1. Please state project objectives and what work was done this quarter to address them:Develop monitoring tools to time management actionsWe have previously reported that captures of N. viridis in cardboard band traps are increased in areas of citrus trees that are mechanically injured as compared with similar but uninjured citrus branches. We subsequently identified volatiles (.-terpinene, citronellal, citronellyl acetate, ß-E-farnesene, a-humulene, and a-E-E-farnesene) that are upregulated in response to damage and confirmed attraction of N. viridis to these volatiles associated with mechanical damage of citrus. Recently, we documented increased captures of various life stages of N. viridis in cardboard band traps baited with a 10 µg/µL concentration of farnesene: ocimene: sabinene blend (in 7:13:17 ratio), as well as those releasing either farnesene or ocimene alone at this same concentration, as compared with the mineral oil (diluent) negative control. These volatiles could be useful for development of an effective monitoring trap for N. viridis, or other control tools. Our current goal is to optimize dosage and blend ratio of volatile components to further increase mealybug captures. We have been attempting to evaluate traps baited with the farnesene: ocimene: sabinene 3-component blend as well as farnesene or ocimene alone at a series of loading concentrations ranging between the 10 µg/µL shown to be attractive previously as compared with higher concentrations of 100 and 1,000 µg/µL. Our hypothesis is that increasing the loading concentrations of volatiles used bait traps will increase captures of mealybugs on traps in the field. Our objective is to conduct the experiments under authentic field conditions because we have already determined which volatiles are involved in attracting the mealybug under laboratory conditions and the investigation has moved toward development of practical tools in the field. The experiment employs cardboard band traps (CCBTs) that are deployed around three branches and trunks of each replicate infested citrus tree. A designated volatile treatment is enclosed in a release capsule, which is then attached to each CCBT. Mealybug captures on traps are recorded over time to determine if volatile treatments affect captures of mealybugs as compared with unbaited controls. Unfortunately, during our previous two attempts in the last quarter to conduct the experiment under field conditions, we were unable to collect reliable mealybug catch data because plots were overprayed with insecticides for management purposes. These management sprays crashed mealybug populations and prevented us from collecting reliable data. Although we will continue attempting to conduct the experiment at this same location, where mealybug populations have been historically high, we have also identified a new site as a backup that is infested with N. viridis mealybugs and that will not be receiving sprays. Therefore, we are confident that we will be able to conduct the above-described experiment and finish this investigation in the next quarter by evaluating higher loading concentrations of our volatile baits. Describe the feeding interactions of lebbeck mealybug with citrus treesWe are continuing to make progress in collecting data on these feeding interactions and are in the process of analyzing the baseline feeding interactions trials Measuring insecticide efficacy and residues on leaves from Citrus Under Protective Screen (CUPS) compared to open groves.For this project we are collaborating with two growers who managed Citrus Under Protective Screen (CUPS): CUPS Chesire and CUPS Groveland. The objective of this study is to replicate insecticidal sprays made in CUPS in an open-air plot located at CREC and compare the residual activity of these sprays between the two systems as well as their efficacy in controlling the hibiscus mealybug. For each insecticide application, we followed the protocol below:Since May 2024, we were able to follow two sprays. Growers used their own materials to spray their CUPS while we used a hand-gun sprayer (PCO Skid Sprayer MCCI100K43HR1M, Chemical Containers) with pump pressure set at 200 psi delivering 99.64 gal/acre to spray the open-air plot. The tank was triple-rinsed between applications.We collected leaf samples the day before the spray as well as 1,3,5 7 and 14 DAT after the spray. One set of leaves were frozen on the day of their collection at -20°C and processed for leaf residues through Ultra High-Performance Liquid Chromatography – Mass Spectrometry (UHPLC-MS). Samples were run against standards to construct a five-point linear curve in a concentration range of 0.5-50 ppm, and then against a five-point standard curve in the range of 5-300 ppb. The concentration represented by the curve was then converted back to µg/g leaf tissue using the exact sample weight.The second set of leaves was used to assess mealybug mortality. Leaf petioles were put in a 1.5 ml Eppendorf tube containing deionized water and secured with parafilm. Leaves were placed into individual Petri dishes and ten 2nd to 3rd instar mealybugs were transferred onto the upper surface of the leaves using a camel hair brush. Petri dishes were sealed with Parafilm and held in a growth chamber under controlled conditions (28 ± 2°C; 70 -80% RH and 12:12 (L:D) h photoperiod). Mortality was assessed under a stereomicroscope by gently probing mealybugs with a fine brush on days 1, 3, 5, 7, and 10 after samples were taken from the field. Only leaf samples from spray #1 have been processed for pesticide residues so far. Small concentrations of spirotetramat were detected before the application was done in CUPS probably due to a previous spray of Movento on March 21st. Spirotetramat was not detected in the open-air grove before the spray. In CUPS, concentrations of spirotetramat reached 7 µg/g leaf tissue 1 and 3 days after the application and decreased to 1.7 µg/g leaf tissue 14 days post-treatment. In the open grove, spirotetramat concentration reached 4.1 one day after treatment but almost nothing was detected 3 days after treatment. 14 days after application spirotetramat concentration was 0.76 µg/g leaf tissue. We noticed that spirotetramat concentration is systematically lower in open grove than in CUPS. Further sprays need to be evaluated to confirm this trend.Survey of natural enemies for N. viridisThe first half of this year’s samples for this objective have been collected. So far, zero parasitoids have emerged from sentinel hosts. 628 Hymenoptera specimens (excluding ants) were found in the yellow pan traps and 272 specimens (43%) were identified as potential parasitoids. Chalcidoidea was the second most frequently detected superfamily. This superfamily contains many potential mealybug parasitoids. The specimens will be identified molecularly and morphologically over the coming months. This experiment will be repeated and expanded in the fall. Rearing of parasitoids of N. viridisThis experiment is still ongoing. We will report our findings in the next quarterly update. 2. Please state what work is anticipated for next quarter:For the upcoming quarter, we will be continuing to work on all objectives outlined in this report from the previous quarter. 3. Please state budget status (underspend or overspend, and why): We are currently on track with our budget 4. Please show all potential commercialization products resulting from this research, and the status of each: Lures may be commercializable if found to be reliable indicators of mealybug activity