2.4. Proboscis Extension Reflex (PER) Test

To investigate the effect of a sublethal dosage of fluvalinate on honey bees, their larvae were treated as described above but with different concentrations of fluvalinate, from 0.0001 to 1 mg/L, equivalent to a total of 0.0004 to 4 ng of fluvalinate received by each larva. Newly emerged honey bees were labeled with different colors on the scutum of the mid-thorax and then put back into a normal colony to experience the same socialization. After 15 days, the honey bees were collected into a plastic box until processing for the experiment. The numbers of adult bees collected in each experimental group were as follows: 30 in the blank unfed group, 30 in the 0.1% DMSO group, 37 in the 4 ng/larva group, 37 in the 0.4 ng/larva group, 25 in the 0.04 ng/larva group, 26 in the 0.004 ng/larva group, and 30 in the 0.0004 ng/larva group. Conventional conditioning of the PER was performed to test the olfactory associative behavior of fluvalinate-treated honey bees. Honey bees were placed in a dark incubator at 25 °C and 40% relative humidity, where they were starved for 4 h before the test. At the third hour of the starving period, the honey bees were anesthetized by being placed in a 4 °C ice bucket for 5 to 10 min. After anesthesia, they were fixed onto 1000 μL pipette tips by a beeswax/resin mixture and left for 1 h until their physiological condition recovered. Cotton swabs dipped in 50% (w/v) sucrose solution were applied to the honey bees’ antennae. Honey bees with a normal PER were tested for their olfactory associative behavior, whereas those that could not produce a PER response were eliminated. Approximately 30 honey bees per group were tested for their olfactory associative behavior after various treatments. The PER training method of Yang et al. [37] was used. The test for PER using odorous stimulation was only conducted once the honey bees associated this stimulation with sugar water. Each honey bee went through 4 associative tests (conditioning 1–4) and the tests were held 20 min apart.