ENTOMOLOGY Identification of Optimization of Sex Pheromones of Lygus hesperus as Practical Lures for Pheromone-baited Traps or use in Control Programs Dr. Kent M. Daane, Robert K. Straser and Noemi Fonseca 137 Mulford Hall Dept. of Environmental Science, Policy and Management University of California Berkeley, CA 94720-3114 [email protected] (559) 646-6522 Dr. David Hall and Dudley Farman Natural Resources Institute Greenwich University England [email protected] Tel +1634 883207 Dr. Jocelyn Millar, Jacquie Serrano, and J. Steven McElfresh Dept. of Entomology UC Riverside Riverside, CA [email protected] (951) 827-5821 Dr. Rodrigo Krugner USDA -ARS Crop Diseases, Pests and Genetics Research Parlier, CA 93648 [email protected] (559) 596-2887 SUMMARY Lygus bugs are serious pests in California strawberry-growing areas. Lygus hesperus is the key Lygus species in strawberries, although Lygus shulli is also present in coastal regions and Lygus elisus in the interior valley. When lygus feeds, it punctures individual seeds, which stops development of the berry near the feeding site and causes irregularly shaped, cat-faced strawberries. Lygus can feed on numerous plant species, which complicates management programs because adults can move into the strawberry field from nearby refuges. 79 2016 RESEARCH PROJECTS Therefore, management of Lygus includes control of weed hosts and monitoring for the appearance of lygus nymphs or adults on nearby alternate host plants as well as strawberries. Insecticide(s) must then be timed to control lygus before they cause significant damage. Currently, sweep nets are often utilized to monitor lygus population presence and density; however, sweep nets are not an effective sampling tool at low lygus densities or when they first move into the strawberry field. Therefore, an effective pheromone-baited trap program that could detect early movement of lygus into strawberries or other susceptible crops would be an important advance. We sought to identify a pheromone blend attractive to L. hesperus. In 2014, we developed pheromonal attractants for L. elisus and in 2015, we identified most of the compounds needed for L. hesperus. In 2015, we refined the L. hesperus pheromone blend. Pheromone was collected from sexually mature lygus and then analyzed by coupled gas chromatography-mass spectrometry (GC-MS). Compounds were identified by matching their mass spectra with database spectra and then confirmed by matching mass spectra and GC retention times on two columns with those of authentic standards. The average blend ratio of likely pheromone components was formulated and then loaded into custom-made dispensers for field-testing in untreated alfalfa fields at the Kearney Agricultural Research and Extension Center, Parlier, CA, where there are abundant lygus. In 2016, we tested two blends; however, although we believe that we have a good identification of the pheromone – we have been less successful at gettingL. hesperus into the traps. This is a stumbling block that keeps this project from moving from basic to applied research. In 2017, we did not request additional funding, but we continued to look at Lygus trapping to determine L. hesperus flight or mating behaviors that might hamper their collection in traps, and re-examine other chemical cues that might be missing from our pheromone formulation. INTRODUCTION The mirid bugs L. hesperus and L. elisus are key pests of many crops, including strawberry (Zalom et al., 2014). Feeding by both nymph and adult lygus bugs causes irregularly shaped cat-faced strawberries (Allen and Gaede, 1963). A key issue in L. hesperus management is the control of weedy hosts and monitoring for lygus as they enter strawberry fields to time insecticide sprays. Lygus adults (and to some extent nymphs) can disperse within and among host plants (Swezey et al., 2013), and for this reason need to be monitored throughout the season. Currently, sweep nets are utilized to sample fields and monitor lygus population presence and density (Zalom et al., 1993). At low densities, especially at the initiation of the reproductive period in spring, sweep nets are not effective for sampling these insects in most crop systems. Researchers have tried other sampling methods and investigated attractiveness of plant volatiles and visual cues for L. hesperus adults and nymphs (Blackmer et al., 2008). Having a tool, such as an effective pheromone-baited trap, that could detect the earliest movement of these bugs, would be an important advance in monitoring lygus bugs. The use of insect pheromones as attractants in monitoring traps has revolutionized sampling methods for many of the major insect pests of agricultural crops in the United States. Pheromone-baited traps provide a simple, effective, and highly selective method for determining the phenology and sometimes the density of insect populations, and are a key factor in providing information for making pest management decisions. Traps and attractant baits are now available for hundreds of insect pest species from different insect orders, such as moths, beetles, and flies. However, to date, the discovery of attractant pheromones for true bug (heteropteran) species has lagged that of insects in other orders, for several reasons. In part, the identification of pheromones for these insects is complicated by the large amounts of volatile defensive chemicals which many of these insects produce (Ho and Millar, 2002, Moreira and Millar, 2005). These defensive compounds are produced in much greater abundance than the pheromone chemicals, and can obscure the pheromone chemicals during analyses. 80 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT ENTOMOLOGY It has been known for decades that female ‘mirid’ bugs, to which Lygus belong, produce sex pheromones to attract males for mating (Ho and Millar, 2002). Over the past decade, David Hall’s group in England has made excellent progress in identifying and developing pheromone blends for several European species (Fountain et al., 2014), including Lygus rugulipennis (Innocenzi et al., 2004, Innocenzi et al., 2005), Lygus pratensis, and two related hemipteran species, Lygocoris pabulinus and Liocoris tripustulatus (Fountain et al., 2014). Because of the similarity in the profiles of volatiles produced by various Lygus bug species, Hall and Millar began a collaborative effort in 2012, to test the European lures for their effectiveness in attracting North American species, such as L. hesperus and L. elisus. First, pheromone compounds and blends had to be identified for both species and then the lures had to be field tested. The preliminary results have been very promising with a strong pheromone developed and field-tested forL. elisus and attractive compounds isolated for L. hesperus. The final goal of this project is to develop effective and practical pheromone-based trapping methods for detection and monitoring of L. hesperus. With this tool, monitoring programs could more easily detect the initial movement of lygus adults into strawberries to better time insecticide applications. Once effective attractants are developed, in addition to monitoring, it may be possible to use them for control tactics such as attract and kill and mating disruption, depending on the potency of the pheromone and its production cost. MATERIALS AND METHODS 1. To determine pheromone components for L. hesperus. Colonies of L. hesperus were maintained from specimens collected in alfalfa and native vegetation. Bugs were reared under long-day conditions (17 light hours) to ensure that they remain reproductively active, as previously used in Millar’s laboratory (Ho and Millar, 2002). Briefly, bugs were maintained on a diet of organically grown green beans, navel orangeworm eggs, and raw sunflower seeds. Eggs were laid in the green beans, which were removed twice weekly and transferred to 2-quart cardboard ice cream cartons with screen lids. The resulting nymphs were held in these containers until they reached adulthood, using the diet described above. Newly emerged adults were sexed and held in single-sex groups for five days before use to ensure that they were sexually mature. To collect pheromone, sexually mature females were transferred to 50 ml glass aeration chambers, as gently as possible to minimize discharge of defensive chemicals. A small piece of green bean was provided as food and to prevent desiccation. The aeration chambers were swept with charcoal-filtered air, and the volatiles produced by the bugs were collected on small activated charcoal traps fastened to the outlet of the chambers. Traps were replaced at the beginning of each light and dark cycle so that the diurnal rhythm of production of the pheromone could be established. Each aeration trial was continued for three days, with a minimum of eight replicates of females of each of the two species (L. hesperus and L. elisus). Controls consisting of green beans only also were aerated, to identify compounds in the extracts that are from the green beans rather than the bugs. In addition, volatiles were collected from sexually mature virgin males of each species for comparison with the extracts of females, i.e., to identify compounds that are female-specific or at the least, produced predominantly by females. Volatiles were recovered from the traps by elution with a small volume of methylene chloride. The resulting extracts were analyzed by coupled gas chromatography-mass spectrometry (GC-MS), and compounds in the extracts were tentatively identified by matching their mass spectra with database spectra. Identifications were confirmed by matching mass spectra and GC retention times on two columns with those of authentic standards. Collections of volatiles were also analyzed by gas chromatography coupled to electroantennographic recording from the antennae of male lygus bugs at NRI to determine which components stimulated receptors on the antenna. 81 2016 RESEARCH PROJECTS In addition, to determine the profiles of defensive chemicals produced by adult bugs, some of which overlap with the pheromone components, the defensive glands were dissected out of sexually mature bugs, extracted in methylene chloride or diethyl ether, and analyzed by GC-MS.