2.3. Pre-Exposure to Herbicides and Bioassays

Since insects could be exposed to residual herbicides by feeding on host plants or by olfactory perception from their surroundings, we treated caterpillars with either volatile herbicides or diet containing herbicides to simulate the natural field conditions. Newly molted fifth-instar caterpillars of H. armigera were fed on a control diet (containing no allelochemicals) exposed to 1 μg volatile herbicides butachlor (BuCh) or haloxyfop-methyl (HLFM), or fed on diet containing 0.5 mg/g herbicides BuCh or HLFM exposing to fresh air for 48 h, and then accumulative mortalities were recorded 60, 120, 180, 240, 300, 360, and 720 min after treated with methomyl (50 μg per caterpillar) and the final mortalities were recorded at 1440 min. The control caterpillars were fed on control diet exposed to fresh air in the volatile induction experiment, and fed on diet containing 0.5 mg/g solvent sterilized water in the feeding experiment. In the synergism analysis, 3 μL cytochrome P450 inhibitor piperonyl butoxide (PBO) was delivered on to the prothorax notum of each caterpillar 1 h before the application of insecticide. Twenty synchronous fifth-instar caterpillars were used for each treatment, and three independent replicates were performed. For toxicity bioassay of carcinogenic mycotoxin aflatoxin B1 (AFB1), third-instar caterpillars were pre-exposed to BuCh or HLFM for 48 h, as mentioned above, and the caterpillars were fed on diet containing 2.5 μg/g AFB1. After that, the weight gain from the third-instar to sixth-instar caterpillars and final mortalities were recorded. Twenty synchronous third-instar caterpillars were used for each treatment, and three independent replicates were performed.

To investigate the effects of exposing to herbicides on growth and development of H. armigera, third-instar caterpillars were used, and the weight gain from the third-instar to sixth-instar caterpillars, pupal weight and pupation rates were compared between the control group and herbicides exposing group. Twenty synchronous third-instar caterpillars were used for each treatment, and three independent replicates were performed.