All experimental procedures were permitted by the Institutional Animal Care and Usage Committees of the Grassland Science College of Gansu Agricultural University (GSC-IACUC-2015-0011). Our experiments were conducted according to their guidelines, which are in accordance with the Guide for the Care and Use of Laboratory Animals (the Constitution of Experimental Animal Ethics Committee of Gansu Agricultural University). All experiments were performed in accordance with ARRIVE guidelines.
Animals and laboratory conditions
Adults of both sexes (three males and three females) were captured in April 2015. Specifically, the animals were captured in Mayin Tan (37°12′N, 102°46′E; Tianzhu Tibetan Autonomous County, China) using live traps25 set at fresh surface mounds. The individuals were then transported to the laboratory and housed in an acrylic box with a pipeline covered with soil. The box and pipeline were covered by black cloth to simulate the dark environment of plateau zokors. The temperature in the room was maintained between 20 and 25 °C. Food was supplied daily and consisted of potatoes, lettuce and carrot. After three days of acclimatization to the laboratory the animals were used in the different experiments. Our laboratory is located 2 km away from the field site. At the end of the experiments all animals were returned to the capture site in good health.
Laboratory testing arena
The experimental setup for the laboratory experiments was as follows (Fig. 1): A transparent Perspex tube (8 cm × 8 cm × 80 cm) was joined to the side of the dark acrylic box (40 cm × 40 cm × 40 cm). A rubber stopper was inserted into one end of the tube to avoid effects from the external environment. Treatment apparatus was placed into the rubber stopper (see “Laboratory treatment apparatus” section, below), and, to avoid the apparatus being damaged by the animals, wire mesh (8 cm × 8 cm × 0.5 cm) was placed about 15 cm from one end of the tube. A mercury thermometer was inserted into the tube in the middle to monitor the tube’s temperature. Between experiments with different animals, the box and tube were wiped with 95% alcohol and then with distilled water.
Laboratory treatment apparatus
A rubber stopper with seven holes was used for plugging one end of the tube (Fig. 2). The oxygen concentration, light, temperature, sound and gas flow were considered in this design.
To avoid the oxygen that was delivered into the tube causing the gas to flow too strongly, become drier, and create a sound, a steel oxygen cylinder and thin hose (0.3 cm in diameter) were selected, and one end of the hose was connected directly to the oxygen cylinder with a humidifier bottle, while the opposite end was inserted into the rubber stopper (Fig. 2). Before beginning the experiment, we allowed the oxygen cylinder to sit for two hours at laboratory temperature to remove any temperature effects. A three-parameter soil gas analyzer (13.05.03Pro, Shanghai SAFE Biotech Co., Ltd, China) was used to monitor the oxygen concentration in the tube (Fig. 2).
The average light intensity—that is, 360 Lux from 8:00 am to 8:00 pm—was measured in the field. One end of a wire was connected to an LED light (1 Watt), and the other end to the power supply (Fig. 2).
The temperature in the burrow entrance in the field was about 3 °C warmer than that at a tunnel depth of 10 cm. As such, one end of a wire was connected to a heater strip and the other end to the electrical power supply (Fig. 2). A thermometer was inserted into the tube to monitor the temperature inside the tube (Fig.1). During the experiment period in the laboratory, we switched on or off to make sure the relatively constant temperature inside the tube. The temperature range inside the tube was 3.2 ± 0.27 °C .
When a burrow is opened, wind whistle can be produced around the burrow entrance. Accordingly, a voice recorder (PCM-D50, frequency response 50 Hz–40 kHz, Sony, Japan) was placed at the burrow entrance in the field to record the burrow-entrance sound, the duration of which was 30 min. In the laboratory, the two ends of a wire were connected to an AVOXIVY loudspeaker (diameter: 5 cm; impedance: 4 Ω; 50 Hz–20 kHz) and a voice recorder, respectively (Fig. 2). The recorded sound was played back with a 60 dB sound pressure level, as measured at the burrow entrance in the field (XL2 sound level meter, Nti Audion, Switzerland). The sound was repeatedly played within one hour.
Gas flow treatment
To avoid ambient atmosphere entering the tube, a negative pressure drainage ball with plastic tube (12 cm long, 2 cm in diameter) connected the tube through a rubber stopper (Fig. 2). The tunnel gas was inhaled by the ball, then we pinched the ball to blow the gas into the tunnel as gas flow treatment.
Field treatment apparatus
For the field experiment, the apparatus consisted of a tube (40 cm long, 8 cm in diameter) and an alarm device. The alarm device was made up of a loudspeaker, two slide rails (15 cm long), two metal plates (approximately 7 cm in length and 3 cm in width), and three coiled metal springs (5 cm long, 2 cm in diameter). The three springs were joined to one of the metal plates, while the other metal plate was fixed on the slide rails. The two metal plates were touched by the plateau zokor when it was plugging, which triggered the alarm device, thus enabling us to know whether or not burrow-sealing behavior was occurring (Fig. 3). The aluminum tube with an oxygen device was embedded into the burrow. The soil covering the tube served as an excellent insulator, buffering the tube from the aboveground temperature (Fig. 4A). A steel oxygen cylinder and thin hose (0.3 cm in diameter) were applied by connecting one end of the hose directly to the oxygen cylinder with a humidifier bottle, and then the opposite end of the hose was inserted into the tube (Fig. 4A). A three-parameter soil gas analyzer (13.05.03Pro, Shanghai SAFE Biotech Co., Ltd, China) was used to monitor the oxygen concentration in the tube (Fig. 4A). Allowing sunlight to enter the burrow, a glass bottle, open at one end but closed at the other, was embedded into the burrow. We also used soil to cover the bottle, and there was a 5 cm gap at the surface (Fig. 4B). The aluminum tube with high thermal conductivity was embedded into the burrow. Again, we used soil to cover the bottle and retained a 20 cm gap (Fig. 4B).
In the laboratory experiment, we tested three males and three females for their responses to each treatment. To avoid generating stress and habituation to treatments, zokors were tested for 12 h each day and there was one hour interval between treatments, and five days interval between round of testing for the same individual (Table 1). We performed a control experiment in which a rod was inserted into the burrow but no further treatment was applied, which allowed us to evaluate whether it was the treatment that was causing the burrow-sealing behavior. Before beginning treatment experiment, each zokor was tested 24 times (12 h × 2 days) under the control experiment. We determined the rod movement as occurrence of burrow-sealing behavior.
In the field experiment, we tested three zokors (one male, two females), and six zokors were caught in the cold season and warm season (three males and three females, respectively). We then fastened radio collars (Ag357, Biotrack, Ltd., UK) to each captured individual to allow us to track the position in foraging tunnels of each zokor. Each zokor was used three times in the experiments under each treatment, and, after finishing each experiment, we changed the position of the foraging tunnel to ensure the test tunnel was not an abandoned tunnel. According to radio-tracking data, the straight-line distance between the test tunnel and the nest for each treatment was about 5 m. We conducted a control experiment that whether plateau zokor move to the test tunnel or not during the time between treatments. In the cold season, from 4 October 2015 to 2 November 2015, the burrow-sealing behavior of each zokor was tested under different treatments during their active time (12:00–18:00) and inactive time (09:00–11:00) for a total of 27 days (Table 2). The same was done in the warm season but for a total of 18 days from 15 May 2016 to 5 June 2016, in which the active time was 14:00–20:00 and the inactive time was 08:00–13:00 (Table 2).
The occurrence of burrow-sealing was recorded as “1”, and non-sealing was recorded as “0”. The frequency of burrow-sealing was the number of times the burrow was sealed divided by the total number of experiments for each treatment26, and we considered the frequency for each individual as a replicate. The latency to reseal the burrow was the period from the start of the treatment to the sealing of the burrow, and we considered each instance of latency to reseal the burrow as a repeat. The latency to reseal the burrow for non-sealing under each treatment was unavailable data and was therefore removed. The presence of a normal distribution in the initial data was determined using the Kolmogorov–Smirnov test. All data followed a normal distribution. A comparison of males and females in their frequency of sealing the burrow and in their latency to reseal the burrow under each treatment was performed with an independent-samples T-test. Multiple comparisons were made for the frequency of burrow-sealing and the latency to reseal the burrow under different treatments by using the least significant difference method at the significance level of P = 0.05. In the field experiment, the number of replicates was fewer than three for frequency and the latency to reseal the burrow, we did not conduct multiple comparisons.
Preliminary statistical analysis of the data was performed using Excel 2013 and SPSS 19.0. All the figures and tables were produced in GraphPad Prism 8.0 and Excel 2013.
Source: Ecology - nature.com