1. Please state project objectives and what work was done this quarter to address them: Objective 1: Evaluate currently available registered insecticides in Florida citrus against DRW In this quarter, we have continued to evaluate the use of Btt against Diaprepes larvae. A laboratory experiment was conducted in preparation for field experiments to determine the effects of concomitant infection of insects by Btt and entomopathogenic nematodes. To optimize conditions, DRW larvae of two ages (6 or 12 wk old) were fed on artificial diet inoculated with either of three different concentrations of Bacillus thuringiensis subsp. tenebrionis (Novodor 3% [AI], [30 mg spores and d-endotoxin crystals per ml product]) (0, 300, or 3000 ppm) for 10 or 21 days. After Btt feeding exposure, beetles were transferred individually to soil, which was inoculated with the entomopathogenic nematode (EPN), Steinernema riobrave, at a rate of 15 injective juveniles (IJs) /cm2. The mortality of 6- or 12-week-old DRW larvae was recorded after three days of exposure to the EPNs based on symptoms of infection, which are presented in Fig. 1. Cadavers of dead DRW larvae were transferred individually to White traps to confirm infection with EPN IJs by detecting emergence of nematodes from killed larvae.No mortality of DRW larvae was observed in the control. Mortality of DRW larvae that were pre-exposed to Btt at both concentrations evaluated (75-87%) was higher than that of larvae exposed to diet free of Bt (54%). There was a trend suggesting that larvae exposed at the higher rate of Bt exhibited more specific symptoms of nematode infection than larvae exposed to the lower rate of Bt. EPN infection of DRW larvae exposed to the higher rate of Btt was higher after 10 (89%) than 21 (72%) days of exposure to Btt. Six-week-old larvae were much more susceptible to EPN than 12-week-old larvae, but are difficult to evaluate under in field conditions due to their small size. Therefore, based on the results of this laboratory investigation, we will expose 12-week-old DRW larvae to Btt at a concentration of 3000 ppm concentration for 10 days in upcoming field trials.Also, nematode symptoms and the presence of infective juveniles in the White traps indicated that larvae infected with Bt provided better conditions for nematode population growth compared to control insects that were not exposed to Btt. More IJs were produced in larvae exposed to 300 ppm of Btt than 3000 ppm Bt. Overall, our results indicate that exposure of DRW larvae to Btt renders the beetle larvae more susceptible to EPN infection. These results suggest that combining Btt with EPN may be more effective for EPM management than either factor alone. Furthermore, the results suggest that the use of Btt against DRW larvae may render beetle larvae more susceptible to naturally occurring EPN in citrus groves. Previous research identified a potential trophic cascade affecting the abundance of Diaprepes root weevil in groves. Elevated soil pH is necessary for the adherence of bacterial endospores of Paenibacillus sp. JF317562 to the cuticle of the native entomopathogenic nematode Steinernema diaprepesi (El-Bori et al., 2005). Large numbers of spores on its cuticle inhibits the ability of the nematode to move through soil and infect DRW larvae. Therefore, the hypothesis is that reducing the soil pH can reduce the number of spores attached to the nematode and thereby increase the infection of weevil larvae resulting in less damage to trees. To study this possibility, preliminary trials using several Paenibacillus species showed that molecular primer-probe sets provided a means of measuring the Paenibacillus sp. JF317562 and S. diaprepesi in soil samples. However, the specificity of the molecular tools remained unproven for species that were not initially tested. In our present survey we detected 126 amplicon sequence variants (ASV) ascribed to at least 55 species of Paenibacillus in the grove, including Paenibacillus sp. JF317562. We also detected more than 50 species of nematodes. The spatial distribution of the Paenibacillus sp. JF317562 ASV was significantly correlated only with those of S. diaprepesi and one other nematode symbiont of gall forming insects. None of the other Paenibacillus ASV were associated with S. diaprepesi. The results confirm that 1) the molecular tool is highly specific for Paenibacillus sp. JF317562 and 2) Paenibacillus sp. JF317562 attaches almost exclusively to S. diaprepesi or perhaps other nematode species associated with insects. Objective 2. Determine the source of DRW infestation and how their dispersal affects management decisions.Diaprepes adults became active in late June 2024, therefore we will report on data from these studies in our next report. 2. Please state what work is anticipated for next quarter: -We will continue to evaluate Btt formulations in the lab and also move into field trials to determine field efficacy. -Mark-recapture studies have begin that will be reported on in the next quarterly report. 3. Please state budget status (underspend or overspend, and why): We were underspent in year 1 due to unforeseen challenges with implementing planned work due to delayed Diaprepes field activity. In the second year of funding, we moving towards being on track with our budget. 4. Please show all potential commercialization products resulting from this research, and the status of each: N/A