data Early-Life Exposure to Invasive Turtles Modified Chronic Stress Response in Tadpoles.xlsx

Ten fragments, each comprising approximately 100 eggs of common frog, were collected from ten freshly laid clutches found in a pool situated between Holubov and Vrábče, South Bohemia, Czech Republic (48.9078633N, 14.3485608E) on March 26, 2023. The collection site was monitored daily to gather same-age, freshly laid egg clutches. Neither the slider nor any other turtle species occurs at the collection locality, so the eggs and their parents were naive prey relative to the turtles.The experiment was conducted in a temperature-controlled laboratory. The embryonal phase was maintained at 12 °C. Following hatching, the temperature was gradually increased from 12 °C to 18 °C over one month. Subsequently, the room temperature was held at 18 °C, with a mean air temperature of 17.7 ± 0.3 °C (± S.D.; hourly measurements) recorded by a Dostman LOG200 PDF datalogger for the remainder of the experiment. For the experiment, glass tanks measuring 100 × 55 × 40 cm were utilized, each holding 220 litres and filled to a 20 cm depth with aged tap water. A Claro 300 filter pump (300 L/hour) was installed in each tank, and the tanks were rinsed three times per week. The tanks were replenished with an equal volume of aged tap water (daily up to 5% of the water volume in the glass tank). The light source was fluorescent tubes (2 x 36 W) with a light regime of 12 h/12 h. To ensure the embryos or tadpoles were only exposed to chemical cues of a predator, not physical contact, each tank was divided by an opaque glass barrier. A 6-cm gap at each end of the barrier with mesh grid allowed water to flow freely between the two compartments. The smaller compartment housed the predator, while the larger one was designated for the eggs or tadpoles. As a predator, four adult red-eared sliders (carapax length: 19 - 24 cm) obtained from Hluboká nad Vltavou Zoo were used. Sliders were fed three times a week with Tetra ReptoMin <i>Gammarus</i> turtle food.To obtain tadpoles with embryonic exposure to predator cues, we divided the frog eggs into six tanks. Two of these tanks contained a slider, while the others were left as control groups without a slider. This 2:4 ratio was chosen to ensure a consistent number of eggs per volume of water while also ensuring a sufficient number of control tadpoles for the next experimental phase. After hatching, tadpoles of all treatments were captured and transferred to six glass tanks (100 tadpoles in each tank). All tadpoles were kept without a slider until the start of the next phase of the experiment. The tadpoles were fed <i>ad libitum </i>with TetraMin aquarium flakes for ornamental fish and Dajana Spirulina &amp; Chlorella Flakes.A controlled experiment was designed with three treatment groups to assess the effects of a slider on tadpole CORT levels. The treatments were E1T1 (Eggs and Tadpoles Exposed) where both the egg and subsequent tadpole stage were subjected to a slider, E0T1 (Tadpoles Only Exposed), where only the tadpoles were exposed to a slider, with the egg stage remaining unexposed, and E0T0 (Control - No Exposure) where neither eggs nor tadpoles were exposed to a slider (serving as a baseline). Each treatment was replicated twice for two distinct age cohorts: 44-day-old and 82-day-old tadpoles.At 44 days of age (Gosner stage 27-28), 80 tadpoles from each of the six rearing tanks were transferred in groups of 40 individuals to six experimental tanks with (E1T1, E0T1) or without (E0T0) a slider, and the CORT response was measured according to the procedure described below. For the E1T1 and E0T1 treatments, we placed sliders in each tank to release kairomones into the water for five days before the second phase of the experiment. The same process was repeated a second time from the other six tanks with tadpoles aged 82 days (Gosner stage 36-37). Regardless of the treatment group, we subjected all tadpoles to the same capture-and-release protocol.<b> </b>CORT level was measured at 0 min as baseline, 2, 20, 60, 360, and 1440 minutes after predator exposure in 44-day-old and 82-day-old tadpoles. At each time point, five tadpoles from each tank were frozen in liquid nitrogen and stored at -80 °C.All methods were carried out following relevant guidelines and regulations. All experimental protocols were approved by the Czech Ministry of Agriculture, Department of Animal Welfare, according to Article No. 15, Section 2 of the act registered under number 9103/2009-17210. Appropriate permission (No. 2/2020) was obtained from the zoo authorities to loan the turtles.