J Chem Ecol (2006) 32: 2163–2176 DOI 10.1007/s10886-006-9137-5

Male-Produced Pheromone of the Green Lacewing, nigricornis

Qing-He Zhang & Rodney G. Schneidmiller & Doreen R. Hoover & Kevin Young & Dewayne O. Welshons & Armenak Margaryan & Jeffrey R. Aldrich & Kamlesh R. Chauhan

Received: 26 January 2006 / Revised: 26 May 2006 / Accepted: 27 May 2006 /Published online: 13 September 2006 # Springer Science + Business Media, Inc. 2006

Abstract Gas chromatographic–electroantennographic detection (GC–EAD) analysis showed that male antennae of the green lacewing, Chrysopa nigricornis Burmeister, the most common lacewing species in the U.S. Pacific Northwest, consistently responded to two compounds in thoracic extracts of conspecific males: 1-tridecene and (1R,2S,5R,8R)- iridodial. These compounds were not detected in extracts of the abdominal cuticle, and no other antennally active compounds were found in the abdominal samples. In field-trapping experiments, traps baited with iridodial significantly attracted large numbers of C. nigricornis males (both western and eastern forms) during summer and early fall, plus a few individuals of conspecific females only in early fall. Iridodial also attracted males of the goldeneyed lacewing, C. oculata Say, and, to a lesser extent, C. coloradensis Banks males. Methyl salicylate (MS), reported as an attractant for both sexes of C. nigricornis and C. oculata, was inactive by itself at the concentration tested in our study, but in a few instances slightly enhanced the responses of Chrysopa spp. to iridodial. However, MS alone and its binary blend with iridodial seemed to attract the hoverfly, Metasyrphus americanus (Weidemann). 2-Phenylethanol, a reported attractant for another lacewing, Chrysoperla plorabunda (Fitch) [= carnea (Say)], did not capture any lacewings. Our assays indicated that the lacewing pheromone, iridodial, loaded onto either rubber septa or as a binary blend with MS in polyethylene bags could last at least 5 wk in the field during the summer season.

Q.-H. Zhang (*) : R. G. Schneidmiller : D. R. Hoover : K. Young : D. O. Welshons : A. Margaryan Sterling International, Inc., 3808 North Sullivan Road, Building 16BV, Spokane, WA 99216, USA e-mail: [email protected]

J. R. Aldrich : K. R. Chauhan USDA-ARS Chemicals Affecting Behavior Laboratory, BARC-West, B-007, 10300 Baltimore Avenue, Beltsville, MD 20705, USA 2164 J Chem Ecol (2006) 32: 2163–2176

Based on this study, a new attractant system for green lacewings is being developed for both domestic and international markets.

Keywords Hoverfly . Attractant . Pheromone . Iridodial . Methyl salicylate . 2-phenylethanol . Nepetalactone . Nepetalactol . 1-tridecene . GC–EAD . Field flight behavioral assay

Introduction

Lacewings () are important predators of aphids and other soft-bodied (New, 1975; Tauber et al., 2000). Furthermore, because of their commercial availability and resistance to insecticides, green lacewings are also among the most commonly released predators for augmentative biological control (Ridgway and Murphy, 1984; Tulisalo, 1984; Aldrich, 1999). However, methods are still needed to retain the predators near augmentation sites and/or to attract wild predators to target areas (Baker et al., 2003). Lacewings are attracted to semiochemicals from different trophic levels, including host plant volatiles (Flint et al., 1979; Zhu et al., 1999, 2005; Hooper et al., 2002); herbivore- induced plant volatiles, such as methyl salicylate (MS; James, 2003a,b, 2006; James and Price, 2004); and sex pheromones of scale insects (Mendel et al., 2003) and aphids (Boo et al., 1998, 2003; Hooper et al., 2002). In particular, some lacewings are attracted to certain isomers of nepetalactone and nepetalactol, which are components of aphid pheromones and are also found in the catnip plant (Lamiaceae: Nepeta cataria L.; McElvain et al., 1941). Recently, we identified a male-produced pheromone from the goldeneyed lacewing, Chrysopa oculata Say, the first pheromone for any lacewing (Zhang et al., 2004). The synthetic pheromone, (1R,2S,5R,8R)-iridodial (IRI; Chauhan et al., 2004), outperformed all other reported attractants for the green lacewings in the genus Chrysopa, and has potential for manipulating lacewings for pesticide-free control of garden and agricultural pest insects (Zhang et al., 2004). Chrysopa nigricornis Burmeister is a large green lacewing distributed throughout the United States (Agnew et al., 1981). Similar to C. oculata, both the adults and larvae are predacious. It is the most common green lacewing species in the U.S. Pacific Northwest and is a useful component of integrated pest management programs in tree and vine crops such as hops (James, 2003a). To develop a pheromone-based attractant for this beneficial insect, we carried out a series of experiments in 2004 that focused on (1) pheromone production and perception in C. nigricornis with gas chromatographic–electroantenno- graphic detection (GC–EAD) and gas chromatographic (GC)–mass spectrometric analyses; (2) field testing the newly identified pheromone and other known lacewing semiochemicals, such as MS, Z,E-nepetalactone (ZE-lactone), Z,E-nepetalactol (ZE-lactol), 2-phenylethanol (2PE), and various combinations of these compounds in garden and orchard environments; and (3) dispenser technology, including dispenser types and the duration of attraction.

Methods and Materials

Adult Insects and Preparation of Extracts

Adult lacewings for GC–EAD and GC–mass spectrometric analyses were collected during summer 2004 from either sticky Delta traps or nonsticky grapefruit-shaped plastic traps in J Chem Ecol (2006) 32: 2163–2176 2165

Spokane, WA. Lacewings recovered quickly from sticky surfaces of the traps and were still viable for EAD recordings. C. nigricornis, C. oculata, C. coloradensis Banks, and the common green lacewing, Chrysoperla plorabunda (Fitch; = carnea) were collected for EAD recordings, but only male C. nigricornis were collected from nonsticky traps for dissection and GC–mass spectrometer. Adults of these lacewings were processed within 6 to 20 hr of capture for GC–EAD and GC–mass spectrometric analyses. Adult male C. nigricornis were anesthetized with CO2, eviscerated under tap water, and their thoracic cuticle was separated from their abdominal cuticle so that each body portion could be extracted individually in 50 μl of methyl tert-butyl ether. No head extracts were made because no glands had been previously reported from the heads of lacewings. All extracts were kept at −20°C until they were analyzed.

Gas Chromatographic–EAD and GC–Mass Spectrometric Analyses

Lacewing extracts and chemical standards were analyzed in splitless mode with a Varian CP-3800 GC equipped with a polar column (CP-Wax 52CB; 1.0 μm film thickness, 30 m × 0.53 mm i.d., Varian, Inc., Middelburg, the Netherlands), and a 1:1 effluent splitter that allowed simultaneous flame ionization detection and EAD of the separated volatile compounds. Helium was used as the carrier gas, and the injector temperature was 220°C. The column temperature was 30°C for 2 min, rising to 240°C at 10°C/min, and then held for 10 min. The outlet for the EAD was held in a humidified airstream flowing at 0.5 m/sec over an antennal preparation. EAD recordings were made by using silver wire-glass capillary electrodes filled with Beadle-Ephrussi Ringer (Zhang et al., 2000) on freshly cut antennae. The antennal signals were stored and analyzed on a PC equipped with a serial IDAC interface box and the program EAD version 2.5 (Syntech, Hilversum, the Netherlands). Antennally active peaks in the lacewing extracts were identified with a GC–mass spectrometer (Varian CP-3800 GC coupled to a Varian Saturn 2000 mass selective detector) operated in split mode (1:10) with a CP-Wax 52CB column (0.25 μm film thickness, 60 m × 0.25 mm i.d., Varian, Inc.), programmed at 30°C for 2 min, increasing to 240°C at 10°C/min, and then held for 10 min. Compounds were identified by comparison of mass spectra and retention times to those of authentic standards. In addition, EAD responses from male antennae of the four species noted above were recorded to a synthetic mixture (100 ng/μl each) containing five C. oculata-produced compounds (1-tridecene, nonanal, nonanol, nonanoic acid, and IRI); three compounds associated with aphid prey [(1R,4aS,7S,7aR)-nepetalactol, (4aS,7S,7aR)- nepetalactone (both sex pheromone components), and (4aS,7S,7aS)-nepetalactone (non- pheromone compound)]; and three herbivore-induced plant volatiles [MS, (Z)-3-hexenyl acetate (Z3HA), and benzaldehyde (BA)].

Chemical Standards

1-Tridecene (97%), 1-nonanol (97%), skatole (98%), MS (99%), 2PE (99%), Z3HA (98%), and BA (99.5%) were obtained from Aldrich Chemical (Milwaukee, WI); nonanal (99%) and nonanoic acid (98%) were from Emery Industries (Cincinnati, OH). (4aS,7S,7aR)- Nepetalactone and (4aS,7S,7aS)-nepetalactone are often referred to as Z,E- and E,Z- nepetalactone, respectively, in the literature (e.g., Hooper et al., 2002). ZE-lactone (98%), E,Z-nepetalactone (96%), (1R,4aS,7S,7aR)-nepetalactol [ca. 90% Z,E-isomer (Hooper et al., 2002) with ca. 2% impurity of iridodial isomers], and the lacewing pheromone, IRI [80%; with 20% of (1R,2S,5R,8S)-iridodial as an impurity] were isolated or synthesized as described by Chauhan et al. (2004). 2166 J Chem Ecol (2006) 32: 2163–2176

Field Trapping

Field-trapping experiments were carried out from early May through the end of September 2004 in either garden or small orchard environments in Spokane by using Pherocon VI traps (Trécé, Inc., Adair, OK) with removable sticky inserts. Traps were hung 1.0 to 1.5 m above the ground on either garden stakes or the branches of cherry (Prunus)/apple (Malus) trees ca. 5 to 10 m apart within each trap line. For each trapping experiment, two sets of traps (each set contained all treatments) were deployed with treatments allocated randomly to their initial trap positions within a set. The treatments were then systematically rotated among trap positions within a set after each replicate so that treatments appeared at least once per location (Latin-square design; Byers, 1991). To minimize positional ef- fects, lacewing collections and trap rotations were carried out when two or more lacewings were caught in any trap. Each replicate lasted several days to 1 wk, depending on lacewing flight activity. The sticky inserts were taken to the laboratory to record species, gender, and catch. Experiment 1 (May 7–August 3, 2004) was conducted to determine potential response to the newly discovered lacewing pheromone [from C. oculata (Zhang et al., 2004) and C. nigricornis (this article)], IRI (5 mg loaded onto rubber septa inserted into a 1.5-ml open plastic centrifuge tube; release rate not determined because of the low quantities emitted and the difficulty of applying the gravimetric method), and three other reported lacewing kairomone attractants, MS [2 g in a 12-mil (≈0.31 mm) polyethylene (PE) bag, Associated Bag Company, Milwaukee, WI, USA; 40 × 50 mm, with felt; with a release rate of ca. 30 mg/day, measured gravimetrically at 20 to 23°C in the laboratory], Z3HA (2 g in 4-ml open glass vial with 1/2 cotton ball; ca. 25 mg/day, measured gravimetrically at 20°C to 23°C in the laboratory), and 2PE [2 g in a 3-mil (≈ 0.076 mm) PE bag (40 × 50 mm) with felt; ca. 5 mg/day], in a full factorial experiment design with a total of 16 treatments (i.e., all individual compounds and all binary, ternary, and quaternary combinations). Each compound was loaded in a separate dispenser; thus, combinations have higher total releases than the individual treatments. Trap set 1 was deployed at a blackberry field (ca. 0.2 ha) in the Strawberry Hill Farm owned by Mr. Fallstorm that was surrounded by mixed pine (Pinus) forests. Trap set 2 was set up in Christensen’s Cherry Orchard (ca. 0.4 ha) with typical agricultural land surroundings. Experiment 2 (May 7–July 7, 2004) was similar to experiment 1, but tested BA [2 g in a 12-mil (≈ 0.31 mm) PE bag (40 × 50 mm) with felt; ca. 35 mg/day] in addition to the previous four individual compounds, along with selected combinations for a total of eight treatments. This study occurred in an urban setting in two smaller residential gardens (Florianovich and Rosengrant) in Spokane Valley, WA (one set of traps in each garden). Experiment 3 (July 28–August 24, 2004), with a total of six treatments, compared the responses to IRI with ZE-lactone, Z,E-nepetalactol, the combination of IRI with MS (5 mg of each compound loaded onto rubber septa), and a commercial product, Benallure™ (Gardens Alive Inc., Lawrenceburg, IN, USA). This study occurred at Strawberry Hill Farm described above. Experiment 4 (June 22–July 27, 2004), with a total of six treatments, tested the aging effect of IRI (5 mg) on rubber septum dispensers (aged from 0 to 5 wk). The dispensers were aged at ca. 20°C to 23°C in the laboratory hood with mild airflow (ca. 0.3 m/sec); the study occurred at Strawberry Hill Farm described above. Experiment 5 (August 5–September 30, 2004) with a total of seven treatments, tested the aging effect of the combination of IRI (5 mg) and MS (1.5 g) in 6-mil (≈0.16 mm) PE bags (20 × 50 mm; with felt; aged from 0 to 5 wk). Fresh IRI (5 mg) rubber septum dispenser J Chem Ecol (2006) 32: 2163–2176 2167

[0(RS)] was included as a positive control. The study occurred at Christensen’s Cherry Orchard described above. Experiment 6 (September 1–30, 2004), with a total of four treatments, tested the response to IRI (5 mg), MS (1.5 g), and their combinations, using PE bag type dispensers (6-mil ≈ 0.16 mm; 20 × 50 mm; with felt; ca. 50 mg/d release for MS; release for IRI unknown). Combinations included IRI + MS-1, where both compounds were loaded in one PE bag and IRI + MS-2, where the two compounds were loaded in separate PE bags. The study occurred at Christensen’s Cherry Orchard described above.

Statistical Analysis

For each experiment, data from the two sets of traps were pooled for statistical analysis because no block effects were found. Because of heterogeneity of variances among treat- ments, trap catch data (number of lacewings caught/trap/wk) were analyzed using the non- parametric Kruskal–Wallis ANOVA on rank test, followed by the Student–Newman–Keuls all pairwise comparison to separate means (Zar, 1984). The total trap catches between C. nigricornis and C. oculata within each experiment were compared by the χ2 goodness of fit test at α = 0.05.

Results

Gas Chromatographic–EAD and GC–Mass Spectrometric Analyses

Gas chromatographic–EAD analyses of extracts of thoracic and abdominal cuticle of male C. nigricornis (both forms: the western form with the entire antenna pale-white; the eastern form with the basal third of antenna dark; Penny et al., 2000) indicated no EAD responses by male antennae to any peaks from abdominal extracts (Fig. 1c), whereas two peaks from thoracic extracts elicited significant antennal responses by males of both forms (Fig. 1c; females were not available for testing). The two EAD-active compounds were identified as 1-tridecene and IRI by comparison of the mass spectra and retention times to those of authentic standards (for details, see Zhang et al., 2004). Other major components from the thoracic extracts were (E)-5-undecene, a C13-hydrocarbon with two double bonds, 2- methylpropanoic acid, N-3-methylbutylacetamide, 2-methyl-1-H-indole, and skatole (3- methyl-1-H-indole), all of which elicited no EAD response (Fig. 1). GC–EAD analyses with male C. nigricornis antennae using synthetic mixtures showed that nonanal, MS, and IRI elicited higher EAD responses than did nonanol, 1-tridecene, ZE-nepetalactone, EZ- nepetalactone, ZE-nepetalactol, BA, or 2PE (Table 1). A similar EAD response pattern was also found for male C. coloradensis; however, its antennal responses to IRI were relatively weak compared with those of C. nigricornis. Antennae of male Cl. plorabunda showed similar EAD responses to most of the compounds in the synthetic mixture, but were unresponsive to IRI and its (8S)-stereoisomer (Table 1).

Field-Trapping Experiments

At least four species of green lacewings, C. nigricornis (both western and eastern forms; >75%), C. oculata (ca. 24%), C. coloradensis (<2%), and Cl. plorabunda (rare) were caught in the various treatments of all experiments. In addition, other beneficial insects, 2168 J Chem Ecol (2006) 32: 2163–2176 such as the brown lacewing, Hemerobius ovalis Carpenter (total catch, 10), the hoverfly, Metasyrphus americanus (Weidemann; total catch, 100), and a few convergent lady beetles, Hippodamia convergens (Guérin-Méneville), were captured in the traps. In experiment 1 (at Strawberry Hill Farm and Christensen’s Cherry Orchard), a total of 495 C. nigricornis males and 192 C. oculata males were caught. More C. nigricornis were captured than C. oculata (χ2 = 134, df =1,P < 0.001). There was an effect of treatment for males of both species (C. nigricornis: H = 215.6, df = 15, P < 0.001; C. oculata: H = 149.7, df = 15, P < 0.001), and traps baited with IRI alone or combined with other test compounds caught more males of both species than did unbaited traps (Fig. 2). MS, Z3HA, and 2PE alone were inactive. No females of either species were captured. In experiment 2 (Florianovich and Rosengrant residential gardens), a total of 10 C. nigricornis males and 106 C. oculata males were caught and there was a treatment effect for both species (C. nigricornis: H = 19.8, df =7,P = 0.006; C. oculata: H = 68.3, df =7, P < 0.001). Again, traps baited with IRI alone or its binary combination with MS or BA were attractive to males of both species, whereas MS, Z3HA, 2PE, and BA were inactive when presented alone (Fig. 3). In contrast to experiment 1, traps baited with IRI alone or mixtures containing IRI caught more C. oculata than C. nigricornis, which may have been because of the habitat differences between the experimental sites (χ2 = 79.1, df =1, P < 0.001). In experiment 3 (Strawberry Hill Farm), 155 C. nigricornis males and 48 C. oculata males were caught. More C. nigricornis were captured than C. oculata (χ2 = 56.4, df =1, P < 0.001). There was a treatment effect for both species (C. nigricornis: H = 56.4, df =5, P < 0.001; C. oculata: H = 44.8, df =5,P < 0.001). IRI attracted more males of both lacewing species than did ZE-lactol (Fig. 4). The number of C. nigricornis males in ZE-

EAD a Skatol iridodial 1-tridecene )-5-undecene E FID ( C13-HC with 2 double with bonds C13-HC 2-methyl-1-H-indole N-3-methylbutylacetamide 2-methyl propanoicacid

b FID

EAD

7:00 9:00 11:00 13:00 15:00 17:00 19:00 21:00 23:00 Retention time (min) Fig. 1 Gas chromatographic–EAD responses of male C. nigricornis (western form) to (a) thoracic and (b) abdominal cuticle extracts of conspecific males hmEo 20)3:2163 32: (2006) Ecol Chem J

Table 1 Electroantennographic detection responses of male Chrysopa and Chrysoperla spp. to pheromone components and other semiochemicals

Chemicals Natural Speciesa sources C. C. C. C. Cl. Cl. oculata nigricornis coloradensis quadripunctata plorabunda rufilabris

Tridecane Unknown ●● ● ● ● ● – 162169 2176 1-Tridecene Lacewing thorax ●● ●● ● ●● ●● ●● (Z)-3-hexenyl acetate Plants; HIPVb ○○ ○ c ○ c Nonanal Lacewing abdomen/plants ●●●● ●●●● ●●●● ●●●● ●●●● ●●●● Benzyaldhyde Plants; HIPV ● ● ●● ● ● ● Nonanol Lacewing abdominal cuticle ●● ● ● ●● ●● ●● Methyl salicylate HIPV ●● ●●● ●●● ●● ●● ●● (1R,2S,5R,8R)-iridodial Lacewing abdominal cuticle/thorax ●●●●● ●●● ● ●●●●● ○ ○ (1R,2S,5R,8S)-iridodial Unknown ●● ●● d ●● ○ ○ 2-Phenylethanol Plants ●● ● ● ● ● (4aS,7S,7aR)-Nepetalactone (ZE) Catnip/aphid sex pheromone ●● d ●●● (4aS,7S,7aS)-Nepetalactone (EZ) Catnip ●● ● ● ● ● (1R,4aS,7S,7aR)-nepetalactol (ZE) Catnip/aphid sex pheromone ●● ●● d ●● ●● ●● Nonanoic acid Lacewing abdominal cuticle ●● ●● ● ●● ●● ●● Skatole Lacewing thorax ○○ ○ ○ ○ ○ Referencese 1, 2 1, 2 2 2 1 2 1 a ● EAD active; ○ EAD inactive at the concentration (100 ng/μl) tested. b HIPV: herbivore-induced plant volatile. c Not tested yet. d Results not clear, more tests needed to confirm. e 1, Zhang et al. (2004); 2, this study. 2170 J Chem Ecol (2006) 32: 2163–2176

8 BC C. nigricornis (N=36) BC C 7 C. oculata (N=36) BC 6 BC 5

spp./trap/wk +SE 4 B b B B

3 b b

Chrysopa b b 2 b b b

1 No. male A A A a A a A a a A a A a a A a 0 MS - + - - - + + + - - - + + + - + IRI - - + - - + - - + + - + + - + + Z3HA - - - + - - + - + - + + - + + + 2PE - - - - + - - + - + + - + + + + Treatment Fig. 2 Captures of male C. nigricornis and C. oculata in traps baited with MS, IRI, Z3HA, 2PE, and all possible binary, ternary, and quaternary combinations, Strawberry Hill Farm, and Christensen’s Cherry Orchard, Spokane, WA, May 7 to August 3, 2004. Bars with the same letter within the same species indicate means that are not significantly different (P > 0.05), Kruskal–Wallis ANOVA on ranks, followed by the Student–Newman–Keuls all pairwise comparison test

5 C. nigricornis (N=8) b C. oculata (N=18) 4 b b

3 spp./trap/wk +SE spp./trap/wk

2

B Chrysopa B 1 a B

No. male No. A a A a A a A a A 0 Blank MS IRI Z3HA 2PE BA MS+IRI IRI+BA Treatment Fig. 3 Captures of male C. nigricornis and C. oculata in traps baited with MS, IRI, Z3HA, 2PE, BA, and selected binary combinations; at Florianovich and Rosengrant residential gardens, Spokane Valley, WA, May 7 to July 7, 2004. Bars with the same letter within the same species indicate means that are not significantly different (P > 0.05), Kruskal–Wallis ANOVA on ranks, followed by the Student–Newman– Keuls all pairwise comparison test J Chem Ecol (2006) 32: 2163–2176 2171

16 D C. nigricornis (N=14) 14 C. oculata (N=14)

12

10 spp./trap/wk +SE 8 C b 6

Chrysopa b B 4

No. male No. 2 a A a Aa A a 0 Blank IRI ZE-lactol ZE-lactone IRI+MS BenallureTM Treatment Fig. 4 Captures of male C. nigricornis and C. oculata in traps baited with IRI, ZE-lactol, ZE-lactone, IRI + MS, and the commercial lacewing/lady beetle attractant, Benallure™ at Strawberry Hill Farm, Spokane, WA, July 28 to August 24, 2004. Bars with the same letter within the same species indicate means that are not significantly different (P > 0.05), Kruskal–Wallis ANOVA on ranks, followed by the Student–Newman– Keuls all pairwise comparison test

Fig. 5 Captures of C. nigricornis, C. oculata, and C. coloradensis in traps baited with aged PE bag dispensers loaded with 5 mg of IRI and 1.5 g of MS compared with the attraction to fresh rubber septa [0 (RS); loaded only with 5 mg IRI] at Christensen’s Cherry Orchard, Spokane, WA, August 5 to September 30, 2004. Bars with the same letter for C. nigricornis indicate means that are not significantly different (P > 0.05), Kruskal–Wallis ANOVA on ranks, followed by the Student–Newman–Keuls all pairwise comparison test. No further pairwise comparisons were made for C. oculata and C. coloradensis because no significant treatment effects were detected by ANOVA 2172 J Chem Ecol (2006) 32: 2163–2176

Fig. 6 Captures of C. nigricornis, C. oculata, and the hoverfly, M. americanus, in traps baited with IRI, MS, and their binary blends in PE bag dispensers (IRI + MS-1: in the same PE bag; IRI + MS-2: in two separate PE bags) at Christensen’s Cherry Orchard, Spokane, WA, September 1 to 30, 2004. Bars with the same letter within the same species indicate means that are not significantly different (P > 0.05), Kruskal–Wallis ANOVA on ranks, followed by the Student–Newman–Keuls all pairwise comparison test. No further pairwise comparisons for M. americanus took place because no significant treatment effects were detected by the ANOVA lactol-baited traps was higher than that in the unbaited traps. ZE-lactone and the commercial attractant, Benallure™, were both inactive. Interestingly, addition of MS to IRI increased the trap catches for C. nigricornis, but not for C. oculata (Fig. 4). In experiment 4 (Strawberry Hill Farm), all traps baited with 5 mg of IRI on rubber septa aged from 0 to 5 wk caught males of both C. nigricornis (59) and C. oculata (32). More C. nigricornis were captured than C. oculata (χ2 = 8.01, df =1,P = 0.005), but there was no treatment effect in the experiment for either species (C. nigricornis: H = 7.9, df =5,P = 0.16; C. oculata: H = 9.04, df =5,P = 0.11). In experiment 5 (Christensen’s Cherry Orchard), traps baited with variously aged PE bag dispensers containing 5 mg of IRI plus 1.5 g of MS caught a total of 1150 C. nigricornis, 109 C. oculata males, 28 C. coloradensis males, 57 M. americanus, and 26 H. convergens. The ratio of eastern/western forms of C. nigricornis in the traps was ca. 1:3. More C. nigricornis were captured than C. oculata (χ2 = 861, df =1,P < 0.001). There was a treatment effect in the experiment for C. nigricornis (H = 30.1, df =6,P < 0.001), but not for C. oculata (H = 1.56, df =6,P = 0.96) or C. coloradensis (H = 10.2, df =6,P = 0.12). All C. nigricornis captured during the August and early September were males; however, from mid to late September, 23 out of the 63 caught (mostly the western form) were females. Trap catches for C. nigricornis seemed to decrease with the lure age (Fig. 5), but this decline was not statistically significant. The 0- to 5-wk-old PE bag dispensers were significantly (five to six times) more attractive to C. nigricornis than were the fresh IRI rubber septa dispensers [0(RS)], although the total loading of IRI was the same. Thus, the PE bag dispensers loaded with IRI and MS should stay attractive for at least 5 wk in the field. J Chem Ecol (2006) 32: 2163–2176 2173

In experiment 6 (Christensen’s Cherry Orchard), a total of 61 C. nigricornis (41 males and 20 females), 18 C. oculata males, and 28 M. americanus were captured during September 2004. More C. nigricornis were captured than C. oculata (χ2 = 23.4, df =1, P < 0.001). There were treatment effects for C. nigricornis (H = 7.8, df =3,P = 0.049) and C. oculata (H = 9.32, df =3,P = 0.025), but not for M. americanus (H = 1.87, df =3, P = 0.60). IRI (5 mg in PE bag dispenser) alone or combined with MS in the same PE bag (IRI + MS-1) or in separate PE bags (IRI + MS-2) were significantly more attractive to C. nigricornis than was MS alone (Fig. 6). No synergistic effect on C. nigricornis and M. americanus was found between IRI and MS; however, the combination of these two compounds in separate PE bags caught significantly more C. oculata than did the IRI or MS alone (Fig. 6). More work is needed to confirm the effects of MS (with or without IRI) on both lacewing species.

Discussion

(1R,2S,5R,8R)-Iridodial was recently discovered as a male-produced pheromone of the goldeneyed lacewing, C. oculata, and is believed to be produced in elliptical glands abundantly distributed between the third and eighth abdominal sternites of the males (Zhang et al., 2004). Our current study indicates that IRI is also a male-produced (females were not available for chemical analysis) pheromone for the green lacewing, C. nigricornis. It elicited an EAD response, and attracted conspecific males into traps. It also attracted some females late in the season. In contrast to C. oculata, IRI was detected in C. nigricornis males from thoracic extracts, rather than from abdominal cuticle extracts. Thus, we expect pheromone-producing glands in the thorax of this species. Another antennally active compound from thoracic extracts of C. nigricornis males, 1-tridecene, was not field tested in the current study because it was reported as one of the defensive compounds produced in the thoracic glands of C. oculata and reduced the numbers of C. oculata males captured in IRI-baited traps (Zhang et al., 2004). In addition to the responses of C. nigricornis and C. oculata, a few males of C. quadripunctata (Zhang et al., 2004) and C. coloradensis (this article) were captured in the pheromone-baited traps. It is not known if these two species also use IRI or its isomers as pheromone components. The low-trap catches might be a result of either extremely low populations in the test areas or missing pheromone component(s) in the treatments. Recently, males of a Eurasian green lacewing species, C. septempunctata Wesmael, were also found to be strongly attracted to synthetic IRI (Zhang et al., 2006). Two other common green lacewing species from the genus Chrysoperla, Cl. plorabunda (= C. carnea) and Cl. rufilabris Burmeister, did not respond to IRI antennally or behaviorally in this and in a previous study (Table 1; Zhang et al., 2004). Methyl salicylate is reportedly an attractant for both sexes of C. nigricornis and C. oculata (James, 2003a, 2006) along with several other beneficial insects (James and Price, 2004). James and Price (2004) also recently found evidence for recruitment and retention of beneficial insects in grapes and hops by using controlled-release dispensers of MS, in Prossor, WA. However, in our study, in Spokane, WA, ca. 300 km northeast of Prossor, MS was inactive alone, but in a few instances enhanced the responses of Chrysopa spp. to IRI. MS alone and its binary blend with IRI attracted another beneficial insect, the hoverfly, M. americanus. Surprisingly, the commercial lacewing/ladybeetle attractant, Benallure™, did not catch any lacewings or other beneficial insects in our experiments. ZE-lactol was slightly 2174 J Chem Ecol (2006) 32: 2163–2176 attractive to C. nigricornis and C. oculata, which might be because of the presence of small amount of IRI as an impurity, whereas ZE-lactone was inactive at the release rate tested. If, indeed, some sympatric Chrysopa species share IRI as a pheromone component, there might be cross-attraction between species, which might undermine species isolation. However, earlier studies on acoustical communication of green lacewings showed that chrysopids produce species-specific, low-frequency, substrate-borne vibrations that guide the conspecifics to one another on a plant (Henry, 1982). Comparative acoustical studies of Chrysoperla versus Chrysopa species indicated that Chrysoperla spp. [C. rufilabris, C. carnea (Stephens), and C. downesi (Smith)] are more dependent on acoustic signals for mating success than are Chrysopa spp. (C. oculata and C. chi Fitch; Henry, 1979, 1980a, b, c). Thus, species of Chrysoperla rely on acoustic communication with no obvious role for pheromones, whereas Chrysopa species communicate with pheromones at long range and, to a lesser extent than Chrysoperla, with species-specific acoustic signals at short range for courtship and isolation from sympatric species. Adults of Chrysopa (sensu stricto) are predacious, whereas adults of Chrysoperla are phytophagous (Principi and Canard, 1984), suggesting that predation in the adult stage somehow favors chemical communication or selects against communication by substrate vibration. Surprisingly, no females of C. oculata were caught during any of our current tests nor in the experiments carried out by Zhang et al. (2004), although antennae from the females were as sensitive to IRI and other male-produced compounds as were the antennae of males (Zhang et al., 2004). Sweep netting and live insect counting at a soybean field in 2004 at Beltsville, MD, showed that IRI not only attracted C. oculata males, but also females to the pheromone-baited plots (Chauhan et al., unpublished data). However, unlike males, the females did not completely approach and enter the traps, possibly because females call males acoustically at close range or females require male-produced, species-specific acoustical signals for close-range communication. Similar to C. oculata, no females of C. nigricornis were captured in IRI-baited traps during the summer season; however, in the early fall (September), some C. nigricornis females were captured in IRI or IRI/MS-baited traps. Our field testing indicated that the lacewing pheromone, IRI, loaded onto either rubber septa or its binary blend with MS in PE bags remained attractive for at least 5 wk in the field during the mid-summer season for both C. nigricornis and C. oculata. Synthetic lacewing pheromone IRI or its combination with MS (the herbivore-induced plant volatile) attracts and retains both male and female lacewings to treated areas, which will probably elevate the local lacewing population (Chauhan et al., unpublished data). Furthermore, the more males attracted to the treated area, the more natural pheromone would be released by males, further strengthening attraction and potentially enhancing biological control. Moreover, IRI and MS or their binary blend also attract many other beneficial insects, such as hoverflies, lady beetles, and predatory bugs (James and Price, 2004 and this article). Based on this study, a new lacewing attractant/trap system is being developed for both domestic and international markets. This product will have potential for manipulating natural or artificially augmented populations of lacewings and other beneficial insects to enhance biological control of garden, agricultural, and forest pest insects.

Acknowledgments We thank Dr. Oliver S. Flint, Jr. (Section of Entomology, Smithsonian Institution, Washington D.C.) and Dr. N. D. Penny (Department of Entomology, California Academy of Sciences, San Francisco, CA) for identifications of lacewings and other insect species; and Mr. Fallstrom, Mr. Christensen, Mr. Florianovich, and Mr. Rosengrant (in Spokane, WA) for allowing us to carry out our field-trapping experiments on their property. J Chem Ecol (2006) 32: 2163–2176 2175

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