Adult (5–8 mm length) killer shrimps, D. villosus, which is an invasive species populating Lake Balaton, were collected locally (Tihany, Hungary) from the littoral region of Lake Balaton, and kept in indoor aquaria filled with aerated, filtered Balaton water under 16–18 h light–dark cycle at room temperature (~16–20 °C) A week before testing, about 250 animals were separated into 15 L containers. In order to ensure/defilade their hideaway, which is characteristic of their lifestyle, small (diameter: 10 mm) ceramic tubes were placed/set into the containers. The shrimps were fed on carrot (Daucus carota subsp. Sativus) ad libitum. Prior to the experiment the animals were starved for the day before the experiment.

The formulated product of a commercially available insecticide APACS 50 WG (clothianidin) was obtained from Arysta Life Science (Budapest, Hungary), and the chemicals for the Bradford assay (Bradford 1976) were purchased from SIGMA.

This assay was carried out with three groups, one control and two treated groups, in each group ten animals were selected. The concentration of APACS 50WG was 3.9 ng l−1 dissolved in Balaton water. The exposition lasted 3 h. Individual animals were placed in 6-well plates, and measurements of swimming behaviour were performed by video tracking system, taking short (90 s) records using a camera. The videos were analysed with Fiji ImageJ 1.52p software. The programme recorded the coordinates and automatically reconstructs the trajectories of the test animals swimming within the ring of the whole of the plate.

The animals were incubated in filtered water containing 2 μM rhodamine B for 60 min (loading). This was followed by a short (3 min) washing period in Balaton water, which allowed the removal of excess dye from the surface of tissues. T0 animals (6 pieces) were immediately frozen providing full uploaded concentration of rhodamine B. The remaining animals (6 pieces) were divided into two groups, individually placed in 100–100 mL low profile bakers. The animals in the control group were kept in filtered Balaton water. The acute (direct) effects of the insecticide were studied on the second group of the animals by incubation in the formulated products (APACS, respectively) dissolved in filtered Balaton water. Treated and untreated animals were frozen following the exposition period (3 h) until further processing. We applied the “accumulation” method of MXR assay (Vehovszky et al. 2018, Smital et al. 2003) where rhodamine B served as a model P-gp substrate. The samples were homogenized in a tissue layer (Tyssuelyzer LT, Qiagen) and centrifuged at 8000 g for 20 min at 4 °C (Biofuge Fresco, HERAUS). The fluorescence of the supernatant (250 μL) was finally measured at λex = 535 nm and λem = 590 nm (Victor 3 plate reader, PerkinElmer) to determine the amount of accumulated rhodamine B in the tissue samples. The protein concentration of the tissue homogenates was also determined in parallel samples applying the Bradford assay (Bradford 1976). The rhodamine B accumulation data were finally expressed as fluorescence units per mg tissue protein.

Data are presented as mean ± SE, and significance was calculated by using Student’s t-test.