of the distance to the closest neighbor in the chamber, with the box representing the 1st quartile (25th percent) and the 3rd quartile (75th percent), the line in the box representing the median, and the Tukey whiskers excluding the outliers. These data were obtained from independent repeats of 30–40 flies per assay: n = 8 repeats for 0.1 and 0.05 mM BPA; n = 7 repeats for vehicle (no BPA), n = 6 repeats for 0.025 mM BPA; and n = 4 repeats for 0.001, 0.01, 0.5 and 1 mM BPA. Non-parametric Kruskal Wallis test was applied to analyze the data for significance (p < 0.0001); Dunn’s rank test comparison of medians indicated those that are different from vehicle, and marked * for p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3. Results 3.1. Abnormal social Interactions in Drosophila following BPA exposure The distance between an individual fly and its closest neighboring flies was used as a measure of social interaction within the group, as previously described [50,52,53]. In a social setting, most Canton-S wild-type flies maintain at least a two body length distance from their closest neighbor (∼0.25 cm) [54]. In OregonR wild-type flies, we found that this distance is a little bit larger, closer to ∼0.5 cm (Fig. 1). Both male and female flies, tested independently, displayed the same patterns, so their results were compiled for each BPA concentration tested. The BPA treatment showed a significant dose-dependent effect on the distance maintained between the flies in a social group. More than 50% of the BPA-treated flies settled less than 0.5 cm from their neighboring flies, whereas the vehicle-treated control flies maintained a distance of more than 0.5 cm. In the vehicle-treated control (no BPA) flies, the upper whisker illustrates the increased values (number of flies) at distances above 1 cm. Furthermore; the range of distance between BPA-treated flies was smaller when compared to the vehicle-treated control flies. BPA-treated flies were significantly closer than the vehicle-treated control flies in a dose-dependent manner.

3.2. Locomotion Locomotion is a major behavior in animals and is linked to their need for exploration, foraging, mating, and escaping predators, thereby providing a valuable behavioral tool. We examined a range of variables of Drosophila locomotion using the Noldus

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Fig. 2. Effect of BPA exposure on the locomotion of Drosophila during the 5-min observation period in the open field assay. The data are represented as scattered plots with mean ± S.E., and the dotted line representing linear regression analysis between the BPA concentration and the parameters examined with the correlation coefficient (r) and p value presented. (A) The mean distance moved by the Drosophila fly during the 5-min observation period. (B) The mean velocity with which the Drosophila fly traveled in the arena. (C) The mean mobility of Drosophila in the arena during the 5-min observation period. (D) The mean angular velocity of Drosophila in the arena. (E) The mean turn angle of Drosophila in the arena. (F) The mean meander of the Drosophila in the arena. (G, H) The mean movement of the Drosophila calculated as the center point of the fly moving or not moving in the arena, respectively. For statistical analysis, *p < 0.05.

EthoVision-XT system, focusing on males, as we did not observe any differences between sexes in the social space assay. 3.3. Exposure to BPA decreases the distance traveled by flies in the open field arena The first variable analyzed by the Noldus EthoVision-XT system was the total distance (cm) that the fly moved during the 5-min observation period (Fig. 2A). Flies treated with 1 mM BPA

(mean ± SE = 0.0096 ± 0.0008; n = 10) traveled a significantly lesser distance (one-way ANOVA, p < 0.05) of the 0.8-cm circular arena when compared to the vehicle-treated (no BPA) control subjects (mean ± SE = 0.012 ± 0.0008; n = 10) during the 5-min trial. There was no significant decrease in the distance moved by the 0.5 mM BPA-treated flies (mean ± SE = 0.010 ± 0.0006; n = 9) when compared to the vehicle-treated control group. Linear regression analysis showed a significant slope (Fig. 2A) for BPA-mediated decrease in the distance (r = −0.434, p = 0.0188).

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3.4. Effect of BPA on the velocity or walking speed of the flies There was no significant difference in velocity between the BPA-treated groups (0.5 mM or 1 mM) and vehicle-control treated (no BPA) group, as shown in Fig. 2B. The 0.5 mM BPAtreated (mean ± SE = 0.2977 ± 0.0203; n = 9) and 1 mM BPA-treated (mean ± SE = 0.2803 ± 0.0228; n = 10) flies did not walk at speeds significantly different from those of the vehicle-treated control flies (mean ± SE = 0.3304 ± 0.0156; n =10). Although there was no significant difference between the groups, linear regression analysis showed a trend toward a slower walking speed in the BPA-treated flies (0.5 mM or 1 mM) than in the vehicle-control group (r = −0.336, p = 0.0745). There was a 10% and 15.2% decrease in the walking speed of the 0.5 mM- and 1 mM BPA-treated flies, respectively, when compared to the vehicle-treated control flies. 3.5. BPA exposure increases mobility in flies As shown in Fig. 2C, there was a significant increase (p < 0.05) in the mobility (independent of the spatial displacement of the fly in the arena) of the 1 mM BPA-treated flies (mean ± SE = 0.1919 ± 0.0260) when compared to vehicle-treated control flies (mean ± SE = 0.1267 ± 0.01168). The 0.5 mM BPAtreated flies (mean ± SE = 0.1667 ± 0.0188) did not show any significant increase in mobility. Linear regression analysis showed a positive slope with a significant increase in the mobility of the flies with BPA treatment (r = 0.420, p = 0.0233). 3.6. Increased angular velocity in the BPA-treated flies In the natural environment, flies constantly change direction in response to external cues, food, or predator. In a closed arena, the BPA-treated flies showed a significant increase in angular velocity when compared to the vehicle-treated control flies (Fig. 2D). One-way ANOVA analysis showed that the 1 mM BPA-treated flies (mean ± SE = 598.9 ± 32.43; n = 10) had a significant increase (p < 0.05) in angular velocity when compared to the vehicle-treated control flies (mean ± SE = 493.6 ± 17.14; n = 10). A significant increase (p < 0.05) was also observed between the 0.5 mM BPA–treated flies (mean ± SE = 584.5 ± 24.16; n = 9) in comparison to the vehicle-treated control flies (mean ± SE = 493.6 ± 17.14; n = 10). Linear regression analysis of angular velocity vs. BPA concentrations showed a positive slope, indicating a highly significant correlation between the angular velocity and BPA concentration (r = 0.494, p = 0.0065) (Fig. 2D). 3.7. Effect of BPA exposure on turn angle in flies The turn angle gives an estimation of the angle at which the fly moves as it reorients its trajectory during its turn. The BPA-treated flies (0.5 and 1 mM) turned at a mean angle of 19.87◦ and 20.33◦ , respectively, whereas the vehicle-treated control flies turned at a mean angle of 18◦ . Although a slight increase in turn angle was observed in the flies treated with 0.5 mM (mean ± SE = 19.87 ± 0.7192; n = 9) and 1 mM BPA (mean ± SE = 20.33 ± 1.054; n = 10) as compared to the vehicletreated control flies (mean ± SE = 18 ± 0.5155; n = 10), it was not statistically significant (Fig. 2E). However, linear regression analysis showed a significant positive correlation (r = 0.376, p = 0.0438) between the turn angle and the BPA concentration (Fig. 2E). 3.8. BPA significantly increases the meander of flies The change in the direction of flies’ movements was calculated as the meander. It showed a similar pattern as the angular velocity and the turn angle. 1 mM BPA-treated flies

Fig. 3. Effect of BPA exposure on grooming episodes during the 5-min observation period. The figure represents the scattered plot data with mean ± S.E. There was a significant increase in grooming by BPA-exposed Drosophila. The linear regression analysis and the correlation coefficient (r) between the BPA exposure and the grooming by the Drosophila are presented as the dashed line and the r on the graph. For statistical analysis, **p < 0.01.

(mean ± SE = 5362 ± 7649; n = 10) showed a significant increase (p < 0.05) in the meander when compared to the vehicle-treated control flies (mean ± SE = 3269 ± 4327; n = 10) (Fig. 2F). There was no significant increase in the meander of the 0.5 mM BPA-treated flies (mean ± SE = 4831 ± 4080; n = 9) as compared to the vehicletreated control flies. The meander showed a significant positive correlation (r = 0.456, p = 0.0128) in the linear regression analysis between the BPA concentration and the degree of meander in the Drosophila (Fig. 2F). 3.9. No significant effect of BPA on the movement of flies During the time spent by the flies in the circular arena, there were periods of activity with intervals of inactivity. For the software to identify whether the fly is moving or not moving, we applied a threshold for spatial displacement to act as a marker of activity (center point moving) and inactivity (center point not moving). One-way ANOVA analysis showed no significant difference in the activity between the 0.5 mM BPA-treated (mean ± SE = 0.695 ± 0.0865, n = 9) as well as the 1 mM BPA-treated flies (mean ± SE = 0.6227 ± 0.0576, n = 10) when compared to the vehicle-treated control flies (mean ± SE = 0.7113 ± 0.0635, n = 10) during the 5-min observation period (Fig. 2G). There was also no significant difference for the period of inactivity between the 0.5 mM BPA-treated (mean ± SE = 0.9496 ± 0.1250) and the 1 mM BPA-treated flies (mean ± SE = 0.9253 ± 0.1313) when compared to the vehicle-treated control flies (mean ± SE = 0.6809 ± 0.0607) (Fig. 2H). However, a pattern of decrease in activity (r = −0.177, p = 0.3565) (Fig. 2G) and an increase in the inactivity (r = 0.295, p = 0.1207) (Fig. 2H), although not significant, was observed between the BPA-treated and the vehicle-treated control flies, which was comparable to the decrease in the distance moved. 3.10. Increase in grooming episodes in BPA-exposed flies Individual male BPA-treated flies showed a significantly increased number of grooming episodes when compared to the vehicle-treated control flies. Fig. 3 shows the scattered plot data with mean ± S.E.M of the three groups (0, 0.5, and 1 mM BPA treatment). A significant increase (p < 0.01) in the grooming of the

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0.5 mM BPA-treated flies (mean ± SE = 25.7 ± 1.795) and 1 mM BPAtreated flies (mean ± SE = 26.7 ± 3.367) was seen when compared to the vehicle-treated control flies (mean ± SE = 13.6 ± 2.083). The linear regression analysis illustrated a significant increase in grooming episodes with an increase in the BPA concentration (r = 0.557, p = 0.0014) (Fig. 3).

4. Discussion The increased prevalence of neurobehavioral disorders such as autism has been of concern recently. This sharp increase could be partly attributed to better diagnosis due to improved diagnostic criteria and increased awareness, but it cannot account for the exponential increase. Research studies into prenatal exposure to environmental contaminants such as pollution, proximity to highways, and maternal occupation, have acquired heightened interest as a possible role in the etiology of autism [55–59]. A recent study of identical twins reported that genetics accounted for only 38% of autism risk, with environmental factors explaining the remaining 62% [60]. In the present study, we identify BPA – induced behavioral alterations in Drosophila melanogaster similar to the human endophenotypes of autism. Drosophila melanogaster is considered a valuable model in the study of human disorders and an important tool for the study of neurodevelopment and behavior [48,61,62]. The rodent models in the study of neurodevelopmental disorders have an advantage due to the presence of increasing number of validated behavioral assays. Successful animal models of human disorder should mimic the genotypic and pathological aspect of the disorders, and also be a strong phenotypic complacent model. Therefore, our behavioral study may also help establish Drosophila as a model to examine disorders with a strong behavioral component. One of the core features of autism and other neurodevelopmental disorders such as fragile X and Angelman’s syndrome is the inability of the individual to interact socially with other individuals [63–65], which is a diagnostic criterion in autism. It is also a common feature in studies using mouse models [66]. Social behavior is the ability of conspecifics to interact, leading to changes in the subsequent behaviors of the individual [67–69]. In a social setting, the individual maintains a personal space or a distance from another individual (personal space boundary), but also a spatial proximity to another individual for effective communication [70]. This social space or the space between two individuals of the same species is seen in most animals such as birds, fish, or locusts. When placed in a social setting, Drosophila tend to arrange themselves uniformly rather than in aggregates or randomly [50,54], and this social interaction in the group leads to learning of higher behaviors from their conspecifics [67,71,72]. Studies have shown that social isolation in Drosophila reduces the fiber number in the mushroom bodies, the functional equivalent of the hippocampus [73]; increases aggression [74,75]; and shortens the life span [76]. When placed in settings where they are allowed to freely interact with other flies, Drosophila flies usually maintain a distance of about two body lengths (1–5 mm) among themselves, similar to other animals [50,54]. This allows for the flies to orient themselves to interact with each other. Thus, interaction between flies follows a non-random pattern [77]. In our study, inter-fly distance was used as a measure for social interaction [52,53,78,79], and we found that when the flies exposed to BPA were placed in a social setting, there was a decrease in the inter-fly distance. This decrease may be due to aberrant social interaction, in which the flies do not maintain the ideal balance of attraction/repulsion and interact inappropriately with each other. Locomotion is the change in the position of the animal in relation to its surroundings. It is a characteristic behavior of all animals and is associated either directly or indirectly with other

behaviors that the animal performs such as foraging, courtship, avoiding predators, and social interaction, as well as learning and memory processes. Exploratory activity allows the animal to gather information about the novel environment, and open field assay is an important test to measure the exploratory activity in animal models of neurological disorders such as depression [80] and autism [66], and is widely used to measure for activity following drug administration [81,82]. Since the locomotor activity is the result of the animal’s ability to react to its immediate environment, such as avoiding predator or mating, it can be regarded as the decisionmaking process of the current state of the animal, as a motivation to either produce the action or not. This activity can be divided into either spontaneous activity or reactivity, suggesting that locomotion might be influenced by genetic and environmental factors [83]. Many individuals diagnosed with autism are also diagnosed with ADHD [84,85], although there is no co-diagnosis of the disorders according to the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria [86]. Rodent models of autism show a deficit in exploratory activity, as studied by the open field test [87,88]. For the open field assay, we chose two of the higher concentrations of BPA (0.5 and 1 mM), which reflected the federally regulated LOAEL (50 mg/kg/day body weight). The open field activity was used to evaluate animals’ locomotor and exploratory behavior using the Ethovision software. Similar tracking has been used for automated tracking in the assessment of children with autism [89]. Prenatal and postnatal exposure to BPA had an effect on the exploratory activity of the flies. There was a decrease in the distance traveled by the BPA-exposed flies, although there was no significant change in the walking speed (velocity) that may alter the distance that they traveled during the course of the assay. Previous evaluations of exploratory behavior in Drosophila demonstrated higher initial spontaneous activity levels that decreased over time [83,90,91], but these higher levels were not observed in our experiment, as we had chosen a smaller duration (5 min) for the assay. Indeed, to ensure that the results were not confounded by other external factors such as fatigue and starvation, we chose a smaller arena (8 mm) and duration (5 min) for the assay. The BPA-exposed flies not traveling the arena stayed at the same place, and performed repetitive movements such as grooming, which was manually observed and correlated with the software’s observation of the increase in the mobility of the animal while remaining at the same place. The legs of Drosophila have a high density of tactile sensory organs known as the sensila trichodea, which the fly preens back into position after it physically comes in contact with another fly. The increased grooming that we observed in the isolated flies can be due to the need for increased sensory stimulation similar to individuals with autism who show an increase in episodes of self-sensory stimulation, auditory or tactile. Restricted repetitive behaviors form a major core of the diagnosis criteria for autism according to the DSM-5 criteria [86]. Children with autism manifest these repetitive behaviors in many forms such as motor stereotypies [92], which may increase during times of increased social and emotional demand [93]. For Drosophila, the open field arena is a novel environment that may be stressful, and the increased grooming can be seen as a strereotypic behavior, similar to that observed in individuals with autism. A similar significant increase in the grooming behavior was observed in the Drosophila model of fragile X syndrome [45]. Cleaning behavior of Drosophila is a complex activity with organized sets of repeated movements that disappear when the animal is left undisturbed [94], but in our study, the BPA-exposed Drosophila continued to do the grooming behavior through the entire duration of the assay. In circular arenas, Drosophila moves with small turn angles [91]. We observed a trend toward an increase in turn angle and a

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significant increase in the angular velocity of movement in the flies with an increase in the BPA concentration. Because of the flies’ innate desire to walk in a straight trajectory either toward an object of relevance such as another fly or food, or away from a predator [95,96], a smaller turn angle may be the result of a fly’s immediate decision to change its trajectory in response to change in the environment (such as the presence of a predator). We therefore hypothesize that the increased turn angle relates to an inability of the BPA-exposed fly to process changes in the environment and react immediately, similar to individuals with autism who have difficulty in their executive decision-making [97,98]. We also observed a tendency for the flies to meander from the path following BPA exposure, as demonstrated by an increase in changes in the direction of their movement. The BPA-exposed flies therefore, reoriented themselves with an increase in the turn angle and in the angular velocity, thereby making a zigzag pattern of movement or navigation. In the environment, movement follows a purpose either toward food, mate or to escape from a predator. This purpose makes the animal follow a path that changes when there is a change in the purpose of the movement. When there is a change in the motivation of the animal, there is a change in the trajectory of the path. Motivation for movement in the novel arena should be foraging, escape, and searching for a mate for the males. The open field assay illustrates the impaired exploratory activity, which may affect their social approach. Behavior is genetically influenced by the activation of molecules in functional regions of the brain as well as by the environment. The environment influences the genome by affecting the development and the activation of the molecules that influence behavior. Thus, the environment can have an effect on social behavior by influencing the genome, thereby a gene-environment interaction in behavioral disorders [99]. The Drosophila model that we employed could be used further to study genetic influences that BPA may execute to provide for the disturbances in the behavior as seen in autism. One of the most significant challenges in studying neurodevelopmental disorders such as autism in animal models is the ability to evaluate behavioral phenotypes that reflect their core behavioral symptoms. In this study, we were able to demonstrate the behavioral deficits in Drosophila when exposed to BPA that are major features of autism. Our model may provide a valuable approach to analyze the effects of environmental agents on behavior, and also to study the role of gene–environment interaction in the neurodevelopmental and neurobehavioral disorders. BPA is a ubiquitous environmental toxin and is shown to lead to behavioral disturbances in Drosophila. In summary, we not only present a model for the behavioral changes following environmental exposure, but also show that a single environmental insult may correlate to the increased incidence of behavioral disorders in specific regions of increased exposure. The present study presents further evidence that environment plays an important role in shaping neurodevelopmental changes. Acknowledgments We thank Dr. Ira Cohen for providing Noldus EthoVision-XT system and his helpful critiques on the manuscript. This work was supported in part by funds from the New York State Office for People with Developmental Disabilities, Autism Research Institute and CUNY – Graduate Center/CSI-CDNDD Program. References [1] Boyle CA, Boulet S, Schieve LA, Cohen RA, Blumberg SJ, Yeargin-Allsopp M, et al. Trends in the prevalence of developmental disabilities in US children, 1997–2008. Pediatrics 2011;127:1034–42.

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