Toxicological Bioassays
This study aimed to investigate the contact toxicity of insecticides against S. invicta workers. Only workers were used in the bioassays. Nine commercial formulations of insecticides were tested in separate experiments. The bioassay arenas were beakers (bottom diameter: 10.4 cm; height: 14.0 cm; Bomei Glass Instrument Factory, Beijing, China) with the inner wall coated with Teflon (500 mL, GUOXIN®, Dongguan, China). For each insecticide, solutions with different concentrations of the active ingredient (0.05, 0.5, 5, 50, 500, and 5000 ppm) were prepared by dissolving the required amount of the insecticide in distilled water, and 170 µL of the solution was added and evenly smeared on the bottom of the beaker (to reach the final amount of 2 µL/cm2). The same amount of distilled water was smeared on the bottom of the beaker as the controls. These beakers were air-dried at room temperature (22 ± 2℃) for 24 h. Right before the experiment, a sterile L-shaped cell spreader (Biglogix 65-1010, BIOLOGIX®, Jinan, China) was inserted into the plastic box containing each S. invicta colony, and workers were allowed to climb up on the surface of the pipette, which was then inserted into the beaker (~ 5 cm above the bottom) and gently shaken until 20 medium workers fall onto the bottom of the beaker. Each concentration of each insecticide was repeated 6 times (one replicate for each colony group). To prevent dehydration of ants, the beakers were placed in plastic boxes with 3 layers of paper towel (moistened using 200 mL distilled water) placed on the bottom, and the plastic boxes were capped to maintain the humidity (Mao et al. 2011). All laboratory experiments were conducted at room temperature (22 ± 2℃). The mortality of ants was recorded at 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, 72 h.
Behavioral Effects of Insecticide-Treated Surfaces on Solenopsis invicta Under Laboratory Conditions
This study aimed to investigate the foraging and particle-covering behaviors of S. invicta workers in response to surfaces treated with insecticides under laboratory conditions. Seventy-two colony groups of S. invicta were used in this experiment. Eight colony groups were collected from the campus of South China Agricultural University (23°17′N, 113°37′E), 32 colony groups were collected from the Zengcheng Teaching and Internship Base of South China Agricultural University (23° 24′ N, 113° 63′ E), and the remained colony groups were collected from Tianlu Lake Park. Nest materials (15–20 L) and ants were transferred to plastic boxes (44 × 29 × 19 cm [L × W × H]) using the similar methods described earlier. Ants were maintained in the laboratory for ~ 1 week before the experiment.
The test for each insecticide was repeated 8 times, and each colony group was tested only once. Before the test, solutions with different concentrations of the active ingredient (0.05, 0.5, 5, 50, 500, and 5000 ppm) were prepared as described above. Plastic squares (50 × 50 mm) were prepared by coating a piece of white cardboard paper with a layer of plastic membrane (Qin et al. 2019), and 50 µL solution was evenly smeared onto the square (the final amount of the solution on each square was 2 µL/cm2). To prepare the untreated (control) squares, the same amount of distilled water was added onto the square and evenly smeared. These squares were air-dried for ~ 8 h. Right before the test, a piece of sausage (10 × 10 × 1 mm, Guangdong Shuanghui Food Co. Ltd., Qingyuan, China) was fixed onto the center of each square using an insect pin (Length = 35 mm, diameter = 0.35 mm). Seven squares either smeared with the active ingredient (0.05, 0.5, 5, 50, 500, or 5000 ppm) or distilled water were placed on the plastic box with randomly assigned orders, and adjacent squares were > 1cm apart from each other (Fig. 1A).
High-resolution pictures were taken for each square at 15, 30, 45, 60 min, and 3, 6, 12, 24 h into the experiment. The number of ants on each square was counted at 15, 30, 45, and 60 min, or until the food was completely transported away from the square within 1 h. The data obtained from different time points were averaged to determine the foraging activities of ants within 1 h (Wen et al. 2020a). The presence of sausages on each square was recorded at 1, 3, 6, 12, and 24 h. Pictures taken at the end of the experiment (24 h) were analyzed to measure the area of squares covered by particles using the regionprops function of MATLAB (MathWorks Inc., Natick, MA, USA). Also, particles on each square were collected and oven-dried at 50℃ for 5 d. The dry weight of particles was measured using a 0.1-mg electronic balance (Mettler Toledo®, Switzerland).
Behavioral Effects of Insecticide-Treated Surfaces on Solenopsis invicta Under Field Conditions
This study aimed to investigate the effect of insecticide-treated surfaces on the foraging and particle-covering behaviors of S. invicta workers under field conditions. The experiments were conducted in Zengcheng Teaching and Internship Base of South China Agricultural University from 19–23 April 2021. Twenty S. invicta mounds were randomly selected, and weeds and rocks around mounds were removed 4 d before the experiments. Solution of each insecticide (500 or 5000 ppm) was prepared, and 50 µL solution was evenly smeared onto the square. For each concentration (500 or 5000 ppm), squares either treated with insecticide or distilled water were placed 20-cm apart from the edge of the S. invicta mound (Fig. 1B). The order and direction of squares were randomly assigned, and adjacent squares were equidistant from each other. Each concentration was repeated 10 times (each mound was tested only once). The average number of foraging ants within 1 h, the presence of food at 1, 3, 6, and 24 h, and dry weight and covered areas of relocated particles at the end of the experiment (24 h) were recorded as described above.
Effect of Particle-Covering Behavior on Toxicity of Contact-Based Insecticides against Solenopsis invicta
Our laboratory and field study showed that S. invicta workers transported significantly more particles onto the squares smeared with fipronil or rotenone compared with the control ones (see results). Herein, we conducted a laboratory study to investigate whether such particle-covering behaviors would reduce the toxicity of fipronil or rotenone against S. invicta.
Six colony groups of S. invicta were collected from Tianlu Lake Park on 24 February 2021, using the same method as described above. Topsoil was collected from the Zengcheng Teaching and Internship Base of South China Agricultural University where S. invicta activities were detected. The soil was oven-dried at 50℃ for > 5 d. The dried soil was crushed using a wooden hammer and sequentially sifted through 0.5-mm and 1-mm sieves (our preliminary study showed that the diameters of most particles relocated by S. invicta workers were ranging from 0.5-1 mm). Before experiments, the required amount of distilled water was added into the soil and thoroughly mixed to prepare the 10%-moisture (w/w) particles.
The experiment for each insecticide contained 7 treatments (Table 2), and each treatment was repeated 6 times (one replicates for each colony group). One hundred and seventy microlitres of insecticide solution (500 or 5000 ppm) or distilled water was evenly smeared on the bottom of beakers with the inner wall coated with Teflon, and air-dried for > 12 h. Certain amounts of soil particles (Table 2) were evenly added onto the bottom of beakers (the density of particles was equal to the mean density of particles relocated onto the squares treated with fipronil or rotenone at the concentration of 500 or 5000 ppm in the laboratory study). Twenty medium S. invicta workers were released into each beaker and maintained in the plastic boxes with moistened paper towels as described earlier. The mortality of ants was recorded at 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, and 72 h for rotenone. Since all ants exposed to fipronil-treated surface (whether covered by particles or not) died within 12 h, the mortality of ants was no longer recorded after that time point.
Table 2
Experiment design to investigate whether particle-covering behavior reduces the contact toxicity of fipronil or rotenone against Solenopsis invicta workers.
Insecticide
|
Treatment
|
Concentration of insecticide solution smeared on the bottom of beakers (ppm)
|
Soil particles added onto the bottom of beakers (g)
|
Fipronil
|
0
|
0
|
|
|
0
|
4.44
|
|
|
3
|
0
|
5.26
|
|
4
|
500
|
0
|
|
5
|
500
|
4.44
|
|
6
|
5000
|
0
|
|
7
|
5000
|
5.26
|
Rotenone
|
1
|
0
|
0
|
|
2
|
0
|
5.48
|
|
3
|
0
|
8.98
|
|
4
|
500
|
0
|
|
5
|
500
|
5.48
|
|
6
|
5000
|
0
|
|
7
|
5000
|
8.98
|