1. Please state project objectives and what work was done this quarter to address them: Over the past quarter, we have made good progress towards the evaluation of pesticides including Bt toxins for the management of Diaprepes. The work represented in the Objective 1 portion of this report is from laboratory trials, which form the basis for moving this testing into groves in the future.Objective 1a: Evaluate currently available registered insecticides in Florida citrus against DRW.InsectsDiaprepes abbreviatus larvae were obtained from a culture at University of Florida’s Citrus Research and Education Center (CREC) in Lake Alfred, FL. This culture was periodically supplemented from collections of adult beetles from citrus groves in Florida. Larvae were reared on an artificial diet developed by Beavers (1982) using procedures described by Lapointe and Shapiro (1999). Larvae used in experiments were 3rd instars.Effect of commercial insecticide formulations on the survival of larval Diaprepes in soil. Morality of larval Diaprepes was determined using a soil assay procedure previously established for evaluating insecticide against the ground inhabiting stage of this pest (Hamlen et al. 1979). Candler sand was used as the substrate and was sieved using a 6-inch N.B.S. #20 sieve (pore size, 841 M) to remove larger particulate matter. The soil was autoclaved and allowed to air dry. Afterward, 9 ml of DI water was added back to the soil to reach approximately 12% moisture content. Approximately 25g of soil was then added to bioassay columns to a depth of 3cm. The columns were constructed from 50mL polystyrene tubes (12.0 cm height, 3.0 cm diameter) that were similar to those described previously by Hamlen et al. (1979). The objective of this experiment was to evaluate insecticides and associated application rates used against Asian citrus psyllid (ACP) to determine if they were also effective against the larval stage of Diaprepes.The insecticides tested and associated rates are given in Table 1. Each insecticide formulation was added to DI water to yield concentrations of 0, 0.27, 2.7, 27ppm and field rate (270 ppm). A total of 1.040 ml of each concentration (or treatment) was pipetted uniformly onto the soil in the bioassay columns. Deionized water alone was used as the negative control. Five neonate Diaprepes larvae were scattered on the surface of the soil per replicate bioassay chamber and a total 12 replicate chambers were established per treatment evaluated. Bioassay units were kept in an incubator held at 25 ± 2 ºC, 50±10 % RH, and 14:10 L:D photoperiod. After 48hr, the number of living and dead Diaprepes larvae recovered in the containment cell at the base of the bioassay column was recorded. Also, soil was thoroughly excavated under a stereo microscope to find any remaining larvae in each chamber to determine morality. The relationship between chemical concentration and larval recovery was determined by probit analysis. Mortality data using field rates were analyzed using a generalized linear model (GLM) with binomial distribution followed by Tukey estimated marginal means, using the package emmeans in R for post hoc comparisons at = 0.05.ResultsAll of the insecticides tested caused significant (F = 9.59, P = 0.0002) mortality of Diaprepes larvae in soil as compared with our negative control treatment (Fig. 1). With Exirel, Sivanto, Danitol, and Delegate applied at field rates, we observed 100% mortality of Diaprepes larvae. These results indicate that the larval stage of Diaprepes is susceptible to a wide variety of insecticide modes of action at rates that were commonly used against ACP not long ago. The LC50 value has thus far only been established for thiamethoxam (active ingredient in Platinum 75 SG insecticide). The LC50 for larval Diaprepes is 0.857 ng/µL (95% FL 0.0267-2.66). We are currently establishing LC50 values for the other insecticides that are being evaluated in order to have baseline values that will allow for monitoring of possible changes in insecticide susceptibility among Diaprepes populations exposed to these insecticides over time.Objective 1b: Pathogenicity of Bacillus thuringiensis subsp. tenebrionis (Btt) against Diaprepes abbreviatus Materials and MethodsPlant material, insects and Bt formulationSix-month-old Cleopatra Mandarin (Citrus reshni Hort., ex Tan.) seedlings purchased from a nursery (Zimmerman’s Tropicals Nursery, FL, USA) were used to evaluate the effects of Bacillus thuringiensis subsp. tenebrionis (Btt) on Diaprepes abbreviatus. Seedlings were planted in 9-cm dia. plastic pots filled with 588.75 cm3 soil-based mix (three parts Peat Moss, two parts Coco Peat, one part Perlite, and one part gravel-sand-soil mixture) and acclimated in a growth chamber at 26°C with a photoperiod of 14:10 (L:D) h, for several weeks prior to the experiment. The experimental formulation (CX-2330) of Bt evaluated was provided Certis USA.Adult Diaprepes root weevils were used to assess the potential Bt translocation in citrus seedlings. Insects provided in May 2023 by the Florida Department of Agriculture and Consumer Services (FDACS, Dundee, FL 33838) were maintained in cages in greenhouse at 27 °C with water and flushing foliage from Citrus Cleopatra Mandarin trees (C. reshni Hort., ex Tan.) that was replaced weekly. Forty Diaprepes root weevils were acclimated in the growth chamber, 10 days prior to initiating experiments. Effect of Btt formulation on leaf damage after root drench applicationTo test if Diaprepes root weevils could distinguish between Btt and non-Btt treated plant, a choice feeding test using a paired design was conducted. One untreated control and one Btt-treated plant were simultaneously placed in the same cage. Each plant was treated with 20 mL of either water (control) or a 3000 µg/mL solution applied as a drench to the roots. Each treatment was replicated 9 times. Two mating pairs of Diaprepes root weevil were placed in each cage, with access to water, and allowed to feed for 7 days without replacing the plants. Feeding on leaves was measured as described by Kok et al. (1992, 2008) using a transparent millimeter square grid at 3, 5 and 7 days after the start of the experiment. Data from the two-choice feeding test was subjected to a t-test (SAS 1999-2001). ResultsWhen provided a choice, Diaprepes root weevils distinguished between Bt-treated and non-treated plants (Fig. 2). Plants treated with the Bt tenebrionis formulation lost considerably less leaf tissue compared to control plants after 3 (t Value= 7.68, p <.0001), 5 (t Value= 5.65, p <.0001), and 7 days (t Value= 4.98, p <.0001; t Value= -8.45, p <.0001) (Fig. 3-4). Objective 2. Determine the source of DRW infestation and how their dispersal affects management decisions.Our team has been trapping for Diaprepes in 5 field locations with known population histories since Spring or Fall 2022, depending on site, prior to receiving funding for this proposal. Within fields, both emergence and interception traps are laid out in a gridded design to capture their activity in space within groves. Additionally, because we have a suspicion that some of the population may be migrating into groves from edges, having developed on roots of oak trees and/or other plants in the adjacent forested areas, we have added traps in these areas. Data presented are from one site with a consistent, though low, population.Tedders (intercept) traps indicated a low level of activity from several groves from August 2022 through January 2023 (Fig. 5), however trap captures did not capture a large enough emergence to enable the mark-recapture studies that we plan to use to better understand movement of Diaprepes within the groves and between the groves and the forested edge. Because trap data have been so low, our team has been walking groves to look for damage or Diaprepes presence. If populations follow last year's pattern, we may not see much activity until August this year. Once sufficient activity is noted for the mark-recapture study, we will begin field implementation.From our trapping data through early June 2023 at one site in Wauchula, we see interesting patterns emerging from the Tedders traps that have captured beetles (Fig. 6). From the spatial analysis, we see both an apparent edge effect as seen in previous sampling efforts, however now the most active edge is on a different end of the grove, closer to the woodline than where populations had been noted previously. We also see hotspots emerging in the grove (Fig. 6), reflecting the propensity of Diaprepes to aggregate.(all figures in word document) 2. Please state what work is anticipated for next quarter: Work on both objectives will continue. While much progress has been made, there is still much to learn to develop better management. For objective 1, we will continue working towards developing the LC50 of effective chemistries and to evaluate the potential efficacy of Btt. For objective 2, our team continues to monitor sites weekly and remains at the ready to initiate the mark-recapture study once populations are high enough to return interpretable data. 3. Please state budget status (underspend or overspend, and why): Our team was recently notified that we are underspent, which was a surprise to us. We suspect that is because we have funds reserved in the supplies for the mark-recapture materials, which must be purchased in close proximity to the time in which the study will be performed. Both the costs of the field materials and the lab assay materials are quite high. 4. Please show all potential commercialization products resulting from this research, and the status of each:All products under evaluation are currently available for purchase, though efficacy for Diaprepes was previously unknown. With the loss and potential future loss of known broad-spectrum chemistries that are efficacious, the knowledge generated by this project to help growers manage this pest challenge and support the use of targeted chemistries in groves affected by Diaprepes is crucially important.