Entomology

Identification of Optimization of Sex Pheromones of 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 -growing areas. is the key Lygus species in , although Lygus shulli is also present in coastal regions and 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 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 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 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.

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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 related hemipteran 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 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.

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

Having determined an optimal blend ratio for L. elisus (2014) and developed a working blend for L. hesperus (2015), we focused on refining and field-testing the optimal blend ratio forL. hesperus (2016 and 2017). The optimal rates initially 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 can be 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. Therefore, in both 2016 and 2017, pheromone blends were reassessed during the season and the next-best-guess pheromone blend was produced in England and sent to be tested in California. Because different blends were for different trials, we present the tested pheromone blends and specific plot designs in the results section.

For all field trials, pheromones were produced and loaded into custom-made dispensers by Hall in England (at NRI) and then shipped to Kearney Agricultural Research and Extension Center (KARE) for field trials in alfalfa, which was a predictable source of Lygus (Figure 1). With each shipment of pheromone, we typically had enough material for two, three-week trial periods. For each trial period, sticky traps (Biolure Delta trap, Suterra Inc., Bend, OR), baited with pheromone lures or the control, were placed in one or two alfalfa blocks (where there were no insecticides for lygus) in a randomized block design, with four to five replicates per treatment. Once deployed, traps were checked every two to three days for a one-week trial period and then the lures were changed. In some trials the traps were checked more frequently, as described in the results section. On each collection date all lygus were collected, taken to the laboratory, and identified to species and adults were identified to gender. For the purposes of this study, only adult maleL. hesperus counts are typically presented. The trials were conducted to match the alfalfa cutting schedule, and before each harvest, both alfalfa fields were 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. In 2016 and 2017 (the current reporting period), there were typically four to five trials, each lasting seven to 21 days, that were conducted fromApril to September.

Additionally, on some trial periods in 2016 and 2017, we added four to five adult femaleL. hesperus to each treatment, using a split plot design with four replicates. 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.

Data were analyzed, summarized across the season and sample dates, by treatment mean with values separate by ANOVA, followed by Tukey pairwise comparisons.

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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.

3. Lygus Mating Behavior

To determine whether L. hesperus uses pheromonal communication combined with vibrational communication, a laboratory study was conducted with the cooperation of Dr. Rodrigo Krugner, USDA – Parlier, CA, following methodologies detailed by Nieri et al., (2017). Experiments were conducted in an arena that provided a uniform background and both reduced airborne noise and observer interference. The arena was a transparent experimental cage (60 × 60 × 80 cm) made of thick acrylic walls, centered inside a chamber formed by 86 × 86 × 98 cm high blackout fabric and sound isolating walls. The arena and chamber were placed on an active vibration isolation table (Model 20-561, Technical Manufacturing Corporation, Peabody, MA). Insect behaviors were monitored via video surveillance (Panasonic Lumix GH4). Vibrational communication signals produced by L. hesperus were recorded using a laser Doppler vibrometer (PDV-100, Polytec, Inc., Irvine, CA) and digitized with Adobe Audition C26 (Adobe Systems, Inc., San Jose, CA) at a 44.1-kHz sample rate and 32-bit resolution. The laser vibrometer focused on a small piece of reflective tape glued to the stem of a potted alfalfa plant placed in the center of the arena.

83 2016 RESEARCH PROJECTS Results 1. To Determine Pheromone Components for L. hesperus.

Extracts of individual bugs were made in ether and analyzed by gas chromatography (GC) and by GC coupled to mass spectrometry (GC-MS). The extracts showed three main components – hexyl butyrate (HB), (E)-2-hexenyl butyrate (E2HB) and (E)-4-oxo-2-hexenal (KA). Hexyl butyrate was the major component, and the relative amounts of the other components were similar in extracts from males or females (Figure 2). Trace amounts of (E)-2-hexenal, 1-hexanol, (E)- 2-hexenyl butyrate (E2HB) and (Z)-3-hexenyl butyrate (Z3HB) were also detected. Volatiles were collected by aeration of individual bugs and trapping on an adsorbent. Analysis of these collections showed the above components predominantly or exclusively in collections from females (Figure 3). The relative amounts of the components were similar to those observed in direct solvent extracts of bugs (Figure 4).

Collection of volatiles separately during the light and dark periods showed the pheromone components were produced during light and dark periods, with a tendency to production more often during the light period (Figure 5). Collections of volatiles from female L. hesperus were also analyzed by GC coupled to electroantennographic (EAG) recording from the antenna of a male bug. Consistent EAG responses were detected to HB, E2HB and KA, but not to (E)-2-hexenal or 1-hexanol or any other components (Figure 6). This work was conducted both in the Millar and Hall laboratories, and both in 2015 and 2016 using L. hesperus from California. The results were very similar leading us to believe that all components of the pheromone blend have been identified. There is a slight possibility that trace components are still unknown (i.e., at rates <<1% of the major component), or that other behavioral signals during the calling process are still unknown, but the primary components have been identified, and then verified in two separate laboratories over two different years.

Figure 2. The amounts of compounds in ether extracts of individual Lygus hesperus by GC-FID analysis on DB-Wax column.

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Figure 3. GC-FID analysis of volatiles from virgin female Lygus 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).

Figure 4. Relative amounts of compounds (HB = 100) in solvent extracts of both sexes, and in volatiles collected from individual virgin female Lygus hesperus over 24 h by GC-FID analysis on DB-Wax column.

85 2016 RESEARCH PROJECTS Figure 5. Amounts of hexyl butyrate produced by individual virgin female Lygus hesperus during successive light and dark periods (each pair of results corresponds to different individual; means as shown in legend).

Figure 6. Analysis of collection of volatiles from virgin female Lygus 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.

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2. To Reconstruct and Field Test the Optimally Attractive Blend of Components for L. hesperus.

A series of trials were conducted in the alfalfa blocks at KARE to determine the adult male L. hesperus response to pheromone blends (primarily in 2016) and to try and optimize the capture of adult males in traps (primarily in 2017).

In 2016, 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 an influence of trial period on trap captures (P < 0.001; df = 1, 1,124; F = 19.252). For simplicity of this presentation, we have combined data from the organic and conventional plots when it did not affect the outcome of the treatments. 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. The May, June and July, 100 sweep samples for male L. hesperus were: 62.7 ± 7.2, 162.7 ± 8.2, and 136.6 ± 6.1, respectively. We also collected through the sweep samples 2,971 adult female and 1,445 nymphs that were not identified to species, but are assumed to be overwhelmingly L. hesperus as well.

In April and May 2016, we tested variants of the pheromone blend, which were based on the best formulations tested in the previous years. The April- and May-initiated trials used a simple two-component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA), a three-component blend of HB, KA and trace amounts of (Z)-3-hexenyl butyrate (Z3HB), 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.

Results show that all pheromone blends captured more L. hesperus than the blank control in the April-initiated trial (P <, 0.001; df = 4, 194; F = 18.138; Figure 7A) and the May-initiated trial (P <, 0.001; df = 4, 275; F = 18.434; Figure 7B). There were also similar treatment differences in both trial periods, with the two-component (HB and KA) and a three component (HB, KA and Z3HB) blend capturing more Lygus than treatments that had the additional trace element of E2HB (with or without Z3HB).

For the June 2016 initiated trial, we used only the two-component (HB and KA) and three-component blend (HB, KA and Z3HB), but included virgin females with both the pheromone lures and the control. The trial was our first attempt to better understand lygus mating behavior with the hypothesis that the pheromone blends were correct but that we couldn’t get male lygus into the traps. Two possibilities were explored in the field in 2016 and 2017. First, that there were vibrational signals produced by either females or males that were used in combination with the sex pheromone; we assumed that because the female sent out the pheromone, she would also either initiate of respond to a male using a vibrational signal. Second, that the density of L. hesperus at the alfalfa field was too high and outcompeted the lures.

To field test the possibility that females sent out a vibrational signal, we compared pheromone-baited lures to virgin females. First, we collected immature lygus, caged the nymphs and fed them alfalfa cuttings until they reached the fifth instar, and then isolated all large nymphs until they reached the adult stage and could be sexed. As with the pheromone blend trials, the experimental design was a complete randomized design, with four replicates, although for both pheromone blends we used a split plot design so that there were two replicates each for the lure and virgin female combination (see plot design Figure 8A), in part because we did not find any difference in trap capture with the addition of Z3HB (Figure 7).

87 2016 RESEARCH PROJECTS Across all lure and control treatments, trap counts were low (<13 L. hesperus males per trap per week). As with other trials, traps with lures had significantly moreL. hesperus males than control traps (Figure 8B), and there was no difference between the two-component and three-component blends tested (F = 6.22; df = 2,13; P = 0.013). Most important, there was no difference in the addition of virgin females: 4.5 ± 1.3 vs. 5.1 ± 1.2 with or without virgin females, respectively (F = 0.26; df = 1,14; P = 0.62). This may have been in part because in this first trial we had a difficult time keeping the female Lygus alive in the small cages in the pheromone traps and, under such stress they may not have initiated mating behaviors.

Figure 7. Lygus hesperus trap captures per day (± standard error) for 2016 trials with (A) April-initiated and (B) May-initiated trials using a two-component blend (HB and KA), a three-component blend of HB, KA and trace amounts of Z3HB, a three-component blend of HB, KA and trace amounts of E2HB, and a four-component blend of HB, KA, Z3HB and E2HB, compared with a control. Different letters above each bar indicated a significant difference among treatment means.

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Figure 8. (A) Experimental design for the addition of virgin female Lygus hesperus (♀) with a two-component pheromone blend (silver), three component pheromone blend (red) and control (white); (B) trap captures per day (± standard error) averaged across all collection days (and female or no female treatments) two-component blend and three-component pheromone blend, compared with a control. Different letters above each bar indicated a significant difference among treatment means. In June and July 2016, we again combined pheromone lures with virgin females. In these trials we used the two-component blend (HB and KA) that had worked in the previous trials, but we changed the release rate. Here, we hypothesized that lower trap captures might be explained by too much or too little pheromone being released. The optimal rates initially 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 can be 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. Treatments were then the ‘standard’ release rate used in all previous trials, half release rate, double release rate, and a standard release rate in a 1 ml pipette bulb (a different pheromone release system); we also rotated the treatment placement every week, during each of the three-week trials (Figure 9).

89 2016 RESEARCH PROJECTS Figure 9. Experimental design for the addition of virgin female Lygus hesperus (♀) with a two-component pheromone blend at a release rate that was standard (silver), half the standard (green), twice the standard (blue), or using a 1 ml pipette bulb (yellow), and control (white).

All pheromone blends captured more L. hesperus than the blank control in the June-initiated trials (F = 13.91; df = 4, 275; P < 0.001; Figure 10A) and the July-initiated trial (F = 18.87; df = 4, 315; P < 0.001; Figure 10B). However, patterns among treatments were more difficult to assess. In the June-initiated trial there were more lygus captured in the double release rate treatment (Figure 10A) but this pattern did not hold in the July-initiated treatment (Figure 10B). The additional delivery method (1 ml pipette) was similar to the standard release rate, but higher than either the half or double release rates.

Figure 10. Lygus hesperus trap captures per day (± standard error) for (A) June-initiated and (B) July-initiated trials using a two-component blend (HB and KA) with different release rates (1X, 0.5X, 2X) and release method (1 ml pipette bulb), compared with a control. Different letters above each bar indicated a significant difference among treatment means.

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With the data combined from both the conventional and organic fields there was no significant different of trap capture with or without the addition of virgin females. However, when the fields were analyzed separately, and data were subjected to a square-root (+1) transformation, there was a slight increase in trap capture when virgin females were added in the June-July trials conventional plot (with ♀♀ 2.0 ± 0.2 males per trap vs. without ♀♀ 1.5 ± 0.2 males per trap; (F = 5.82; df = 1, 138; P = 0.017). The impact of the addition of virgin females to the control or lure treatments was not found in any other trials. As previously mentioned, the captured females were under considerable stress and although water (in a plastic vial) and alfalfa cuttings were provided, many of the females were dead after 2-3 days in the trap.

Lure longevity and the daily capture rate will be important for development of a commercial product. We used the July-initiated trial in 2016, combining data from the conventional and organic fields, to determine lure longevity – or how long before the pheromone was either gone from the lure, was at such a low dose that it was no longer active, or had degenerated due to heat and was no longer attractive. 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. The results show a general pattern of reduced trap capture after the first week (Figure 11). Surprisingly, the double release had poor trap capture on the first sample days and then increased to match the standard release either in the lure or the 1 ml pipette.

Figure 11. Lygus hesperus trap captures per day (± standard error) for July-initiated trials using a simple two-component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) at a standard release rate, a half release rate, a double release rate and a standard release rate in 1 ml pipette bulbs.

91 2016 RESEARCH PROJECTS In 2017, we shifted our focus to the study of lygus behavior to determine why trap captures were lower than expectations. As mentioned previously, one hypothesis was that the pheromone was working – but we could not get the Lygus males into the traps. In 2016 we attempted to isolate this mating behavior by adding virgin females to pheromone baited and control traps, but had little impact on trap captures, possible because the captured females did not behave normally and initiate mating behaviors. We used the conventionally managed alfalfa field at KARE (no insecticides for lygus), which consistently had higher L. hesperus densities (compared with the organic field). Biolure Delta traps with the HB and KA (10:6 mg) baited lures or a no pheromone control were deployed and checked every day, removing any lygus captured, to test the possibility that captured males (which often take many hours or days to die in the trap), gave off an alarm pheromone. The trial ran from May 2 to June 9, 2017, with the pheromone lures replaced on May 22, 2017. Periodically, we also made sweep samples around the traps to see if more males were in the trap vicinity, as compared with the control.

Results showed consistently higher trap catches in the pheromone baited treatment than the control, and that the lures remained active for about 10 to 12 days (May 3 – 16 and May 23 –June 5, 2017) (Figure 12A). Capture rates were similar to those from previous year’s trials, suggesting that dead or dying male lygus in the traps were not emitting a volatile that dramatically altered the results. More importantly, we took 10 sweep samples around each trap (pheromone-baited and control) twice each week of the trial period. There was no difference in the number of adult L. hesperus around traps with or without pheromone-baited lures (Figure 12B).

Figure 12A. Lygus hesperus trap captures per day (± standard error) for May-initiated trial in 2017 using a simple two-component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) at a standard releaser rate, and with the dead or dying Lygus removed every day.

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Figure 12B. The number of adult L. hesperus (± standard error) collected in 10 sweep samples each on all four sides of each trap.

To determine if there was a flight period during the day, we used the same split plot design with four replicates as described previously, but collected trapped lygus every hour from 8 a.m. to 4 p.m. New lures were placed in treatment traps on each day. The trial was conducted for five days, from June 12 – 16, 2017. Results show highest trap captures early in the morning and then during the peak of the daily heat (3 p.m. to 4 p.m.) (Figure 13). One flaw in this design was that we expected higher catches earlier in the day (7 a.m. – to10 a.m.) and focused on this time period. The trial may be repeated extending the work into the evening (until midnight).

Figure 13. Lygus hesperus trap captures per day (± standard error) for an August-initiated trial in 2017 using a simple two-component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) at a standard releaser rate, and with the dead or dying Lygus removed every hour (7 a.m. to 4 p.m.).

93 2016 RESEARCH PROJECTS In the final 2017 field trial, we tested whether there was competition betweenL. hesperus and the pheromones (e.g., too many competing signals to get the adult males into the traps). Five treatments were used to vary the density of lygus near the pheromone lure. We began after the alfalfa was mowed, raked and baled – thus leaving only five small strips of tall alfalfa (or berm) with everything else mowed close and supporting few if any lygus. The distance between each alfalfa strip was 30.5 m (100 ft). The treatments were virgin female traps as a natural lure, placed in the tall alfalfa on the berm, the HB:KA pheromone, and a no-pheromone control also on the berm, and pheromone-baited traps placed 7.6 m (25 ft) and 15.2 m (50 ft) away from the berm (and the Lygus population). There were four replicates in a randomized block design, and the lygus were collected every two to three days during the trial that ran from August 2 to 18. Here, we have grouped all trial dates for the data analyses.

Results showed that the pheromone baited traps pulled in significantly moreL. hesperus than the control or the live virgin females, and regardless of the distance from the berm – where almost all the Lygus were found, the pheromone baited traps captured the same number of adult male L. hesperus (F = 15.48; df = 4, 228; P < 0.001; Figure 14). The results do suggest that the pheromone traps are working, and that they pulled in lygus from their berm location, but for some reason we are still not capturing the hundreds per day per trap that would be expected for a highly active sex pheromone.

Figure 14. Lygus hesperus trap captures per day (± standard error) for an August-initiated trial in 2017 using a simple two-component blend of hexyl butyrate (HB) and (E)-4-oxo-2-hexenal (KA) at a standard release rate, with traps placed on an alfalfa berm with the lygus population, or 25 and 50 ft away on newly mowed alfalfa, as compared with a no-pheromone control and traps baited with three to five virgin female L. hesperus.

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3. Laboratory Trial of Lygus Mating Behavior

Working in Dr. Krugner’s laboratory with specialized equipment, Ms. Fonseca observed and recorded male-female pairs of L. hesperus. First, and most exciting, both male and female L. hesperus use a vibrational signal (or sound) that appears to bring a male-female pair closer to each other on the plant, likely after they are drawn to the site with the aid of the pheromone. After localizing the female on the plant, the male and female exchange vibrational signals until they mate. Therefore, it appears that in this species vibrational signals are crucial components of mate localization and courtship. Signals are generated by a rapid dorso-ventral movement of the abdomen. During signal emission, the abdomen does not appear to touch the plant, which indicates that signal waves are transmitted to the plant through the legs. The vibrating behavior can be viewed at the website: https://youtu.be/0CfXsc3x_g8

This finding has obvious implications for the pheromone and for our studies. For example, brown marmorated stink bugs use both chemical and vibrational communication to achieve mating, suggesting that combining both chemical and vibrational lures into a trap may improve trap performance (Mazzoni et al., 2017). Through the recording of the videos we also found that with this species, under the experimental conditions we provided, mating was not an easy behavior to elicit. First, young virgin females did not initially mate but had to be held for a few days until they became reproductively active – this may be one reason why our field studies with virgin females were similar to the no-pheromone control (2016 trials and Figure 14). Second, males may not enter the traps without a vibrational signal, although this does not explain why we did not get higher male counts surrounding the traps (Figure 12B).

We will continue to explore the possible manipulation of vibrational signals and how this might influence the practical use of pheromones for monitoring L. hesperus. For example, does L. elisus also use vibrational signals and if so, why were they so easily captured with their pheromone? Will a different trap type improve the trap captures – for example can a vibrational signal be incorporated into a trap economically?

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 ofLygus 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 from three interlocking insights. First, Hall’s group showed that very careful collection of odors from individual, undisturbed females was required 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 and irritant properties, 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.

95 2016 RESEARCH PROJECTS On the basis of this extraordinary progress, and because of the similarity in volatiles produced by various Lygus bug species, we began collaboration with 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 three to 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 focused on mating behavior. In all studies the two-component pheromone blend attracted more L. hesperus males than controls or virgin females. We also showed that we could attract males 50 ft away from the nearest source of bugs at the same density as traps placed in the source (where the Lygus were located on the berm). We also have a better understanding of Lygus flight periods (afternoon), although this will be studied again in 2018 to confirm these findings. Perhaps most important is the finding that a vibrational signal is used by both males and females to achieve mating. However, this does not explain why adult males are not found in the vicinity of the pheromone- baited traps and this needs to be further studied.

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References

• Allen, W.W. and S.E. Gaede. 1963. Relationship of Lygus bugs and thrips to fruit deformity in strawberries. Jour- nal of Economic Entomology 56: 823-825.

• Blackmer, J.L., J.A. Byers, and C. Rodriguez-Saona. 2008. Evaluation of color traps for monitoring Lygus spp.: Design, placement, height, time of day, and non-target effects. Crop Protection 27: 171-181.

• Fountain, M., G. Jastad, D. Hall, P. Douglas, D. Farman, and J. Cross. 2014. Further studies on sex pheromones of female Lygus and related bugs: development of effective lures and investigation of species-specificity. Journal of Chemical Ecology 40: 71-83.

• Graham, H.M. 1988. Sexual attraction of Lygus hesperus Knight (: Miridae). Southwestern Entomolo- gist 13: 31-37.

• Ho, H.Y. and J.G. Millar. 2002. Identification, electroantennogram screening, and field bioassays of volatile chemi- cals from Lygus hesperus Knight (Heteroptera: Miridae). Zoological Studies 41: 311-320.

• Innocenzi, P.J., D. Hall, J.V. Cross, and H. Hesketh. 2005. Attraction of male European , Lygus rugulipennis to components of the female sex pheromone in the field. Journal of Chemical Ecology 31: 1401- 1413.

• Mazzoni, V., J. Polajnar, M. Baldini, M., M.V. Rossi Stacconi, G. Anfora, R. Guidetti, and L. Maistrello, L. 2017. Use of substrate-borne vibrational signals to attract the Brown Marmorated Stink Bug, Halyomorpha halys. Jour- nal of Pest Science 90: 1219-1229.

• Moreira, J.A. and J.G. Millar. 2005. Short and simple syntheses of 4-oxo-(E)-2-hexenal and homologs: Phero- mone components and defensive compounds of . Journal of Chemical Ecology 31: 965-968.

• Nieri, R., Mazzoni, V., Gordon, S.D., and Krugner, R. 2017. Mating behavior and vibrational mimicry in the glassy- winged sharpshooter, Homalodisca vitripennis. Journal of Pest Science. 90: 887-899.

• Strong, F.E., J.A. Sheldahl, P.R. Hughes, and M.K. Hussein. 1970. Reproductive biology of Lygus hesperus Knight. Hilgardia 40: 105-133.

• Swezey, S L., D.J. Nieto, J.R. Hagler, C.H. Pickett, J.A. Bryer, and S.A. Machtley. 2013. Dispersion, distribution, and movement of Lygus spp. (Hemiptera: Miridae) in trap-cropped organic strawberries. Environmental Entomolo- gy 42: 770-778.

• Zalom, F.G., C. Pickel, C., D.B. Walsh, and N.C. Welch. 1993. Sampling for Lygus hesperus (Hemiptera: Miridae) in strawberries. Journal of Economic Entomology 86: 1191-1195.

• Zalom, F.G., M.P. Bolda, S.K. Dara, and S. Joseph. 2014. Insects and Mites. UC IPM Pest Management Guide- lines - Strawberry. University of California ANR Publication 3468.

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