A male-produced aggregation-sex pheromone of the beetle Arhopalus rusticus (Coleoptera: Cerambycidae, Spondylinae) may be useful in managing this invasive species

The longhorned beetle Arhopalus rusticus (Coleoptera: Cerambycidae, Spondylinae) is a common species in conifer forests of the Northern Hemisphere, but with global trade, it has invaded and become established in New Zealand, Australia, and South America. Arhopalus rusticus is a suspected vector of the phytopathogenic nematode, Bursaphelenchus xylophilus, the causative agent of pine wilt disease, which is a major threat to pine forests worldwide. Here, we report the identification of a volatile, male-produced aggregation-sex pheromone for this species. Headspace odours from males contained a major male-specific compound, identified as (2 S, 5E)-6,10-dimethyl-5,9-undecadien-2-ol (common name (S)-fuscumol), and a minor component (E)-6,10-dimethyl-5,9-undecadien-2-one (geranylacetone). Both compounds are known pheromone components for species in the same subfamily. In field trials in its native range in Slovenia, (S)-fuscumol was significantly more attractive to beetles of both sexes, than racemic fuscumol and a blend of host plant volatiles commonly used as an attractant for this species. Fuscumol-baited traps also caught significant numbers of another spondylidine species, Spondylis buprestoides (L.), and a rare click beetle, Stenagostus rufus (De Geer). The pheromone can be exploited as a cost-effective and environmentally safe tool for detection and monitoring of this invasive species at ports of entry, and for monitoring the beetle’s distribution and population trends in both endemic and invasive populations.


Results
While field testing a series of known cerambycid pheromones (AZK, unpub. data), we noticed that A. rusticus beetles were being caught specifically in traps baited with racemic fuscumol and its acetate, which was the first indication that the alcohol and/or its acetate might be pheromone compounds of this species. Live males and females were returned to the laboratory for collection of headspace volatiles. Three out of five extracts of headspace volatiles collected from males were dominated by two peaks, and no other insect-produced compounds were consistently present (Fig. 1). The major peak was identified as (E)-6,10-dimethyl-5,9-undecadien-2-ol (fuscumol) (diagnostic ions: m/z 196, 178, 109, 69) ( Fig. 2A), and a second, minor peak as (E)-6,10-dimethyl-5,9-undecadien-2-one (geranylacetone, diagnostic ions: m/z 194, 69,43) (Fig. 2B). The two compounds were released in about a 4:1 ratio (between 25% and 28% in the three extracts). Comparable extracts from females did not contain either of the two compounds, nor did they contain any female-specific compounds. The fact that only two compounds were consistently present in significant quantities in extracts of males suggested that these two components might be potential pheromone candidates. The absolute configuration of fuscumol in the extracts was determined with a chiral stationary phase Cyclodex B GC column. Because the alcohol enantiomers were not resolved, the insect-produced extract was acetylated (Fig. 3A). Reanalysis of the derivatized extract showed that the retention times of the acetylated insect-produced compound and (S)-fuscumol acetate matched exactly, whereas the retention time of the (R)-enantiomer was markedly different (Fig. 3), confirming that the insects produced exclusively the (S)-enantiomer of fuscumol.

Discussion
The results of this study expand our knowledge of the pheromone chemistry within the cerambycid subfamily Spondylinae. The identification of the A. rusticus pheromone provides the basis for development of effective methods for monitoring and managing this, and possibly other pest and invasive Arhopalus species that may vector serious phytopathogens such as the pinewood nematode. The pheromone of A. rusticus should provide a sensitive, cost-effective, and environmentally safe tool for early detection and rapid response in order to prevent its establishment and spread in non-native areas. In regions where this species is endemic, data on its abundance and population trends from pheromone-based trapping would provide the information necessary to make informed pest management decisions. The analytical and bioassay data presented here showed that (S)-fuscumol is the major and likely only component of the male-produced aggregation-sex pheromone of A. rusticus. The role of geranylacetone, the minor component identified in extracts from A. rusticus males, remains uncertain, because the blend of fuscumol with geranylacetone, in the ratio tested was less attractive to females than fuscumol alone, and actually caused attraction of A. rusticus females to drop significantly compared to fuscumol alone, suggesting a deterrent effect for females. In further studies aimed at optimizing the attractant lure for practical applications we will also test different ratios of a minor pheromone component, (geranylacetone), on attraction to the major component. Geranylacetone has been demonstrated to be an intermediate in the biosynthesis of fuscumol in two spondylidine species 29 , and it may have some role in mediating heterospecific attraction among co-occurring and closely related Arhopalus species, as has been shown for the minor components of pheromone blends of some other cerambycid species [30][31][32][33] .
Both sexes of A. rusticus were attracted to fuscumol indicating that this compound is an aggregation-sex pheromone 34 , as is the case for all other species in the subfamilies Spondylinae and Lamiinae that use fuscumol or fuscumol acetate as pheromone components 26 . However, the sex ratio of adults attracted to racemic or (S)-fuscumol was highly female biased. Whereas this might suggest that A. rusticus females are more strongly attracted to fuscumol than males, but, there is little information on the natural sex ratio, and sex ratios in traps may also be influenced by factors such as asynchronous maturation, mating status, different orientation mechanisms, and different distribution www.nature.com/scientificreports www.nature.com/scientificreports/ patterns between the sexes 22,35 . However, for possible control programs based on pheromones, a lure that strongly attracted females would be more advantageous than a male-biased attractant, because removing females from a population would likely result in larger effects on the subsequent generation than removing males 36 .
Similar numbers of A. rusticus were caught in traps baited with either racemic or (S)-fuscumol, suggesting that the (R)-enantiomer does not inhibit attraction of this species. This is useful for practical purposes because it means that the relatively cheap racemic compound, rather than the much more expensive (S)-enantiomer, can be used as an effective lure for detection and management of A. rusticus. Because pheromone structures are frequently conserved within related cerambycid taxa, it is also possible, and even likely, that other Arhopalus species will be found to produce and respond to fuscumol or closely related analogs. For example, males of the North American congener Arhopalus productus LeConte have been shown to produce (S)-fuscumol and geranylacetone (JGM, unpub. data). There is also increasing evidence that the fuscumol motif is actually shared more broadly. For example, within the same tribe (Asemini) as A. rusticus, Asemum nitidum LeConte produces and is attracted to (S)-fuscumol and geranylacetone 37 , Asemum caseyi Linsley produces and is attracted to geranylacetone 37 , and several Tetropium species produce and are attracted to (S)-fuscumol 37,38 . Furthermore, during our bioassays, we caught significant numbers of Spondylis buprestoides, in a different tribe (Spondylidini), suggesting that this pheromone motif may be conserved throughout the pheromones of the subfamily Spondylidinae.
To date, several studies 23,[39][40][41][42][43][44][45][46][47] have demonstrated significant attraction of spondylidine species, and specifically Arhopalus species, to ethanol (released as a by-product of anaerobic degradation of damaged or dead trees 48 , and/ or other host plant volatiles such as α-pinene (a defence compound) produced by conifers against insects and pathogens 49-51 , for review see 26 . We found that a mixture of ethanol and α-pinene was attractive to both sexes of A. rusticus, but the host volatiles were not as attractive (at least for females) as the racemic or (S)-fuscumol lures. However, plant kairomones may strongly influence the attraction of insects to their sex or aggregation pheromones (e.g. [52][53][54][55][56]. For example, α-pinene was found to strongly synergize attraction of Tetropium fuscum to its aggregation-sex pheromone (fuscumol) 43 . Thus, combining host plant volatiles with fuscumol may increase attraction of A. rusticus. This will be tested in ongoing trials aimed at optimizing lures and their release rates, traps, and trapping protocols for this species.
The capture of significant numbers of the click beetle Stenagostus rufus in traps baited with a blend of fuscumol and geranylacetone suggested that these compounds might be mimicking the sex pheromone of this species. In particular, a number of click beetle sex pheromones have terpenoid motifs 57 , and very recently, fuscumol acetate was shown to be an excellent mimic of methyl dihydrofarnesoate, the sex pheromone of two North American click beetle species in the genus Cardiophorus 58 . However if the compound were a sex pheromone we would expect that males would be attracted. Alternatively, predatory click beetles have been shown to exploit the pheromones of heterospecifics as a means of finding their prey. For example, the click beetle Elater ferrugineus L. is attracted to the pheromone of its prey, the scarab beetle Osmoderma eremita Scopoli 59 . In particular, larvae of S. rufus, have often been associated with larvae of longhorn beetles, such as A. rusticus and Rhagium inquisitor, Spondylis buprestoides, and Strictoleptura rubra, living together on the same stage of decaying wood, and attacking the www.nature.com/scientificreports www.nature.com/scientificreports/ cerambycid larvae 18 . This further indicates that the pheromones of cerambycids might function as kairomones for predatory click beetles, mimicking cues or signals associated with hosts, and particularly, oviposition hosts, rather then mimicking sex pheromone of click beetles. S. rufus is on the European Red List, (European IUCN Red List) currently listed as "LC" (least concern), but found locally in small populations, and nationally or regionally classified as threatened or near threatened in several European countries, such as Italy, Norway, Sweden, Finland, and others [60][61][62] . Thus, our study also provides an important first step in investigation of the chemical ecology of this under-recorded and vulnerable insect species.
There are a number of possible practical applications for the A. rusticus pheromone, alone or in combination with host plant volatiles. First, the pheromone could be used as a standardized and targeted sampling tool in studies of the biology and life cycle of this nocturnal species. Previous studies have relied on more timeand effort-intensive visual surveys and manual collections of adults from logs randomly selected in the field, or have used generic host plant volatile attractants and light traps to sample Arhopalus species (e.g. 22,23,63 ). Pheromone-based trapping would also enable long-term and large-scale field surveys that could, in combination with mark-release-recapture experiments, improve our understanding of the dispersal behaviour of A. rusticus, provide more reliable estimates of population size, population dynamics, and seasonal phenology in relation to biotic (e.g. predation by click beetles) and abiotic factors (e.g. climate change, storms and fires), and clarify habitat preferences [64][65][66] .
Second, pheromone-baited traps could provide effective and economically viable tools for surveillance and early detection of A. rusticus and possibly other invasive Arhopalus species around ports of entry, and particularly around warehouses and other shipping facilities where sealed shipping containers are opened for distribution of their contents. For example, Arhopalus was found to be one of the five most frequently intercepted cerambycid genera in six US ports during the inspection of solid wooden packing material 67 . Several recent studies have highlighted the effectiveness of pheromone-baited traps for sampling cerambycids and detecting incursions of exotic species (e.g., 27,28,68 ). Pheromone-baited traps would also facilitate the ongoing monitoring efforts to study the distribution and rate of expansion of A. rusticus populations as it continues to spread in new areas of the world which it has invaded 10,14,22 .
Third, fumigation is commonly employed as a phytosanitary treatment for export logs to prevent the spread of target pests into new areas of the world 69 . However, there is heavy pressure to reduce or eliminate the use of fumigants such as methyl bromide, a known ozone destroyer 70,71 . In this context, pheromone-baited traps, along with other environmentally benign measures (e.g. heat treatment, light trapping) may have a role in assessing the need for and efficacy of phytosanitary treatments for quarantine risk management of post-harvest export logs 72 .
Fourth, the A. rusticus pheromone could be exploited in management strategies for beetle-vectored phytopathogenic microorganisms such as the pinewood nematode Bursaphelenchus xylophilus 45 , which is causing severe ecological and economic damage in conifer forests in areas of Asia and Europe, which it has invaded, and the nematode is under quarantine within the European Union [73][74][75] .
The growing evidence that fuscumol and its analogues appear to be conserved pheromone structures within both the subfamilies Spondylidinae and Lamiinae (e.g. 27,[76][77][78][79][80][81] ) also has major implications for invasion biology. Specifically, it is a prerequisite for reproduction that males and females encounter each other and mate, but the sexes are less likely to locate one another successfully at low densities (i.e. Allee effect 82 ). Here, we highlight the concept of the Allee effect in mating success, and the dynamics of biological invasions and consequently in risk assessments of invasive species 26,83 in the context of A. rusticus and related species that use fuscumol or related compounds as their pheromones. The studies cited above have shown that fuscumol is a common pheromone motif among cerambycid species from several continents. When an exotic species using fuscumol-type compounds as its pheromone arrives in a new country, it is possible and even likely that some of the native species will produce the same or very similar pheromone compounds. Through shared pheromones, these endemic species may create a barrier to invasion by disrupting the ability of the invader to find a mate, thus hindering reproduction, population growth, and establishment of the invader 84 .
Extracts were analysed at the University of California, Riverside, by coupled gas chromatography-mass spectrometry (GC-MS), in splitless mode using an HP 6890 GC coupled to an HP5973 mass selective detector (Hewlett-Packard, now Agilent, Santa Clara, CA, USA). The GC was fitted with a DB-17 column (30 m × 0.25 mm × 0.25 μm film; J &W Scientific, Folsom CA, USA), and the oven temperature was programmed from 40 °C for 1 min, 10 °C min −1 to 280 °C, with helium carrier gas. Compounds were conclusively identified by matching their retention times and mass spectra with those of authentic standards.
To determine the absolute configuration of the male-specific compound fuscumol, an aliquot of an extract was acetylated by adding 100 µl of acetyl chloride solution (1% v/v in dry methylene chloride) and 100 µl of pyridine solution (20 µl + 1-2 mg dimethylaminopyridine in 1 ml dry methylene chloride). The mixture was stirred for 1 hr at room temperature. The excess acetyl chloride was destroyed by addition of ethanol (5 microliters), followed by stirring for an additional 1 hr. Most of the methylene chloride was then removed by blowing down under a stream of nitrogen. The sample was then partitioned between 1 ml of 1 M aqueous HCl and 1 ml hexane, vortexing for 30 sec, followed by removal of the top hexane layer, and washing it with 1 ml of saturated aqueous NaHCO 3 solution. After drying over anhydrous Na 2 SO 4 the hexane layer was concentrated under a stream of nitrogen and analyzed by GC on a chiral stationary phase Cyclodex B column (J&W Scientific, 30 m × 0.25 mm id × 0.25 um film) in splitless mode, with the oven temperature programmed from 50 °C for 1 min, then ramped at 3 °C to 220 °C. Authentic standards of (R)-and (S)-fuscumol acetate were analysed under the same conditions, and as final proof of the configuration of the insect-produced compound, it was coinjected with the (S)-fuscumol acetate standard, resulting in a single peak.
Pheromone solutions were prepared in advance and kept at −20 °C until needed. Lures were loaded with a pipette at the field site immediately before being deployed. Lures were suspended with wire in the central open area of custom-made flight-intercept panel traps as described above, suspended from trees. The traps were painted with Fluon emulsion (Insect-a-Slip Insect Barrier-Fluon, Bioquip Products Inc., Rancho Dominguez, CA, USA) to render trap surfaces slippery. Trap collection cups (white plastic, 8 cm diameter × 17 cm height) were filled with 200 ml of saturated aqueous NaCl solution to preserve captured beetles. For all experiments, traps were placed 20-25 m apart in transects, suspended from tree branches at a height of 1.5-2 m on randomly selected tree species (e.g. Picea, Abies), and treatments initially assigned randomly to traps. Traps were checked once weekly (total 10 trap checks) and at each check, the traps were rotated one position down the transect, to control for position effects.
Statistical analyses. Field bioassay replicates were based on temporal (i.e. 10 trap checks) and spatial replications (i.e. five). Differences between mean numbers of beetles, males and females separately, caught per treatment blocked by site and date, were tested using the nonparametric Friedman's test (because data violated the equal variances assumption of ANOVA 87 ) followed by the Conover multiple comparison test 88 , and corrected by the Benjamini-Hochberg procedure 89 . Replicates that contained no specimens were dropped from analyses. The exact binominal test 90 was used to test whether the sex ratio differed significantly from 0.5. All statistical analyses were conducted with R software, version R 3.5.0 (Copyright (C) 2018 The R Foundation for Statistical Computing Platform 91 ).

Data availability
Data are the property of the National Institute of Biology (NIB), maintained on government computers, and are of public record. Bioassay data are and the specimens of A. rusticus and other species are retained by the authors at NIB and are available by request.