Acute Exposure to Arsenic Affects Cognition in Drosophila melanogaster Larvae

INTRODUCTION

Arsenic (As) is a toxic heavy metal that can be found naturally almost everywhere [1]. Exposure to arsenic has been linked to a significantly increased risk of a wide variety of disorders, such as type II diabetes, atherosclerosis, high blood pressure, myocardial infarction, keratosis, anemia, melanoma, bladder cancer, lung cancer, and cognitive impairment [2]. Consumption of inorganic arsenic is a major contributor to an increased death rate in arsenic-endemic regions [3-5]. Arsenic poisoning is a chronic systemic disease that can develop after extended exposure to arsenic in the environment, including the air, soil, water, and foods. Arsenicosis has sparked alarm among people worldwide, and it is a problem with people's health that should not be disregarded. Arsenic poisoning poses a risk to more than 300 million people around the world, including residents of the United States, Mexico, Chile, Hungary, Bangladesh, Thailand, India, and China [6].

Various epidemiological studies have established a correlation between prolonged exposure to arsenic and cognitive impairment as well as other neurobehavioral impairments [7-11]. In rat models, arsenic, which is also a powerful neurotoxin, leads to hippocampal-dependent behavioral impairments [12]. Arsenic exposure in animal research models has been shown to produce significant changes in the functioning of the hippocampus [13]. Cognitive impairment has a negative impact on an individual's overall quality of life and can disrupt the activities that constitute a normal day. It is also related to a large number of adverse consequences, such as an increased chance of death and catastrophe [14].

The vinegar fly, Drosophila melanogaster, commonly known as a fruit fly has been an invaluable organism for the study of learning and memory, as well as the discovery of new genes and their functions [15]. For the past few years, a significant amount of progress has been made in unraveling the fundamental mechanisms that drive olfactory learning and memory in Drosophila [16, 17]. The various neurotransmitter systems like dopaminergic, GABAergic, and glutamatergic play significant roles in learning and memory [18-20]. The fruit fly is highly sensitive to odors, and the availability of robust behavioral paradigms to measure olfaction, learning, and memory makes it a useful model to study the effects of various substances on its cognitive behavior. The larvae of fruit flies are simpler in terms of neural complexity and have been used in various experiments including behavioral assays.

Making a decision is a high-level cognitive process that needs the evaluation of available options, which may be based on the decision-own maker's preferences, and prior experiences [21, 22]. Based on cognitive ability, behavioral assays are designed to determine the response of Drosophila larvae. The Drosophila larvae could also be trained and the memory formed could be studied further. In the present study, we have investigated the effects of acute exposure to arsenic in third-instar Drosophila larvae and measured their olfaction and learning ability in a time and dose-dependent manner using behavioral assays.

MATERIALS AND METHODS

Fly care and husbandry

Wild-type flies of the Oregon R+ strain were reared on cornmeal media and maintained at 25⁰C on a 12 h light/dark cycle in a BOD incubator. The media comprised high-grade 8 gm/l agar, 15 gm/l yeast extract, 80 gm/l corn, 20 gm/l, dextrose, and 40 gm/l, sucrose. Antifungal and antibacterial agents such as 4 ml/l propionic acid and 0.6 ml/l orthophosphoric acid, respectively, were added after the media cools down to room temperature. All these chemicals were obtained from HiMedia (Mumbai, India).

Chemicals

Treatment of larvae involved the use of sodium arsenite (NaAsO2) with a purity of 90% (MW 129.91 g/mol). Different concentrations of sodium arsenite solution were made in a 5% sucrose solution used as the solvent. Monopotassium phosphate (KH₂PO₄), calcium chloride (CaCl₂), sodium chloride (NaCl), potassium chloride (KCl), disodium phosphate (Na₂HPO₄), and Polyethylene glycol – 6000 (PEG 6000) were used for larvae isolation. High-grade chemicals from Himedia were used for the above-mentioned experiments. The highest-quality ethyl acetate (EA) odorant and the mineral oil (diluent) were purchased from Sigma-Aldrich.

The treatment and behavioral experiments were conducted in glass Petri dishes with a diameter of 90 mm purchased from Borosil, India. Faber-Castell paint brushes with fine and soft bristles were used for handling the larvae. To isolate the larvae, strainers with fine mesh were purchased from the neighboring market.

Larvae isolation and sodium arsenite treatment

An average of 150–200 fruit flies were transferred in fresh corn meal media bottles, which were then put into the BOD incubator (25⁰C) to lay eggs. After 20 hours of egg laying, the bottles were made fly-free by transferring all the flies to another bottle, and the bottles with eggs were kept at a controlled temperature of 25⁰C for 3 days to continue development in the BOD incubator. The early third instar larvae were developed from eggs in around 72 h. For neurobehavioral investigations using behavioral assays, early third-instar larvae were used. These larvae were isolated by collecting the top layer of corn media gently in a strainer with the help of a soft paintbrush, taking care to prevent injury to the larvae. For the purpose of separating the larvae from the media, the coarse media containing the larvae was transferred into a vial containing 30 percent PEG-6000 solution (300 g of PEG 6000 dissolved in 1000 mL distilled water). Due to the difference in their relative densities, the media debris sank to the bottom of the vial while the larvae floated to the top. To remove the PEG solution sticking to the larval body, the top layer of the vial was emptied into the strainer and rinsed gently with running water. The larvae were then collected into a Petri plate with 0.5 mL of Ringer's solution after being removed from the cornmeal media and PEG solution [23, 24], maintaining osmotic balance to keep larvae from drying out while behavioral assays were being performed. Ringer’s solution comprised 128 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl₂, 0.9 mM Na₂HPO₄, and 0.37 mM KH₂PO_4.

Larvae were treated with specific concentrations (1 mM and 1.5 mM) of arsenic after being harvested from media bottles as third-instar larvae. A Petri plate with a thin layer of agar was prepared by pouring 20 mL of a 1 percent Agar solution. 2.5 mL of the sodium arsenite solutions of the required concentrations were poured onto the agar layer to treat larvae. These concentrations were selected based on the study of the effect of arsenic as reported by Rizki et al. [25] on Drosophila. The harvested larvae were put onto an Agar Petri plate containing sodium arsenite solution and were treated for 17 hours. These treated larvae were then tested for olfaction, trained, and tested for memory formation.

Behavioral experiments

Larval plate assay

Odors induce varying behavioral responses in Drosophila via the sensitive olfactory system. The olfactory response of both untreated and arsenic-treated third instar larvae was quantified using larval plate assay. This assay was adapted from Khurana et al. [26]. The Petri plate contained a thin layer of 1 percent agar solution prepared in Ringer’s solution. About 10 ml of the 1 percent agar solution was poured into the Petri plate. Two small round filter discs were placed diametrically opposite within the two arcs of 20 mm from the edge of the Petri plate (Figure 1). On each paper disc, 20 µl of the ethyl acetate (EA) odorant diluted in mineral oil (at 10^-2 dilution) was poured.

Approximately 50 larvae were placed at the center (S zone) of the Petri plate (90 mm) just before pouring the odor. The larvae started crawling toward the odor, after 2 minutes, the photos were taken to discern the number of larvae in various delineated zones, and the response index (RI) was calculated. The larvae were also counted manually for confirmation.

Larvae learning and memory assay

training

The treated and untreated larvae were trained as described by Honjo & Furukubo-Tokunaga, 2006 [27]. Untreated larvae were trained in two ways. The first set was trained in 1 ml of distilled water (DW) while the second set was trained in 1 ml of 1 M sucrose. Both DW and 1 M sucrose were spread on freshly prepared 1 percent agar Petri plates used as training plates. A filter disc was placed on the inner side of the lid and 10 µl of neat odorant (EA) was poured onto it. With the help of a paintbrush, the larvae were transferred from Ringer’s solution to the training Petri plate. Immediately after the larvae were transferred, the training Petri plate was covered with the lid with the odorant. The Petri plate was left undisturbed for 30 minutes. Larvae got exposed to odor and sucrose simultaneously and associated sucrose with odor (Figure 2). After training for 30 minutes, the larvae were transferred to a plate containing distilled water to rinse off any sucrose or odor residue that still adhered to their bodies. After rinsing thoroughly, the trained larvae were transferred onto the testing plate for testing their learning and memory using larval plate assay as described earlier in this paper. Arsenic-treated larvae were trained similarly as the second set of larvae mentioned above were trained with sucrose-associated odor.

Testing after training

The olfactory response of both naïve and arsenic-treated third instar larvae were quantified at 30 min time intervals for 90 min. This was the modified assay of Khurana et al., 2009. The response indices (RI) of trained treated and untreated larvae were calculated at 30 min of intervals for 90 min. The appetitive learning index (LI) was calculated using the following formula: