The experiments were conducted at the Laboratory of Plant Protection, Shanghai Academy of Landscape Architecture Science and Planning (SALASP), Shanghai, China. Boles of American sweetgum infested with A. suncei were collected from Lingang New Town, Pudong District, Shanghai, China (121.92° E, 30.93° N). The adult beetles of A. suncei used in the experiments were obtained from these infested tree boles and stored in plastic containers [26 (link)]. Healthy boles of American sweetgums (8~10 cm DBH), which were collected from the Geqichun nursery, Pukou District, Nanjing, Jiangsu province (118.55° E, 32.09° N), were cut into 20 cm segments, sent to SALASP, and sealed with wax and paraffin to prevent water evaporation.
Fresh logs were mixed with infested logs, and when new adults started emerging, newly colonized segments (with signs of boring dust and entrance holes) were removed and placed vertically in plastic containers (21 × 12 × 11 cm) with 200 mesh gauze and kept in incubators under controlled temperature and humidity conditions, and the date was recorded. Two circular holes with a diameter of 7 cm were made on the opposite sides of each container. Twelve replicate containers were kept in incubators (MIR 350H, Sanyo Electric Co., Ltd., Osaka, Japan) with 25 ± 1 °C, 70% relative humidity (RH), and complete darkness.
Four formulas of artificial diets were tested for rearing larvae of A. suncei (Table 1 ). The phloem powder was obtained from healthy American sweetgum which was ground and sieved through 0.9 mm mesh and then dried in an oven at 72 °C for 48 h. The compositions were formulated based on previous research on other bark beetle species [19 (link),27 (link)].
The yeast powder and sucrose were mixed in 25 mL of distilled water in a beaker while heated in a hot water bath at 52 °C and stirred until dissolved. Then the agar, inositol, Wechsler salt, multivitamin, cholesterol, ascorbic acid, potassium sorbate, and methylparaben were dissolved, adding 15 mL of distilled water in the beaker under the same condition. The wheat germ powder, microcrystalline cellulose, bark and phloem powder, and 10 mL of distilled water were then added to the solution and stirred. The mixture was fluffy in the beaker. The final mixture in the beaker was wrapped, sealed, and stored at 4 °C in the refrigerator for the beetle rearing test. The artificial diet was not solid in the fridge.
Using a chisel, eggs were collected from excavated galleries in American sweetgum logs. Eggs were transferred gently to a sheet of black filter paper in glass Petri dishes (100 × 20 mm) using a moist sable brush (size 000). In total, 394 eggs were collected for the artificial diet test. In an attempt to minimize contamination from the original galleries, all eggs were soaked in 75% alcohol for 10 s in glass Petri dishes (100 × 20 mm) using a sable brush (size 000), and rinsed with sterile water in glass Petri dishes for 10 s. All eggs were sterilized one by one. They were individually placed at the bottom of 1.5 mL Eppendorf tubes which were then filled with 1 mL of the artificial diet for the growth of A. suncei larvae. When the artificial diet was heated to 25 °C, the artificial diet could be used for rearing. We used a spoon to fill Eppendorf tubes with artificial diet and squeezed the artificial diet with a glass stirring rod. Once the tubes were filled, they were all kept under the same conditions and left until the adults emerged.
Fresh logs were mixed with infested logs, and when new adults started emerging, newly colonized segments (with signs of boring dust and entrance holes) were removed and placed vertically in plastic containers (21 × 12 × 11 cm) with 200 mesh gauze and kept in incubators under controlled temperature and humidity conditions, and the date was recorded. Two circular holes with a diameter of 7 cm were made on the opposite sides of each container. Twelve replicate containers were kept in incubators (MIR 350H, Sanyo Electric Co., Ltd., Osaka, Japan) with 25 ± 1 °C, 70% relative humidity (RH), and complete darkness.
Four formulas of artificial diets were tested for rearing larvae of A. suncei (
The yeast powder and sucrose were mixed in 25 mL of distilled water in a beaker while heated in a hot water bath at 52 °C and stirred until dissolved. Then the agar, inositol, Wechsler salt, multivitamin, cholesterol, ascorbic acid, potassium sorbate, and methylparaben were dissolved, adding 15 mL of distilled water in the beaker under the same condition. The wheat germ powder, microcrystalline cellulose, bark and phloem powder, and 10 mL of distilled water were then added to the solution and stirred. The mixture was fluffy in the beaker. The final mixture in the beaker was wrapped, sealed, and stored at 4 °C in the refrigerator for the beetle rearing test. The artificial diet was not solid in the fridge.
Using a chisel, eggs were collected from excavated galleries in American sweetgum logs. Eggs were transferred gently to a sheet of black filter paper in glass Petri dishes (100 × 20 mm) using a moist sable brush (size 000). In total, 394 eggs were collected for the artificial diet test. In an attempt to minimize contamination from the original galleries, all eggs were soaked in 75% alcohol for 10 s in glass Petri dishes (100 × 20 mm) using a sable brush (size 000), and rinsed with sterile water in glass Petri dishes for 10 s. All eggs were sterilized one by one. They were individually placed at the bottom of 1.5 mL Eppendorf tubes which were then filled with 1 mL of the artificial diet for the growth of A. suncei larvae. When the artificial diet was heated to 25 °C, the artificial diet could be used for rearing. We used a spoon to fill Eppendorf tubes with artificial diet and squeezed the artificial diet with a glass stirring rod. Once the tubes were filled, they were all kept under the same conditions and left until the adults emerged.
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