Work done this quarter (continued on Word document)(1a) Develop monitoring methods to time management actionsWe have developed methods for following the seasonal populations of lebbeck mealybugs and are working with 7 growers throughout central Florida to monitor populations. Based on data collection through the end of May 2021, there is some consistency in the onset of population development, with mealybug populations increasing in March and April concurrent with early fruit development. Lebbeck mealybugs appear to be highly attracted to developing fruit and establish populations around the calyx at fruit set. In our study and in a fruit drop study by a colleague, we are seeing high levels of fruit drop related to the mealybugs not related to physiological fruit drop. While we are not quantifying it in our phenology study, Dr. Vashisth has added this quantification to her fruit drop trials.Along with population monitoring, we are evaluating the potential use of virgin females as a lure for males with the intention of using these data to develop a targeted lure in the future to help reduce field populations and detect cryptic populations before they increase to damaging populations. No data yet, this method is in the trouble shooting stage.(1b) Adjuvant screeningPreliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 10 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with mealybugs attached. 9 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control. (1c) Evaluate promising materials in open grove setting: We began a field trial at a 10-acre commercial citrus planting that was heavily infested in 2020. First insecticide applications occurred as a pre-bloom prophylactic spray of a systemic material, with a second spray planned in the end of April.(1d) Ant Management We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Four experimental treatments were tested and compared to an untreated control: Chlorpyrifos drench, Clinch ant bait (Abamectin), Extinguish ant bait (S-methoprene), and spot treating ant colonies with hot water. Fire ant abundance was assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with pecan sandie baits. Natural enemy abundance was assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods took place every 2 weeks.Currently, all treatments have been applied and we are conducting follow-up surveys to determine their efficacy. Preliminary data shows that both insecticidal baits and hot water treatments significantly reduced fire ant colony abundance, while the chlorpyrifos drench had no effect on colony abundance.Impacts on mealybug predator population development is currently being quantified now that populations are starting to establish.(1e) Evaluate management options for IPCsEntomopathenognic Fungi (EPF) data shared last quarter. More work to come.(2a) Predator assessmentsPredators who directly consume prey:The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, Hippodamia convergens, and Chrysoperla carnea have been screened using no-choice assays to determine if they will feed on larval lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught and lab reared predators have also been screened, including Harmonia axyridis, larva of the genus Ceraeochrysa (colloquially called trash bugs), Diomus austrinus, and Coccinella septempunctata. Both adult and larval C. montrouzieri readily feed on lebbeck mealybug larvae and ovisacs, as do larval Ceraeochrysa. Larval Chrysoperla carnea and adult Diomus austrinus consistently consume mealybug larvae, but do not consume ovisacs. Adult Orius insidiosus, early instar Adalia bipunctata, and adult and larval Harmonia axyridis do not feed on lebbeck mealybug larvae or ovisacs. Adult Hippodamia convergens, Coccinella septempunctata, and Adalia bipunctata do feed on some lebbeck mealybug larvae, but appear to attack and kill the larvae more than fully consuming them. Neither feed on ovisacs. From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae may act as natural biological control in the grove. Chrysoperla carnea may be useful for controlling mealybug larvae, but not reproductive adult females.Parasitoids:Limited numbers of Anagyrus pseudococci, a commonly used parasitoid for mealybugs in greenhouses, were tested to determine if they would parasitize lebbeck mealybug adults and ovisacs. A. pseudococci adults were placed in arenas with adult lebbeck mealybugs, or ovisacs and left for 40-48 hrs. Surviving parasitoids were then transferred to adult Citrus mealybugs and given an opportunity to parasitize them, to act as a positive control. Mealybugs were checked for parasitism after 16 days. The majority of parasitoids died in arenas with lebbeck mealybugs, and none parasitized adults or ovisacs. However, half of those that survived went on to parasitize Citrus mealybug adults. These preliminary results suggest that A. pseudococci will not readily parasitize lebbeck mealybug, however further trials with more individuals and life stages of mealybugs are needed.Gut content assays of field-collected potential predatorsSpecies-specific primers for lebbeck mealybugs and a DNA extraction protocol have been established and used in feeding bioassays with the predator mealybug destroyers (Cryptolaemus montrouzieri). Using DNA extracted from the predators after they fed on the mealybug in PCR, we were able to model the length of time that the mealybug DNA fragment associated with our primers is detectable in mealybug destroyers. We found that the lebbeck mealybugs amplicon was detectable in 100% of mealybug destroyers for 4 hours after feeding, in 40% of mealybug destroyers up to 56 hours after feeding and was no longer detectable in mealybug destroyers 60 hours after feeding. Field collections were executed in mealybug infested groves August 2019 through December 2020. Species identified as potential predators of lebbeck mealybugs were selected for DNA extraction and PCR using the lebbeck mealybugs primers. So far we have found that green lacewings have the lowest abundance but the highest rate of positive detection of lebbeck mealybugs DNA. Field-collected mealybug destroyers has the next lowest abundance and the next highest rate of positive detection of lebbeck mealybugs DNA. The most abundant predators are spiders, but which have a far lower rate of positive detection of N. lebbeck mealybugs DNA. The spider which tests positive most frequently is the jumping spider, Hentzia palmarum. Predatory flies in the family Dolichopodidae will also be screened for lebbeck mealybugs DNA using our primers. (2b) Determine how to implement mealybug management concurrent with other pest management programsStarting in July 2021 for field efficacy then planning to test programs in Spring 2022(2c) Determine what insecticide chemistries inhibit feedingWe have completed EPG documentation of lebbeck mealybug to determine wavelength correlations on one host plant and will be starting on a second host plant once plants are voided of insecticides (3-4 month holding process in the greenhouse to). Baseline feeding interaction data are necessary for a minimum of 3 host plants prior to including insecticides to look at feeding inhibition and the ability to kill the adult female in her ovisac. (2d) Develop tools to minimize spreadKilling crawlers with isopropyl alcoholWe tested different concentrations of isopropyl alcohol to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches, and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points. Using steam to kill adult mealybugs and ovisacsAdult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs. Using a steam cabinet on-station, we steam treated adults and ovisacs at 100, 120, and 130 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. At 100 degrees F for all times points, adult mealybug and ovisac mortality was not significantly different than the control. At 120 degrees F for 5 minutes, mortality was also functionally 0%. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes. At 130 degrees F, mortality was 100% for all time points. This week we have begin testing ovisacs embedded in IPCs, which are insulating the ovisacs, requiring higher temperatures and longer time points. 2. Please state what work is anticipated for next quarter:(1a) Seasonal population monitoring will occur at 7 groves for one calendar year. Virgin female lure traps will be deployed once seasonal rains end.(1b) Adjuvant screening will continue to determine optimal adjuvants to work in synergism with insecticide sprays. We plan to take the most effective adjuvants and add them to an insecticide with good, but not great, efficacy on its own.(1c) We will continue treating and scouting the grove we have started a management comparison trial at.(1d) The ant management project will continue throughout the upcoming quarter with the addition of monitoring for predatory insect establishment.(1e) IPC management trials will continue, incorporating conventional materials for management and spray penetration by tractor mounted sprayers (this was delayed from our work plan last quarter by unexpected challenges in establishing infestation).(2a) Working with FDACS, we have permission to deploy sentinel infested materials to screen more broadly for predators that may be present in the system which we missed with haphazard rearing from infested grove sites. We plan to do this starting in late summer 2021, once higher populations have established, which would attract a higher abundance and diversity of predatory insects.(2c) Feeding mechanisms will continue to be worked out using EPG with the future plan to determine if we can interfere with their feeding via chemical intervention leading to reduced offspring production.(2d) We will continue evaluating sanitation options and are planning to look at vehicle & equipment sanitation in the next quarter. 3. Please state budget status (underspend or overspend, and why): on target