Entomology

Identification of Optimization of Sex Pheromones of hesperus as Practical Lures for Pheromone-baited Traps or use in Control Programs Dr. David Hall and Dudley Farma Natural Resources Institute Greenwich University England [email protected] Tel +1634 883207

Jocelyn Millar, Jacquie Serrano, and J. Steven McElfresh Dept. of Entomology UC Riverside Riverside, CA [email protected] (951) 827-5821

Peter Goodell UC IPM Kearney Agricultural Research & Extension Center 9240 So. Riverbend Ave. Parlier, CA [email protected] (559) 646-6515

Robert K. Straser Maynhia Yang Alejandro I. Hernandez Jesus Ceja Kent M. Daane 137 Mulford Hall Dept. of Environmental Science Policy and Management University of California Berkeley, CA 94720-3114 [email protected] (559) 646-6573 [email protected] (559) 646-6522

81 2015 RESEARCH PROJECTS Summary Lygus bugs are a serious pest in California -growing areas. is the key lygus species in , although Lygus shulli is also present in coastal regions and L. 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. Therefore, management of lygus includes control of weed hosts and monitoring for the appearance of lygus nymphs or adults on nearby alternate host plants and strawberries. Insecticide(s) must then be timed to control lygus before they cause significant damage. Currently, sweep nets are utilized to monitor lygus population presence and density; however, sweep nets are not an effective sampling tool at low lygus densities or the initiation of their reproductive period. 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 fields at the Kearney Agricultural Research and Extension Center, 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 getting L. hesperus into the traps. This is a stumbling block that keeps this project from moving from basic to applied research. In 2017, we will not request additional funding but we will continue to look at lygus trapping to determine L. hesperus flight and mating behaviors 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 the strawberry field in order to time insecticide sprays. Lygus nymphs and adults can disperse within and among host plants as they move to new floral buds and seeds (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, sweep nets are not effective for sampling these in most crop systems. Researchers have tried other sampling methods and investigated attractiveness of plant volatile 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 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 () species has lagged behind that of insects in other orders,

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for a number of 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.

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 (Innocenzi et al., 2004, Innocenzi et al., 2005), , and two closely related species, Lygocoris pabulinus and 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 in order 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 L/7 D) to ensure that they remain reproductively active, using methods previously worked out 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 of the bug. 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.

83 2015 RESEARCH PROJECTS 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. Co-Principal Investigators Millar and Hall have extensive libraries of such standards from previous work with Lygus and other true bug species.

Collections of volatiles were also analyzed by gas chromatography coupled to electroantennographic recording from the antenna of a male lygus bug at NRI in order to determine which components stimulated receptors on the antenna.

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.

Figure 1. Tested pheromone blends were loaded into plastic dispensers by Dr. Hall’s team at the University of Greenwich and shipped to the Kearney Agricultural Research and Extension Center (Parlier, CA) and attached inside a triangular sticky trap attached to a small wooden stake. The trials were conducted in two alfalfa fields with high populations of L. hesperus.

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Figure 2. Amounts of compounds in ether extracts of individual L. hesperus by GC-FID analysis on DB-Wax column.

Figure 3. GC-FID analysis of volatiles from virgin female L. hesperus on a polar DB-Wax column ((E)-2- hexenal 5.28 min; 1-hexanol 7.29 min; hexyl butyrate 8.25 min; (E)-2-hexenyl butyrate 9.04 min; (E)-4-oxo-2-hexenal 10.5 min).

85 2015 RESEARCH PROJECTS Figure 4. Relative amounts of compounds (HB = 100) in volatiles collected from individual virgin female L. hesperus over 24 h by GC-FID analysis on DB-Wax column.

Figure 5. Amounts of hexyl butyrate produced by individual virgin female L. hesperus during successive light and dark periods (each pair of results corresponds to different individual; means as shown in legend).

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2. To reconstruct and field test the optimally attractive blend of components forL. hesperus.

Having determined the average blend ratio of likely pheromone components produced by each species, blends were formulated by Co-Principal Investigator Hall, and loaded into custom-made dispensers for field testing. In 2014, we determined the optimum blend ratio for L. elisus, and in 2015 and 2016, we focused on the optimum blend ratio for L. hesperus, including the total number of components that are both necessary and sufficient for attraction, and the optimal ratio of those components.

Having determined an optimal blend ratio for L. elisus (2014) and developed a working blend for L. hesperus (2015), we focused on refining the optimal blend ratio for L. hesperus (2016). Selected blends and doses were determined by Co-Principal Investigators Hall and Millar. The first batch of pheromone blends were based on the laboratory GC-MS analyses (Objective 1) and field trials in 2015. In April 2016, five treatments (four pheromone blends and the control) were loaded into custom-made dispensers at NRI and shipped to Kearney Agricultural Research and Extension Center (KARE) for field trials, using alfalfa as a predictable source of lygus. The first shipment contained enough material for two, two to three week trial periods (initiated in April and May), to match the alfalfa cutting schedule. For each trial period, sticky traps baited with the dispensers were placed in two alfalfa blocks (no insecticides for lygus) in a randomized block design, with five replicates per treatment. Once deployed, traps were checked three times per week during each alfalfa cutting- to-harvest cycle. On each sample date, all lygus were collected, taken to the laboratory, and identified to species. One week before harvest, each block was sampled using a sweep net (100 sweeps per block) and the number and species of lygus were recorded to compare trap counts with standard sampling programs.

Additionally, on two trial periods we added four to five adult femaleL. hesperus to each treatment, using a split plot design. The insects were collected using a sweep net, sorted by gender, and females were isolated and fed on cut alfalfa until they were used. Small plastic vials with organdy covered lids were used to house the bugs, which were placed inside each sticky trap and against the trap so that any vibrational cues would be picked up by the trap and wooden pole holding the trap in place.

In particular, the optimal rates that Dr. Hall determined for lygus species in England will almost certainly need to be modified for California, where the climate is warmer, lygus densities can be higher, and the strawberry season is longer. With custom dispensers, this can be readily done by adjusting one or more of the variables: total dose loaded into the dispenser; the ratio of the inert carrier material to the active pheromone components; and the aperture of the release device. In the second set of trials, release rates were tested. Pheromone blends were reassessed from the April and May trials and the best pheromone blend was loaded into custom dispensers at half-standard rate, standard rate and double standard rate, and an additional treatment was the pheromone blend at standard rate but deployed in a different release device (a 1 ml pipette bulb). The treatments were deployed and tested in June and July as described above.

Throughout trials, weather data were gathered from nearby weather stations. Data were analyzed, summarized across the season and sample dates, by treatment mean with values separate by ANOVA, followed by Tukey pairwise comparisons (P. <0.05). For a more accurate treatment separation, data will be analyzed using a general linear model, with sampled data set as a categorical value (basically an ANOVA test that takes into account changing sample densities across different sample dates).

87 2015 RESEARCH PROJECTS Results In April and May, we tested variants of the pheromone blend, which were based on the best formulation tested in 2015. The April and May trials used the same blends, and both trials lasted 14 weeks. Overall, we trapped 3,468 male L. hesperus (3.08 ± 0.10 per trap per two-day collection period), compared with only 53 L. elisus (0.05 ± 0.01 per trap per 2-day collection period). This is a great advance since 2014, when we were catching primarily L. elisus. These data also suggest that the pheromone blends tested are now more attractive to L. hesperus; for example, sweep net counts showed the sampled sites had a 51.7: 1 ratio of male L. hesperus to L. elisus, whereas pheromone traps had a 775: 1 ratio of male L. hesperus to L. elisus.

Across all five trial periods and treatments (n = 1127), the average capture was 3.08 ± 0.10 L. hesperus per trap per collection period. However, each collection period lasted for either two days (e.g., Monday to Wednesday) or three days (e.g., Friday to Monday); for this reason, we transformed data to captures per day for all other analyses.

Across all trial periods, there were slightly more L. hesperus captured in the organic plot (1.43 ± 0.07 per trap per day) compared with the conventional plot (1.23 ± 0.06 per trap per day) (P = 0.027; df = 1, 1,124; F = 4.914) and there was a significant influence of trial period on trap captures (P < 0.001; df = 1, 1,124; F = 19.252). However, for this report we have combined data sets from these two fields because all treatments and start dates were the same for the April and May trials and the June and July trials, and the overall results and statistical outputs did not change.

In two trial periods, we added four to five adult female L. hesperus to each treatment. This was done because trap captures remained lower than our expectations and we decided to test whether the presence of adult females might make any of the lures more attractive, assuming there was a vibrational or other signal that we were not including in the reconstructed pheromone blends. However, paired t-test showed no significant difference in trap captures with the treatment versus treatment and adult females (t = -1.43, df = 22, P = 0.166). For this reason, these data were also combined for the complete analyses.

The April- and May-initiated trials used a simple two component blend of HB and KA (Silver treatment), which was the best tested blend in 2015. An additional component (Z3HB) was added in trace amounts to HB and KA to form the second treatment (Red), and another trace element (E2HB) was added with HB and KA for the third treatment (Blue) and all four were added to the final blend (Green). All pheromone blends captured more L. hesperus than the blank control in the April-initiated trials (P <, 0.001; df = 4, 194; F = 18.138; Figure 6) as well as the May-initiated trial (P <, 0.001; df = 4, 275; F = 18.434; Figure 6). There were also similar treatment differences in both trial periods, with Silver (HB and KA) and Red (HB, KA and Z3HB), capturing more Lygus than Blue and Green treatments that had the additional trace element of E2HB (with or without Z3HB).

For the June- and July-initiated trials we used only the combined HB and KA, but we changed the release rate and the delivery method. Treatments were then Silver (standard release rate), Green (half release rate) Blue (double release rate) and Yellow (standard release rate in a 1 ml pipette bulb). All pheromone blends captured more L. hesperus than the blank control in the June-initiated trials (P <, 0.001; df = 4, 275; F = 13.910; Figure 7A) and the July-initiated trial (P <, 0.001; df = 4, 315; F = 18.871; Figure 7B). However, patterns among treatments were more difficult to assess. In the June-initiated trial there were more Lygus captured in the Blue (double release rate) treatment (Figure 7A) but this pattern did not hold in the July-initiated treatment (Figure 7B). The additional delivery method (Yellow - 1 ml pipette) was similar to Silver (same release rate and Yellow), but higher than either the half (Green) or double (Blue) release rates.

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The daily capture rate will be important for development of a commercial product. It will also be important to determine the effective field lifetime of the lures. We combined data from the first (April and May) and second (June and July) trial periods. The results were similar across all treatments, and showed a pattern of reduced trap captures the longer the lure was left in the field. However, even on the last day of the trial period, there were more Lygus captured by the pheromone blends than the control.with sampled data set as a categorical value (basically an ANOVA test that takes into account changing sample densities across different sample dates).

Figure 6. Analysis of collection of volatiles from virgin female L. hesperus by gas chromatography coupled to electroantennographic detection (GC-EAD) using the live antenna of a male bug. Upper trace is EAD, lower trace is GC. EAD responses (*) to HB, E2HB and KA but not to 1-hexanol at 8.08 min.

89 2015 RESEARCH PROJECTS Figure 7. Lygus hesperus trap captures per day (± standard error) averaged across all collection days for (A) April-initiated and (B) May-initiated trials using a simple two component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) (Silver treatment), a three-component blend of HB, KA and trace amounts of (Z)-3-hexenyl butyrate (Z3HB) (Red), a three component blend of HB, KA and trace amounts of (E)-2-hexenyl butyrate (E2HB) (Blue) and a four component blend of HB, KA, Z3HB and E2HB. Different letters above each bar indicated a significant difference among treatment means

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Discussion It has been known for decades that female bugs in the family , to which Lygus bugs belong, produce sex pheromones to attract males for mating. For example, Strong et al., (1970) showed that female L. hesperus produce a volatile sex pheromone, which attracted males in both laboratory and field bioassays, and these findings were later confirmed by Graham (1988), who also demonstrated that females of other North American Lygus species (e.g., L. lineolaris, L. elisus) produced pheromones which attracted males as well. A large number of volatile compounds have been identified from extracts of lygus bugs of both sexes (Ho and Millar, 2002), but most or all of these compounds appear to be defensive compounds. However, over the past decade, David Hall’s group in England has made excellent progress in identifying and developing pheromone blends and lures for several European species, including Lygus rugulipennis (Innocenzi et al., 2004, Innocenzi et al., 2005), Lygus pratensis, and two closely related species, Lygocoris pabulinus and (Fountain et al., 2014). These results came about as a result of three interlocking insights. First, they showed that very careful collection of odors from individual, undisturbed females was required in order to obtain extracts that contained only the female pheromone components. The second key insight was the realization that a component that previous workers had assumed to be strictly a defensive compound, on the basis of its structure, was actually a key component of the pheromone. Third, they showed that the insects were very sensitive to the blend ratio and release rate of the pheromone, and developed lures that released a relatively constant blend at a relatively low release rate for periods of several weeks.

On the basis of this extraordinary progress, and because of the similarity in volatiles produced by various Lygus bug species, we began collaboration with Dr. Hall in 2012, testing some of his lures developed for European Lygus species in California. The results were very promising. In 2013, we identified a blend that was extremely attractive to L. elisus. In 2014, iterations of this blend captured 100 fold more L. elisus that the control, in a field where there was a large L. hesperus population and sweep net samples collected few or no L. elisus. In 2015, the goal was to identify a pheromone blend attractive to L. hesperus. In March-April trials we again showed the L. elisus pheromone blend was still a powerful attractant, and new blends captured two to three times more L. hesperus than the control and only a few L. elisus. Later in 2015 we refined the pheromone blend and captured five to eight times moreL. hesperus than the control. Our results are consistent with those obtained by Byers et al., (2013), but we have further optimized the blends and developed practical lures for field use.

In 2016, we re-analyzed the L. hesperus pheromone components in both UC Riverside and University of Greenwich laboratories. We tested the major and trace components and showed consistent attraction to our reconstructed blends. However, trap captures are still lower than we expected. For example, with so few L. elisus in the field that we had a difficult time collecting them with a sweep net, we still captured 3-50 per trap per day in out 2014 and 2015 trials. In contrast, with a large population of L. hesperus – so large that our control traps collected adults – we were not getting the same level of responses in trap captures. Moreover, this relatively weak response is even more puzzling because L. elisus and L. hesperus are closely related and we assumed that their field behaviors and responses to their species-specific pheromone blends would be similar. This continuing relatively weak response is a stumbling block and at this point, we cannot recommend that the L. hesperus blend/trapping system is ready for grower use as a sensitive method of monitoring this species. Because of this ongoing problem, in 2017, we will not request additional funding but will continue to work with Lygus trapping, to determine L. hesperus flight and mating behaviors and re-examine other chemical cues that might be missing from our pheromone formulation, as well as possible vibrational cues that might be important in bringing bugs right onto traps.

91 2015 RESEARCH PROJECTS Figure 8. Lygus hesperus trap captures per day (± standard error) averaged across all collection days for (A) June-initiated and (B) July-initiated trials using a simple two component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) at a standard releaser rate (Silver treatment), a half release rate (Green), a double release rate (Blue) and a standard release rate in 1 ml pipette bulbs (Yellow). Different letters above each bar indicated a significant difference among treatment means.

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Figure 9. Lygus hesperus trap captures per day (± standard error) for (A) April and May-initiated using a simple two component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) (Silver treatment), a three component blend of HB, KA and trace amounts of (Z)-3-hexenyl butyrate (Z3HB) (Red), a three component blend of HB, KA and trace amounts of (E)-2-hexenyl butyrate (E2HB) (Blue) and a four component blend of HB, KA, Z3HB and E2HB; and for and (B) June- and July-initiated trials using a simple two component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) at a standard releaser rate (Silver treatment), a half release rate (Green), a double release rate (Blue) and a standard release rate in 1 ml pipette bulbs (Yellow).

93 2015 RESEARCH PROJECTS References

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