High-Throughput Screening of Psychotropic Compounds: Impacts on Swimming Behaviours in Artemia franciscana

Abstract

Animal behaviour is becoming increasingly popular as an endpoint in ecotoxicology due to its increased sensitivity and speed compared to traditional endpoints. However, the widespread use of animal behaviours in environmental risk assessment is currently hindered by a lack of optimisation and standardisation of behavioural assays for model species. In this study, assays to assess swimming speed were developed for a model crustacean species, the brine shrimp Artemia franciscana. Preliminary works were performed to determine optimal arena size for this species, and weather lux used in the experiments had an impact on the animals phototactic response. Swimming speed was significantly lower in the smallest arena, whilst no difference was observed between the two larger arenas, suggesting that the small arena was limiting swimming ability. No significant difference was observed in attraction to light between high and low light intensities. Arena size had a significant impact on phototaxis behaviours. Large arenas resulted in animals spending more time in the light side of the arena compared to medium and small, irrespective of light intensity. The swimming speed assay was then used to expose specimens to a range of psychotropic compounds with varying modes of action. Results indicate that swimming speed provides a valid measure of the impacts of behaviour modulating compounds on A. franciscana. The psychotropic compounds tested varied in their impacts on animal behaviour. Fluoxetine resulted in increased swimming speed as has been found in other crustacean species, whilst oxazepam, venlafaxine and amitriptyline had no significant impacts on the behaviours measured. The results from this study suggest a simple, fast, high throughput assay for A. franciscana and gains insight on the impacts of a range of psychotropic compounds on the swimming behaviours of a model crustacean species used in ecotoxicology studies.

Keywords: artemia; behaviour; behavioural ecotoxicology; ecotoxicology; psychotropics.

Figure 1

Dimensions and location of light and dark zones for (A) small arena, acrylic plate 1, (B) small arena, acrylic plate 2, (C) medium arena, acrylic plate 1, (D) medium arena, acrylic plate 2, (E) large arena, acrylic plate 1, (F) large arena, acrylic plate 2.

Figure 2

Experimental design for A. franciscana exposure and behavioural analysis. The 12-well plates were loaded in duplicate to provide 12 replicates per treatment. The procedure was repeated for each of the four compounds.

Figure 3

Mean velocity of A. franciscana between arena sizes in (A) 2-min and (B) 10-s time bins. Error bars represent 95% confidence. Asterisks indicate significant differences between arena sizes. For the 10-s data, asterisks indicate significant differences in velocity between large and medium arena only. Significance level * p ≤ 0.05.

Figure 4

Percent duration A. franciscana spent in the light zone during 3-min dark and 3-min light phases between small, medium and large arenas when exposed to light at (A) 100% intensity and (B) 5% intensity. Error bars indicate 95% confidence. Asterisks indicate significant differences between arena sizes. Significance level * p ≤ 0.05.

Figure 5

Mean velocity of A. franciscana following exposure to fluoxetine between (A) treatment, (B) light phase, (C) length of exposure, and (D–F) interactions between treatment and light phase across the three lengths of exposure. Error bars represent standard error. Asterisks indicate significant differences from post hoc analysis. * p ≤ 0.05, ** p ≤ 0.001.

Figure 6

Mean velocity of A. franciscana following exposure to oxazepam between (A) treatment, (B) light phase, (C) length of exposure, and (D–F) interactions between treatment and light phase across the three lengths of exposure. Error bars represent standard error. Asterisks indicate significant differences from post hoc analysis. * p ≤ 0.05, ** p ≤ 0.001.

Figure 7

Mean velocity of A. franciscana following exposure to amitriptyline between (A) treatment, (B) light phase, (C) length of exposure, and (D–F) interactions between treatment and light phase across the three lengths of exposure. Error bars represent standard error. Asterisks indicate significant differences from post hoc analysis. * p ≤ 0.05, ** p ≤ 0.001.

Figure 8

Mean velocity of A. franciscana following exposure to venlafaxine between (A) treatment, (B) light phase, (C) length of exposure, and (D–F) interactions between treatment and light phase across the three lengths of exposure. Error bars represent standard error. Asterisks indicate significant differences from post hoc analysis. * p ≤ 0.05, ** p ≤ 0.001.