The following progress has been made toward Objective 2 of this proposal: -Objective 2: An insecticide resistance management protocol will be 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. Thiamethoxam (Platinum 75 SG) has been one of the most overused neonicotinoids for Asian citrus psyllid (ACP) management. Among insecticides currently in use for Asian citrus psyllid, resistance is greatest for neonicotinoids, like thiamethoxam. First, we investigated the possible mechanisms involved in thiamethoxam resistance and the stability of thiamethoxam resistance in the laboratory by establishing populations of field collected resistant ACP. Second, we investgated how to reverse a resistant population back to susceptibility in the field by implementing rotation schedules. Investigations were conducted in areas that were previously treated with consecutive neonicotinoid applications and where the resistance ratio (RR) to thiamethoxam reached between 1266.20 and 1395.00 fold compared with a susceptable population. Finally, we developed a protocol that can be implemented in the field to reverse resistance to thiamethoxam in areas where it is found. First, RNA was extracted using a total RNeasy mini prep kit from adult ACP collected from specific areas where resistance to thiamethoxam was present at two locations: Lake Alfred and Wauchula, FL. A laboratory susceptible population was used as a control for comparison. For each population, ten tubes containing ten ACP adults were used for RNA extractions. The quality of RNA from each sample was assessed on a Nano Drop 2000 Spectrophotometer using A260/A280 ratios, at approximately 2.0. Thereafter, 500 ng of total RNA from the two insecticide resistant populations and the laboratory susceptible population were used for cDNA synthesis. Relative expression of three CYP4, three GST, and one EST gene(s) in each population was compared by qPCR using SYBR Green Power Up PCR master mix in an ABI 7500 Real Time PCR system. The production of the gene specific product and absence of primer dimers was verified by 1% agarose electrophoresis in Tris-acetate EDTA buffer with gel red. qPCR was performed in a 15 µL reaction volume containing 7.50 µL of SYBR green PCR master mix, 1 µl of cDNA, 0.45 µL of each forward and reverse primers and 5.60 µL of nuclease free water. Amplification cycles consisted of an initial denaturing step at 95°C for 10 min, followed by 40 cycles of 95°C for the 30s, 60°C for 30s and 72°C for 30s, and final melting curve step. Three biological replicates were performed for each gene. Actin was used as a reference gene, and to normalize changes in specific gene expression to the ACP laboratory colony. Transcription levels of three CYP, three GST, and one EST genes that are potentially involved in metabolic resistance to insecticides were compared between the laboratory and field selected strains. Second, recovery to susceptibility was determined for two ACP populations where thiamethoxam resistance was present by quantifying the LC50 value of every generation after constant selection for neonicotinoid resistance was ceased. The treatments (rotational schemes) were: dimethoate followed by cyantraniliprole, fenpropathrin, and diflubenzuron (Rotation A) and fenpropathrin followed by dimethoate, cyantraniliprole, and imidacloprid (Rotation B). The third treatment consisted of thiamethoxam followed by clothianidin, thiamethoxam, and imidacloprid (no mode of action rotation). After week 13, the order in rotation A was thiamethoxam, spinetoram, fenpropathrin and abamectin+thiamethoxam. In rotation B, the order was diflubenzuron, dimethoate, abamectin+thiamethoxam, fenpropathrin, and spinetoram. The third area where only neonicotinoids had been applied, received a recovery order consisting of: fenpropathrin, dimethoate, spinetoram, cyantraniliprole, and diflubenzuron. Insecticides were diluted in water at maximal label rates and applied by air-blast sprayer delivering approximately of 100 g of carrier/acre. Adult ACP were collected in the field for the Lake Alfred and Wauchula sites and placed into 60 × 60 × 90 cm insect-proof cages on four citrus plants and maintained in a greenhouse under the rearing conditions. The initial population in each cage contained at least 400 adult ACP from each no rotation treatment and location. In the field, we modified the rotation schedule to cease neonicotinoid applications in plots that indicated high levels of neonicotinoid resistance. After each application, the toxicity of thiamethoxam to ACP was determined to assess how levels of resistance changed over time. The resistance ratios decreased from 1266.20 to 21. 57 and from 1395.00 to 28.71 after four applications of the recovery mode of action rotation order at Lake Alfred and Wauchula, respectively. Under laboratory conditions, the resistance ratios decreased from 1266. 20 to 28.86 and 1395 to 36.71 for Lake Alfred and Wauchula populations, respectively, after five generations of no insecticide exposure. The qPCR analysis showed that expression of CYP4C67 was significantly increased in both resistant populations relative to the laboratory susceptible population. We also showed that resistance in ACP to thiamethoxam declined significantly in the absence of selection pressure under laboratory conditions and rotation application in field. Both laboratory and field investigations indicated susceptibility to thiamethoxam fully recovered after five generations. Three main mechanisms of insecticide resistance typical among insects are: metabolic detoxification, target site modification, and reduced penetration/increased excretion. Previous studies reported five cytochrome P450s (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1, and CYP4G70) analyzed in this investigation that are known to be upregulated after ACP obtain sub-lethal dosages of imidacloprid. In addition, CYP4 genes have been previously implicated in ACP resistance to imidacloprid. There are several cases where up-regulation of one or several detoxification enzyme genes has been attributed to neonicotinoid insecticide resistance or insecticide detoxification among insects. In the present investigation, populations of ACP exhibiting high neonicotinoid resistance were associated with upregulated expression of CYP4C67, implicating the gene product in neonicotinoid resistance in ACP. We compared the expression of CYP, GST and EST genes between known neonicotinoid resistant and susceptible ACP. Similar to the trend in CYP4 expression, GST and EST genes generally exhibited overexpression in insecticide resistant ACP as compared to susceptible counterparts, although these differences were not always statistically significant. Our findings suggest that populations of ACP that exhibited resistance to neonicotinoids in Florida also showed evidence of overexpression of genes implicated in metabolic detoxification. Further research is necessary to determine whether these differences among field populations of ACP have become genetically fixed. Our current focus is on developing an RNA-seq based database to further understand the mechanism(s) underlying thiamethoxam resistance in field selected populations of ACP. Our results revealed that ACP populations develop high levels of resistance to thiamethoxam under continuous selection by label rate applications in cultivated citrus. A high level of resistance occurred following only 3-4 consecutive neonicotinoid sprays and within five egg to adult generations and was associated with subsequent product failure. We also showed that resistance in ACP to thiamethoxam declined significantly in the absence of selection pressure under laboratory conditions and when modes of action rotation was implemented under field conditions. Recovery to a susceptable state under rotation in the field was more rapid than under no selection in the laboratory population. These results suggest that thiamethoxam resistance is likely unstable under the field conditions. Collectively, our results indicate that rotation of thiamethoxam with insecticides from other chemical classes, including cyantraniliprole, fenpropathrin, dimethoate, spinetoram and diflubenzuron effectively mitigates neonicotinoid resistance in areas where ACP are managed with insecticides.