RProceedingsesponse of invasiveof the H longhoawaiian rEnntomological to phe Societyromones (2019) on H 51(2):15–23awaii 15

Response of Invasive Longhorn Beetles (Coleoptera: ) to Known Cerambycid Aggregation-Sex Pheromones in the Puna District of Hawaii Island

R. Maxwell Collignon,1,2* Matthew S. Siderhurst,2 Jocelyn G. Millar,3 and Dong H. Cha1 1USDA-ARS, Pacific Basin Agricultural Research Center, Hilo, Hawaii, USA. 2Eastern Mennonite University, Harrisonburg, Virginia, USA. 3University of California Riverside, Riverside, California, USA. *[email protected]

Abstract. The Queensland longhorn borer (QLB; Acalolepta aesthetica [Olliff 1890]; Coleoptera: Cerambycidae: Lamiinae: Monochamini) and plumeria long- horn borer (PLB; Lagocheirus obsoletus [Thomson 1778] = Lagocheirus undatus [Voet 1778]; Coleoptera: Cerambycidae: Lamiinae: Acanthocini) are invasive longhorn species that have become established on the island of Hawaii. Both QLB and PLB are polyphagous. Known hosts of QLB include cacao, citrus, kukui, and breadfruit in Hawaii, and QLB are known to attack live, healthy trees. Currently the beetle occurs in the Puna district of the island, but its range is expanding. PLB is a pest of plumeria and other ornamental plants throughout the state of Hawaii and elsewhere. As a first step towards developing a monitoring tool for these invasive beetles, we tested four known aggregation-sex pheromones of cerambycids in this subfamily—monochamol, fuscumol acetate, fuscumol, and geranylacetone—that have proven effective for attracting more than 30 lamiine species in different areas of the world. When tested in panel traps, these compounds individually and in a blend attracted 9 QLB total, which was not significantly different than the 5 QLB captured in solvent control traps. In contrast, traps baited with one of the tested compounds, fuscumol acetate, captured significantly more PLB than solvent blank control traps. We discuss future research directions for developing attractants using chemical ecology approaches to monitor QLB and PLB.

Key words: invasive species, longhorn beetles, pheromone trapping

Longhorn beetles (Coleoptera: Ceram- high likelihood of cerambycid species be- bycidae) are ecologically and economi- ing introduced into novel habitats (Haack cally important, both as beneficial native 2006). in forest systems (Haack and Byler All known examples of volatile phe- 1993, Grove 2002, Teale and Castello romones from cerambycids in the sub- 2011, Ulyshen 2014) and as invasive pests family Lamiinae are male-produced in recipient habitats (Haack et al. 2010, aggregation-sex pheromones that attract Parry and Teale 2011). Because larvae both sexes, with multiple species often can persist cryptically within wooden “sharing” pheromones. So far, four basic commodities shipped internationally structures have been identified or impli- (e.g., lumber, wooden shipping materials, cated as aggregation-sex pheromones for and finished wooden products), there is a ~30 lamiine species (Hanks and Millar 16 Collignon et al. 2016). These structures include fuscumol beetle (Anoplophora glabripennis and [(E)-6,10-dimethyl-5,9-undecadien-2-ol], Anoplophora chinensis, respectively; the corresponding ester fuscumol acetate Cerambycidae: Lamiinae: Monochamini). [(E)-6,10-dimethyl-5,9-undecadien-2- The plumeria longhorn borer (PLB) is yl acetate], geranylacetone [(E)-6,10- known from the southern United States, dimethyl-5,9-undecadien-2-one], and the Caribbean, Central America, South monochamol [2-(undecyloxy)ethanol] America, and the South Pacific Islands (e.g., Fonseca et al. 2010, Pajares et al. (Mondaco 2008). It was first recorded in 2010, Mitchell et al. 2011, Wickham et al. Hawaii in 1890 (Gressit and Davis 1972). 2014, Ryall et al. 2015, Hughes et al. 2016, The beetle is polyphagous, with recorded Meier et al. 2016, Bobadoye et al. 2018). hosts in the genera Plumeria, Manihot, Fuscumol, fuscumol acetate, and gerany- Bursera, Euphorbia, Ficus, Forestiera, lacetone are also pheromone components Jatropha, Spondias, Araucaria, Capsi- for cerambycid species in the subfamily cum, Gossypium, Hibiscus, and Yucca Spondylidinae (e.g., Silk et al. 2007, Hal- (Mondaco 2008). The species is a pest loran et al. 2018). of concern on Oahu (MM Ramadan, The Queensland longhorn borer (QLB) pers. comm.). As a first step towards Acalolepta aesthetica (Olliff 1890) developing monitoring and possibly and the plumeria longhorn borer (PLB) control tactics for these invasive species, Lagocheirus obsoletus (Thomson 1778) we field tested the four generic lamiine (= Lagocheirus undatus [Voet 1778]) are pheromones described above—fuscumol, two invasive cerambycid species from the fuscumol acetate, geranylacetone, and subfamily Lamiinae (tribes Monochamini monochamol—as possible attractants for and Ancanthocini, respectively) that have these two invasive species. Here, we report become established in Hawaii. QLB is the results of those tests. native to Australia and was first collected from the Puna district of Hawaii island in Methods and Materials 2009 (Matsunaga and Chun 2018). The Field bioassays testing fuscumol, large beetles (up to 4.5 cm body length) fuscumol acetate, geranylacetone, and are polyphagous: confirmed hosts include monochamol. At three sites in the upper ulu (breadfruit; Artocarpus altilis), kukui Puna district of Hawaii island, we de- (candlenut; Aleurites mouccanus), queen ployed black cross-vane, panel intercept sago palm (Cycas circinalis), Citrus spp., traps coated with Fluon (Alpha Scents, and cacao (Theobroma cacao). Gunpow- Portland, Oregon, USA; Graham et al. der tree (Trema orientalis) and avocado 2010) hung from the lower branches of (Persea americana) are two other pos- various tree species; beetles were live sible hosts, but these require verification trapped for use in laboratory assays. All (Matsunaga and Chun 2018). Congeners three sites were in a 4 km2 area (center: of QLB have been shown to be attracted 19°34’11.2”N 154°59’57.7”W). The first to monochamol in China (Wickham et two sites were on properties in residen- al. 2014), and to pheromones from the tial areas (i.e., surrounded on all sides subfamily Cerambycinae in Australia by other residential properties), one with (Hayes et al. 2016). QLB can success- several kukui, citrus, avocado, and plu- fully attack live, apparently healthy trees meria among other trees on the property (SG Chun pers. comm.; RMC pers. obs.), (two spatial replicates), and the other with a behavior shared with the polyphagous kukui, ulu, cacao, and plumeria among Asian and citrus longhorn other trees on the property (one replicate). Response of invasive longhorn beetles to pheromones on Hawaii 17 Both residential sites had active infesta- fuscumol (50 mg/ml), racemic fuscumol tions of QLB in about 20–30% of possible acetate (50 mg/ml), geranylacetone (25 mg/ host trees on each property (RMC, pers. ml), a blend of these three compounds (at obs.); hosts were abundant in the neighbor- the same concentrations as the individual- hoods as well. The third site was situated compound treatments), and blank solvent on a cacao farm with a few interplanted control. The pheromones were dispensed citrus trees and was surrounded by jungle from low-density polyethylene bags (0.05 (one replicate). This third site had active mm wall thickness, 5 cm x 7.5 cm; Fisher infestations of QLB in approximately Scientific, Milwaukee, Wisconsin, USA) 10–20% of the cacao trees at any given with 2-propanol as the solvent; 1 ml of time (RMC, pers. obs.). Infestations were solution of a given treatment was loaded being actively managed via hand-removal into a lure. Lures were changed weekly. by the farmer (the cacao trees were ap- Monochamol and geranylacetone were proximately 5 cm in diameter and thus had used at a concentration of 25 mg/ml rather relatively thin bark, so beetle damage by than 50 mg/ml because they are single late-instar larvae could be diagnosed by isomer compounds, in contrast to the two looking for soft spots along the bark). enantiomeric isomers present in racemic At the two residential sites, due to the fuscumol and fuscumol acetate. All phero- heterogeneous distribution of host and mones were purchased from Bedoukian non-host trees, traps with different treat- Research (Danbury, Connecticut, USA). ments were set up amid potential host Collected beetles were sexed based trees, whereas the cacao farm site allowed on antennal length and abdomen size for for traps to be deployed in banana trees QLB (males have antennae ~2–2.5x body (not a known host for either species) ~20 m length and less robust abdomens, whereas away from the citrus trees and ~50 m from females have antennae ~1.5x body length the cacao trees (QLB-hosts), to limit posi- and much rounder and more robust ab- tional effects. Traps were placed roughly domens), and based on presence (male) 10–15 m apart in linear transects for each or absence (female) of antennal spurs on of the spatial replicates at the three sites, the tip of the 4th antennal flagellomere for with a treatment randomly assigned to PLB (IP Swift, pers. comm.). We did not each trap. At the site in which there were have the sexing characteristics available two transects, the transects were 20 m for PLB for the first experiment and begin- apart. Traps were checked approximately ning of the second experiment, thus some weekly, and the order of treatments along beetles are listed as ‘unsexed’ in Table 1. a given transect was re-randomized after Voucher specimens of both species have each check. been archived at U.S. Pacific Basin Agri- Two different subsets of candidate pher- cultural Research Center (Hilo, Hawaii), omone compounds were tested individu- and are available upon request. ally or in a blend in two consecutive field Statistical analyses. The field trapping trapping experiments over the three sites. experiments were spatially and temporally The first experiment ran from 17 April replicated, with temporal replicates repre- through 14 June 2018, and tested three senting the number of times trap catches treatments: racemic fuscumol acetate (50 were counted. Temporal replicates where mg/ml), monochamol (25 mg/ml), and no beetles of a given species were trapped blank solvent control. The second experi- in any treatment were removed from ment ran from 14 June through 30 August analyses, as zero-catch days were typi- 2018, and tested five treatments: racemic cally associated with inclement weather. 18 Collignon et al. Table 1. Total number of QLB (Acalolepta aesthetica) and PLB (Lagocheirus obsoletus) captured in traps baited with generic aggregation-sex pheromones (fuscumol, fuscu- mol acetate, monochamol, and geranylacetone) known for the cerambycid subfamily Lamiinae. Solvent blank was 2-propanol. The blend consisted of fuscumol, fuscumol acetate, and geranylacetone. QLB PLB Treatment Female Male Female Male Unsexed

Experiment 1 Solvent blank 3 1 0 0 0 (17 Apr– Fuscumol acetate 1 2 0 0 7 14 Jun 2018) Monochamol 0 1 0 0 1

Experiment 2 Solvent blank 1 0 1 0 0 (14 Jun– Fuscumol 1 0 0 0 0 30 Aug 2018) Fuscumol acetate 1 1 1 2 6 Geranylacetone 1 0 0 0 0 Blend 0 1 1 1 2

Because of low trap catches, species were ≥ 0.15; exp. 2: p ≥ 0.99; Figs. 1 and 2). analyzed by the total number of beetles A total of 8 unsexed PLB were trapped caught to increase statistical power (Table in the first experiment (4 replicates without 1). Data were analyzed with a max-t test zero-catch days), and 3 females, 3 males, (Herberich et al. 2010). This test is ro- and 8 unsexed individuals in the second bust to departures from assumptions of experiment (10 replicates without zero- normality and homogeneity of variances, catch days; Table 1). In both experiments, a common occurrence with trap catch fuscumol acetate was significantly more data. These analyses were conducted in R attractive (16 PLB total) than the solvent (3.4.2; The R Foundation; R Core Team, controls (1 PLB total) (exp. 1: p = 0.02; 2017). exp. 2: p = 0.037; Figs. 3 and 4), whereas no other treatments were significantly Results different from the solvent control. Field assays testing fuscumol, fuscu- mol acetate, geranylacetone, and mono- Discussion chamol. A total of 4 female and 4 male None of the traps baited with the four QLB were trapped in the first experiment known cerambycid pheromones captured (5 replicates, once zero-catch days were more QLB than control traps. This was removed from the analysis), and 4 and 2 somewhat surprising, in part based on the respectively in the second experiment (3 generic nature of these compounds in the replicates without zero-catch days; Table attraction of other species of lamiines (see 1). There was no significant difference in Introduction), but also because Acalolepta trap catches between the solvent controls formosana (Breuning 1935), a species (5 QLB total) and any of the pheromone closely related to QLB, was found to be treatments (5 total QLB in fuscumol attracted to monochamol in China (Wick- acetate traps, and 1 each in the other ham et al. 2014), using trapping methods pheromone treatments; Table 1) (exp. 1: p similar to those used in this research. Response of invasive longhorn beetles to pheromones on Hawaii 19

Figure 1. Mean (± SE) numbers of QLB (Acalolepta aesthetica) caught in traps baited with solvent control, fuscumol acetate, or monochamol (experiment 1). There were no significant differences between the treatments and the solvent control (max-t test, p > 0.05).

Figure 2. Mean (± SE) numbers of QLB (Acalolepta aesthetica) caught in traps baited with solvent control, fuscumol, fuscumol acetate, geranylacetone, or a blend of the three compounds (experiment 2). There were no significant differences between the treatments and the solvent control (max-t test, p > 0.05).

In contrast, the bioassay data indicated significantly more beetles than con- that fuscumol acetate may be an attract- trols. Based on the lower trap catches ant pheromone candidate for PLB, with in the blend of fuscumol+fuscumol fuscumol acetate baited traps catching acetate+geranylacetone versus the fus- 20 Collignon et al.

Figure 3. Mean (± SE) numbers of PLB (Lagocheirus obsoletus) caught in traps baited with solvent control, fuscumol acetate, or monochamol (experiment 1). Means with an asterisk are significantly different than the solvent control (max-t test, p < 0.05).

Figure 4. Mean (± SE) numbers of PLB (Lagocheirus obsoletus) caught in traps baited with solvent control, fuscumol, fuscumol acetate, geranylacetone, or a blend of the three compounds (experiment 2). Means with an asterisk are significantly different than the solvent control (max-t test, p < 0.05). cumol acetate alone, fuscumol and/or is commercially available and relatively geranylacetone may be antagonistic. inexpensive to deploy. Furthermore, based From a management standpoint, this on recent results with other lamiine spe- is a useful discovery for monitoring or cies showing increased lure efficacy to control of PLB, because fuscumol acetate naturally produced enantiomeric ratios Response of invasive longhorn beetles to pheromones on Hawaii 21 (Hughes et al. 2016, Meier et al. 2016, compounds released by QLB and PLB, Silva et al. 2019), it is possible that PLB identification of attractive kairomone produces only one enantiomer of fuscumol compounds released from host plants, acetate, or a nonracemic mixture of the food and symbiotic microbes, and optimi- enantiomers. Thus, future research will zation of ratio and release rates of those evaluate whether correct enantiomeric chemicals to develop effective monitoring blend could improve lure efficacy. and potential behavioral control methods Because QLB were not attracted to any to manage QLB and PLB in Hawaii. of the four compounds tested here, even though we knew they were present due to Acknowledgments continued new infestations of host trees in We thank Stacey G. Chun of the Hawaii the vicinity and the continuous trapping of Department of Agriculture for sharing his QLB adults in low numbers in the various findings regarding QLB, helping scout treatments (including solvent blank treat- out field sites, and acting as initial liaison ment traps), this suggests that (1) QLB between the authors and the property does not use attractant pheromones at all, owners and East Hawaii Cacao Growers (2) its pheromone consists of compounds Association (EHCGA). We thank Patrick other than those tested, or (3) one of the Merritt, president of the EHCGA for help- enantiomers in the racemic blends or the ing coordinate our efforts with EHCGA ratio tested was antagonistic. The first members. We also thank the property possibility seems unlikely because, of the owners and EHCGA farmer who allowed several hundred cerambycid species that us to conduct field assays on their prop- have been examined for pheromone use, erties. We also thank Eastern Mennonite species in only one genus (Phoracantha University students Hannah Walker and Newman 1840, in the subfamily Ceram- Leah Lapp for assistance in the field. 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