Objective 1: Compare the effectiveness of biological control in commercial orchards in FL and determine if abiotic factors or management practices influence effectiveness. Use this knowledge to improve the effect of biological control. We have continued to investigate the contribution of natural enemies on population regulation of Asian citrus psyllid (ACP) by measuring psyllid mortality of psyllids in response to natural enemies, where sentinel psyllids were deployed monthly on tree branches with or without screen exclusion cages. During the previous quarter, we have been able to process and analyze abundant data collected during previous quarters. We found that the mortality of uncaged ACP, where natural enemies had access to flush with eggs and nymphs, was higher than of caged ACP at organic sites during all seasons analyzed i.e. Summer, Spring and Fall. These results demonstrated that natural enemies reduced ACP population in organic management groves with no chemical input, which is consistent with previous investigations employing exclusion cages to measure the impact of biological control. As anticipated, psyllid mortality was not different between caged and uncaged sentinel psyllids in conventionally managed groves that received sprays of neurotoxins during the majority of our monitoring period. These results suggest that natural enemies may have contributed to regulation of ACP populations in organic groves more so than under intermittent use of conventional insecticides. We measured populations of ACP under both management regimes and recorded more ACP in areas treated intermittently with conventional insecticides than in organic blocks, suggesting that the level of insecticide input was insufficient to adequately reduce ACP populations. Furthermore, populations of natural enemies densities may have been impacted by the intermittent insecticide applications made in the conventional groves. Natural enemies population were similar in groves sprayed intermittently with conventional insecticides vs. groves managed organically and without use of chemical inputs for ACP. In both management groves, we found six different natural enemies including predators such as ladybeetles (Coccinellidae), syrphid flies (Syrphidae), green lace wings (Chrysopidae), long-legged flies (Dolichopidae), and spiders (Aranae) and parasitic wasp T. radiata (Eulophidae). Among these natural enemies, spiders were the dominant group of predators observed in both management groves. The mortality of ACP in an open cages in the exclusion experiment was higher even though other natural enemies were relatively fewer which may have been caused by abundant spiders. Another dominant natural enemy observed were Dolichopodids, which are predaceous; these especially feed on adult psyllids. The predatory Dolichopodid flies were found in higher numbers after spiders in both management sites. Ladybeetles are an important predator that contributes to mortality of ACP and were the third most frequently observed natural enemy in this study. During our study period, the abundance of ladybeetles was also not affected by the management regime. Tamarixa radiata is one of the key natural enemies of ACP, but we found very few wasps in both organic and conventional groves and occurrence of parasitism was rare. In addition to parasitism, female T. radiata also kill a proportion of psyllid nymphs by host feeding. The low numbers of Tamarixia could be due to indirect effects of predators such as Coccinellidae and Dolichopodidae. In previous work conducted in Central Florida, T. radiata was found to suffer mortality (64-95%) due to predation of parasitized nymphs by ladybeetles. Dolichopodids also compete for resources with Tamarixia and eat them when encountered during their searching behavior. Objective 2. Revise insecticide resistance management for psyllid IPM in new plantings 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. ACP has developed resistance to neonictinoids in some groves in 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. In the current study, we took three genotypes (groups where individuals within groups are genetically similar, but where the groups differ from one another) of ACP with varying levels of resistance and susceptibility to neonictinoids, and comparatively investigated them. Specifically, we performed a detailed transcriptional analysis comparing all of the ACP genes between resistant and susceptible populations. By figuring out which of these differences are important to development of resistance, we can better understand how the resistant populations changed because of resistance development and therefore what we need to do to reverse it. First, we collected psyllids from these areas, extracted their DNA, and conducted a data analysis called RNA-seq. We identified what are called differentially expressed genes (DEGs) between the resistant populations and our susceptable baseline (‘wild type’) population. These DEGs indicate differences between the resistant and susceptable populations. We found a total of 388 DEGs that distinguish the resistant Lake Alfred population from a known susceptable ACP population and 368 DEGs that distinguish the Wachula resistant psyllids from the susceptible population. After identifying the differences between resistant and susceptable populations (the DEGs), we determinted the molecular function of the DEGs using a Web Gene Ontology Annotation Plot. The Gene Ontology (GO) classifications were used to predict and identify the specific functions for the identified DEGs. In other words, we determined what type(s) of protein differences are produced by the genetic differences between susceptable and resistant ACP. This tells us what the genes do for the insect. Three hundred genes were annotated in this manner for the Lake Alfred population and 290 DEGs were assigned a function for the Wauchula insecticide resistant population. The cellular process, including cell and catalytic activity terms contained 120, 94, and 61 DEGs in Lake Alfred and were dominant in each of the three main categories such as biological process, cellular component, and molecular function. Likewise, 104, 84, and 59 DEGs were assigned to cellular proces, cell, and catalytic activity in the Wauchula population. The functions of some genes involved in metabolic processing, immune system processing, enzyme regulator activity, receptor activity, and catalytic activity were specifically related to expression of insecticide resistance development in ACP. Third, we conducted a procedure called functional enrichment analysis to identify which DEGs were significantly enriched, statistically, in the gene ontology GO using the Kyoto Encyclopedia of Gene and Genomes (KEGG). We did this to visualize the functional involvement of DEGs (differences between resistant and susceptable ACP) in various biological pathways in psyllids. Pairwise comparisons (Lake Alfred vs Wauchula) of the KEGG annotated proteins revealed two proteins absent in Lake Alfred, namely purine-nucleoside phosphorylase, and nicotinamide mononucleotide adenylyltransferase which are critical for the insects ability to metabolize (break down) nicotinates and nicotinamides (like neonicotinoid insecticides). In addition, three proteins that are in the cytochrome P450 family, including glutathione S-transferase and carbonyl reductase 1, were found present in high levels the Lake Alfred populations. In insects, these genes are specifically responsible for metabolism of xenobiotics, including insecticides. For the Wachula population, we identified one glutathione S-transferase that distinguishes this population from normal susceptable ACP populations. Glutathione S-transferases are known specific proteins in insects that break down insecticides, including pyrethroids. To summarize, we identified very specific differences between two populations of ACP in Florida that show significant resistance to several insecticides as compared with wild type susceptable ACP. We know that some of these genes are specific to neonicotinoids, which is congruent with our findings that neonicotinoids are most often the mode of action exhibiting significant resistance, but also found that more general detoxifying enzyme groups are more active in resistant psyllids. These results suggest that our current recommendations for rotation of five modes of action should continue working in Florida as a method for reversing and preventing resistance and that the situation does not appear to have worsened. However, the finding that the majority of the transcripts that we identified in this investigation are novel, and have not been previously implicated in insecticide resistance mechanisms in ACP and other insects, are surprising. Our new results suggests that resistance development to neonicotinoids, such as thiamethoxam, in ACP is more complex than previously described. Our new results implicate the participation of a broad array of novel resistance genes and possible resistance mechanisms working in concert. We continue conducting more detailed quantification of expression of the selected genes we have identified for the first time. These results will be incorporated into our insecticide rotations protocols, and if they improve resistance management, they will be incorporated into our spray recommendations for FL citrus.