Laboratory behavioral tests of mainland and island populations of Podarcis siculus
Description
In accordance with the permit of the Ministry of Environment and Energy (Class: AP /L612-07 I 18-481 65; No: 517-07-l-l-l-18-4), and the permit of the Ethical committee of the Faculty of Science, University of Zagreb, Croatia (No: 251-58-10617-18-16), animals (10 males from each location) were captured by noosing, and transported to the laboratory in cloth bags. Since the population on islet Pijavica is strictly protected, this was the maximum number of animals we were permitted to collect. In the laboratory, animals were maintained in individual terraria (40x30cm) with peat as a substrate, fed every two days with crickets (Acheta domestica), that were supplemented with calcium and vitamins, with water available ad libitum. The light cycle was the same as the natural cycle (14:10 h, light: dark). Room temperature varied between 22°C during the night and 30° C during the day, to simulate natural day-night temperature variation. A plastic tube with one entrance was put in each terrarium to serve as a hiding place. These tubes were also used as containers for transferring the lizards from their terrarium to the experimental setup, to reduce handling stress. Lizards were allowed to habituate to laboratory conditions for one week before being subjected to behavioral testing. The experiments were conducted in accordance with the Directive of the European Parliament and of the Council (2010/63/EU) and the Croatian Animal Protection Law (“Narodne Novine”, 102/17 and 32/19). All efforts were made to reduce the number of animals used and to ensure animal welfare.
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Novel environment and novel object exploration The laboratory tests of activity in the novel environment, and novel object exploration were performed in an open field apparatus (Sahgal, 1993), a square Plexiglas box (50x50x50 cm) with an open top. The testing was performed under a temperature of 30°C, between 9:00 and 17:00 hours. Each lizard was exposed to the open field two times a day (once in the morning, and once in the afternoon) during the two consecutive days. The order of lizards entering the experiment for each of the four trials was randomized with an online program (Research randomizer). Each animal was transferred from its terrarium using the earlier mentioned tube, and was gently shaken out of the tube to the center of the open field apparatus. Recording of animal behavior started after two minutes of acclimatization period, and lasted for 15 minutes. At the end of each trail, the lizard was coaxed with a wooden stick to move back into the tube for transfer to its home terrarium. After each test, the apparatus was wiped with 30% alcohol and air dried. First and fourth trial were recorded with a camera from above and analyzed using Noldus Ethovision XT 13. Exploration of the novel environment was determined on the first trial by measuring time spent in movement (TM, in s), total distance covered (TDC, in cm), and percentage of time spent in the central part of the apparatus (%C). The second and third exposures to the open field were performed to familiarize the lizard to the open field apparatus. On the fourth trial, we tested exploration of a novel object (a piece of rubber laboratory glove) placed in the in the center of the apparatus, in a now familiar environment. Behavior towards the novel object was determined by measuring latency to approach the novel object (LANO, in s) and time spent exploring the novel object (TENO, in s). The values of TDC, TSM and %C were also used as a measure of general activity.