Objective 2. Revise insecticide resistance management for psyllid IPM in new plantings An insecticide resistance management protocol is being developed for young tree protection in Florida citrus that: 1) could be deployed in areas where insecticide resistance is already present and reduce ACP populations and 2) would allow return to normal susceptibility levels for insecticides that have been compromised in effectiveness due to resistance. ACP has developed resistance to neonictinoids in some Florida citrus groves. Therefore, we assessed the mechanism(s) conferring resistance in these ACP populations, compared to the highly susceptible laboratory colony of ACP. Knowing the mechanisms allows development of methods to reverse the problem where it exists and prevent it where it has not yet developed. Previously, we determined that resistance to the chemical fenpropathrin (pyrethoroid insecticide) occurs among populations of Asian citrus psyllid (ACP) after continuous exposure to this insecticide for 10 egg to adult generations of ACP. We also conducted a risk assesment for resistance development to this insecticide in Florida, and compared relative expression of the gene CYP6A2-1 between insecticide resistant (RR) populations of ACP and a laboratory susceptible population (SS). Our results indicated that continuous exposure of ACP populations to fenpropathrin can generate high levels of resistance (100 fold increase) and that this is, in part, caused by increased expression of cytochrome P450 (CYP) genes. Cytochrome P450 genes aid in metabolic detoxification and are one of the mechanisms contributing to fenpropathrin resistance in ACP. In our most recent experiments, we used the same populations of ACP that are known to be resistant to fenpropathrin to assess the stability of fenpropathrin resistance in ACP. We compared ACP populations with differing initial frequencies of resistance to understand the reversal process back to susceptability. In addition, we performed molecular analysis of fenpropathrin resistance by targeting the specific receptor that confers fenpropathrin insensitivity and also conducted biochemical analysis to determine how metabolic detoxification of fenpropathrin differs between resistant and susceptable populations of ACP. First, we collected ACP from commercial citrus groves that exhibit resistance to fenpropathrin (RR) and crossed (mated) these psyllids with ACP from the laboratory susceptible population (SS). The ratios of crossed populations that were established were 100RR+0SS, 75RR+25SS, 50RR+50SS, 25RR+75SS and 0RR+100SS. The five populations were set up on March 23, 2020 in a greenhouse environment without exposure to insecticides. Thereafter, we performed bi-monthly leaf dip bioassays assesing insect mortality in response to insecticide exposure to assess the stability of fenpropathrin resisitance over time. The leaf-dip bioassays were performed on June 12 and July 29, 2020 for the five crossed populations that were established. The bioassays determined the level of resistance to fenpropathrin in each population. These laboratory bioassays included 5 to 10 concentrations of each insecticide tested with 3-4 replications per concentration. Mortality counts of the insects were taken 48 h after being transferred into a room under the same environmental conditions used for insect rearing. Our results showed no consistent changes in susceptibility of the established strains that consisted of 100% initially resistant individuals (100RR and 0SS cross) or 100% susceptible individuals (0RR and 100SS strain) after four months of rearing without insecticide exposure. In the resistant population (100RR and 0SS cross), resistance to fenpropathrin remained very high (RR > 100), while sensitivity of the susceptible population (0RR and 100SS strain) did not change over 4 months. However, resistance was not stable in populations of ACP that resulted from cross breeding of 25% resistant (RR) and 75% susceptible (SS) and 50% RR and 50% SS. In the case of the populations that were established with different initial frequencies of resistant ACP, fenpropathrin resistance declined over the course of four months in the absence of selection pressure. These results indicate that if a population of ACP develops resitance to pyrethroids in the field, this resistance can be bred out of the populaton through an influx of susceptible genes, if susceptible psyllids breed with the resistant individuals. Second, we worked to identify the specific knockdown (kdr) gene that confers resistance to fenpropathrin by cloning and sequencing genes from the laboratory population and then comparing them to the field population. For pyrethoids like fenpropathirn, a common mechanism confering resistance is a mutation in the sodium channel caused by differences in the kdr gene. Identification of specific mutations in the sodium channel gene required extracting genomic DNA from ACP samples from the SS population and then conducting PCR on three regions of the sodium channel gene designated as regions highly responsive to amino acid substitutions (V410, V930 and F1530). Primers for reach region were designed 100 base pairs (bp) up and downstream of the predicted substitution site (site of the mutation). PCR products were purified using a universal DNA purified kit, and the purified product was ligated into the pGEMT-easy vector. The recombinant plasmid was cloned into JM109 competent cells. The competent cells were spread on Luria-Bertani (LB) solid medium, selected based on color, and cultured to turbidity in LB broth media. We used gel electroporesis to analize the gene sequence. We found gene regions containing the V410, V930, and F1530 positions, and the specific amino acids containing substitutions within the kdr gene were amplified in the susceptible (SS) population. The results suggest that fenpropathrin resistance is likely unstable under field conditions, primarily due to the presence of localized susceptible ACP populations throughout Florida that periodically interbreed with those populations of ACP that develop resistance to fenpropathrin when sprays are not appropriately rotated. Also, the gene regions of the sodium channel that contain the V419, V930, and F1530 positions may have a role in resistance to pyrethroids in ACP. In future studies, we will continue to monitor insecticide resistance in populations of ACP in Florida citrus groves, as well as, the expression of the CYP6A2-1 gene using laboratory and field selected populations. Also, we will extract genomic DNA from 20 individuals from all five cross-resistance and susceptible populations after six or eight-months of monitoring insecticide resistance levels. While analyzing the three regions (V410, V930, and F1530) of the sodium channel gene that undergo mutation, we will determine how the mutations lead to fenpropathrin resistance. The ultimate goal is to understand how the mechanisms of resistance [metabolic totoxification vs. target site (sodium channel)] insensitivity interplay to confer stability of fenpropathrin resistance in ACP. If we can figure out how each mechanism or the combination of the two influences stability of resistance to this very important class of insecticides, we could then more effectively destabilize it, so that it can be quickly reversed in cases where resistance to pyrethroids shows up in the field.