Changes to volatile profiles of arborvitae, Thuja occidentalis, from drought and insect infestation: olfactory cues for the cypress bark beetle, Phloeosinus aubei

The cypress bark beetle, Phloeosinus aubei (Coleoptera, Curculionidae, Scolytinae) prefers to build breeding galleries in decaying host trees. However, volatile cues specific to decaying or actively infested trees have not yet been reported. Any such information would be useful for the management of this invasive, wood-boring species that has become established across Europe in the temperate zone and is now a key pest of ornamental Cupressaceae in nurseries and urban landscapes. Our objective was to reveal key components in host volatiles specific to stressed trees, which could be potential signals for the pest of its suitability for colonisation. Volatile profiles of arborvitae, Thuja occidentalis ‘Smaragd’ suffering from either dehydration or fresh infestation of P. aubei was collected and compared to that samples from healthy control trees. Analyses of volatiles by gas chromatography coupled to an electroantennographic detector revealed substantial differences between healthy, dehydrated and infested trees. Structural elucidation of the major antennally active components revealed that both α-pinene and α-thujene were prevalent in the volatiles of infested trees, whilst α-thujone was the major component in volatiles of healthy trees. In volatiles collected from trunks housing fresh P. aubei female nuptial chambers, sharp increases of β-pinene, myrcene, limonene, and p-cymene were noticed, alongside trace amounts of camphene and fenchene. Volatiles collected from fresh frass showed a similar profile, but with a somewhat smaller amount of limonene. Fenchone was present in each type of volatiles, however, only in low amounts. Further studies should be directed to reveal the behavioural role of these components, which could be helpful in developing kairomone-based techniques for monitoring the flight of the pest in stands of scale-leafed trees.


Introduction
For host plant selection of most bark beetle species, health status is just as important as the tree species. Some bark beetle species are able to colonise healthy trees, others live in dead logs, whilst many others prefer trees in intermediate stages of decline (Toffin et al. 2018;Schlyter and Birgersson 2000, and references therein). Drought is known to promote attraction of pine-leafed associated bark beetle species (Faiola and Taipale 2020). Little specific attention has been paid to the long-range, kairomone-triggered attraction of bark beetles associated with scale-leafed conifers. A study in Oregon revealed that a bait composed of natural essential oils, acetone and ethanol attracted Phloeosinus scopulorum Swaine and P. serratus (LeConte). In this study, ethanol was primarily responsible for the attraction, mimicking the odour of declining western juniper, Juniperus occidentalis var. occidentalis Hook (Hayes et al. 2008). Whilst not thoroughly studied, a similar process seems likely for the invasive cypress bark beetle, Phloeosinus aubei Perris, (Coleoptera, Curculionidae, Scolytinae), which is now causing economically alarming outbreaks in the temperate zone of Europe. The life cycle of the historical populations Communicated by Günther Raspotnig.
1 3 includes female beetles searching for declining host trees to make breeding galleries in April-May. Adults of both sexes visit healthy trees for boring small overwintering tunnels from August to October (Bozsik and Szőcs 2017).
P. aubei is specialised to various tree species of the plant family Cupressaceae (Balachowsky 1949;Mendel 1984). Whilst there are detailed phytochemical studies on the essential oil contents of scale-leafed conifers (Piovetti et al. 1981;Pauly et al. 1983;Adams 1998;Pierre-Leandri et al. 2003;Wang et al. 2022), only a few reports focussed on the value of the volatile organic compounds, VOC, as potential hostplant kairomonal cues for P. aubei. Bozsik et al. (2016) reported more than twenty VOCs from isolated (detached) twigs of American arborvitae, Thuja occidentalis L. cv. 'Smaragd' ('Emerald'), each of which elicited antennal response from P. aubei. In that study, however, the freshly cut twigs originated from otherwise healthy trees, and therefore represented an immediate response to an acute physical stress factor, rather than a declining tree, suffering from chronic stresses. In a more recent study, sixteen components were found in the VOC profile of healthy T. occidentalis 'Smaragd', which elicited antennal response from either males or females of P. aubei, and of the cypress jewel beetle, Ovalisia (Lamprodila/Palmar) festiva L. (Coleoptera: Buprestidae, Chrysochroinae) (Bozsik et al. 2022). The cypress jewel beetle is another upcoming, invasive woodboring pest of Cupressaceae, which attacks both healthy and declining trees. Also, the specific components in the volatile profile of declining trees remain unknown, which are crucially important cues used by P. aubei for searching for suitable host for breeding galleries.
Determining these unknown cues is interesting from practical point of view, because of the growing economic importance of P. aubei. Originally confined to the Mediterranean area and Asia Minor (Balachowsky 1949), P. aubei reached the Carpathian Basin, probably years ago (see Endrődi 1959) along the Illyria (West Mediterranean) migration route (Varga 2010). After apparently several decades of latency, as observed in other invasive insect species (Kozár et al. 1995;Kozár 1997), P. aubei suddenly devastated young T. occidentalis 'Smaragd' in ornamental tree nurseries around Szombathely, SW/W Hungary (Rakk and Bürgés 1994;Reiderné-Saly and Podlussány 1994). Proceeding northeasterly from this region, P. aubei spread all over Hungary, in the subsequent years (Both and Farkas 2005). On a European scale, the spread occurred along various directions, reaching for example Hessen, Germany (Dern 1976), East Germany (Sobczyk and Lehmann 2007), the Netherlands (Moraal 2010), Romania (Olenici et al. 2015) and the United Kingdom (Winter 1998). The recent spread of P. aubei across the temperate zone of Europe, was followed by serious economic losses of ornamental evergreen trees of Cupressaceae in urban green areas (Mooral 2010; Fiala and Holuša 2019).
At present, neither kairomone-nor pheromone-baited traps are available for monitoring the flight of P. aubei because of the insufficient knowledge of the chemical communication of the pest. In order to support the development of kairomone-baited traps for P. aubei, this study is aiming to reveal the key olfactory signals in the volatile profile of stressed T. occidentalis trees.
Our model system represents an example of human assisted exposure of vegetation in urban habitats to settlement of an invasive species in a system pre-adapted to be susceptible to such significant damage. Notably, the Mediterranean cypress, Cupressus sempervirens L., is the primary host plant of P. aubei in the Mediterranean region but is not native to the temperate zone of Europe. Moreover, only the common Juniper, Juniperus communis L. represents the family of Cupressaceae in Hungary (Soó 1964). Additionally, cultivars of several scale-leafed conifers, in the genera Thuja, Chamaecyparis, Cupressocyparis and Cupressus, are frequently planted as ornamental trees in gardens and city parks throughout Europe. Horticultural activities may have also provided a travel route for P. aubei. Interestingly, the arrival of the pest was not followed immediately by an explosive invasion. Instead, it was preceded by a long latency period. When rapid population build-up was accompanied by alarming tree declines in ornamental tree nurseries, plant protection techniques proved to be incapable to cope with this pest. Proper monitoring systems were not available, because the chemical ecology of P. aubei was largely unknown, which remains the case today.
For the development of kairomone-based monitoring, it was first described how P. aubei became adapted to the temperate climate (Bozsik and Szőcs 2017). Next, we began to study host plant kairomones intensively (Bozsik et al. 2016(Bozsik et al. , 2022. Our present study fits within the latter line of research. We now report on the changes to composition of volatiles emitted by the host as they are affected by either draught or fresh infestation by P. aubei. In the present study, we focussed on the components apparent in stress-induced volatiles that can be perceived by P. aubei, rather than trying to chemically identify all potential kairomone compounds.

Selection of host trees for sampling volatiles
Two types of stressed T. occidentalis 'Smaragd' trees were chosen, representing the major stress factors. As irrigation poses a challenge in some tree nurseries, trees suffering from drought, but not infested by P. aubei were selected for volatile collection. As a second treatment, well-maintained trees infested by P. aubei that were making nuptial chambers were included in the studies. For comparison, volatiles were also collected from healthy, un-infested trees.
Tree specimens originated from Tahi Tree Nursery ( GPS: 47.7653704536209 N,19.06798750162125 E). Selected trees were each approximately 4-5 years old, 1.5 m height and 5 cm diameter at 1 m above ground level. They were carefully taken out with ground ball and transported to the laboratory of the Plant Protection Institute, Centre for Agricultural Research (Budapest, Hungary, GPS: 47.54803661427715 N, 18.93472731113434 E) and replanted into flowerpots. They were kept under natural conditions. Replantation was made 2 months prior the experiments.
To have a preliminary insight into volatile profiles between of cultivars beyond T. occidentalis 'Smaragd', we also included T. occidentalis 'Brabant', a variety also highly preferred by P. aubei (Bozsik et al., unpublished observations). For similar reasons to broaden the range of host samples, the congener T. plicata 'Excelsa' was also studied. These taxa are, in general, all heavily attacked by P. aubei. However, the tree specimens, taken for the studies, were free from infestation, and looked healthy. Samples of the trunks of these varieties were isolated, as describe above for the healthy T. occidentalis 'Smaragd'.

Experimental insects
P. aubei adults were collected in Tahi Tree Nursery and in Prenor Tree Nursery (Szombathely, Vas-county, GPS: 47.26419949232103 N, 16.59957200288773 E) from T. occidentalis 'Smaragd' twigs, during their overwintering season. Specimens were isolated by sex and kept under ambient conditions in T. occidentalis 'Smaragd' twigs, in an open-wall greenhouse of the Plant Protection Institute.

Volatile collection from intact trees
Single healthy undamaged, intact twigs of the live T. occidentalis 'Smaragd' trees were covered by chemically inert oven bags (Alufix GmbH, Wiener Neudorf, Austria), fastened 25 cm back from the tip. Headspace volatiles were collected by 1.5 mg charcoal CLSA filters (Brechbühler AG, Schlieren, Switzerland), with a flow rate of 2.6 L min −1 for 4 h, under a closed loop system, using a DC12 rotary vane pump (Fürgut GmbH, Tannheim, Germany). The procedure was carried out under ambient conditions, when the temperature was 23-24 °C. Volatiles from the filter were washed by n-hexane (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) three times. The total volume of extracts was 50 μl. Extracts were kept in a − 40 °C freezer until analysed.

Volatile collection from cut logs
To roughly imitate drought-afflicted trees, an approximately 20 cm long, and 5 cm diameter part of the T. occidentalis 'Smaragd' trunk was cut from a living tree and kept under ambient conditions for 4 months (from December to April). Volatiles from the declining, semi-dried piece of trunk were collected using the same method as described above. Oven bags were used to isolate the undamaged bark of the trunk from the mechanically injured parts. Also, cut surfaces were excluded from sampling to avoid collecting volatiles induced directly by mechanical wounding.
As an experimental replication, an adjacent part of the same trunk was sampled by the same method (see Supplementary Materials).

Volatile collection from trees, freshly infested by P. aubei females
Infestation may have cascading effects on volatiles emitted from the attacked tree. As a specific response to infestation caused by P. aubei, the tree may alter the composition of volatiles, but the females inside their tunnels may also contribute by emitting pheromones. As a result, the infested tree may become more attractive to further colonisation. In order to obtain an overall picture of the volatile profile of newly infested T. occidentalis, we sampled trunks with fresh nuptial chambers containing females. Thirty-two unmated female P. aubei adults were introduced within an approximately 20 cm long by 5 cm diameter trunk section of T. occidentalis 'Smaragd' in a 4 L glass jar. All the females prepared nuptial chambers under the bark of the trunk. One month after the first P. aubei females started to make their nuptial chambers, headspace volatiles of the infested trunks were collected by the same method as above. For comparison, a volatile collection from nine unmated females in nuptial chambers was made by the same methods as above.
As an alternative method, volatiles were sampled directly from the lumen of the female-occupied nuptial chambers. A tip of a Pasteur-pipette was tightly inserted into the tunnel. The sampling proceeded by an open loop system, using the same type of absorbent within the pipette. The duration of sampling was 3 min per nuptial chamber. Altogether 7 nuptial chambers were sampled by this method.
Volatile collection from the frass of P. aubei females 320 mg of freshly produced frass from P. aubei female nuptial chambers in T. occidentalis 'Smaragd' was collected and isolated in a baking bag. Throughout we will use the term of "frass" to collectively represent, chewed dust, excreta and other debris associated with gallery construction. Subsequently the headspace volatile collection procedure was carried out.

Volatile collections from T. occidentalis 'Brabant' and T. plicata 'Excelsa'
For comparing the volatile constituents of the two Thuja species, T. occidentalis 'Brabant' and T. plicata 'Excelsa', headspace volatiles of intact, healthy twigs with leaves were collected by the same method as above. The only methodological difference was a 3-h timespan for volatile collection.

Analysis of volatile collections by gas chromatograph coupled to an electroantennographic detector
For comparing the volatile constituents of healthy, declining and infested T. occidentalis 'Smaragd', gas chromatographic (GC) analyses equipped with electroantennographic detector (EAD) were used. A 6890 N gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a 30 m × 0.32 mm × 0.25 μm film thickness DB-WAX column (J&W, Scientific, Folsom, CA, USA) was used for separation of the volatile components. The column was linked to an electroantennographic detector (SYNTECH, Hilversum, the Netherlands) for simultaneous analyses with the flame ionisation detector (FID) of the GC. Extracts were injected in split-less mode. The oven temperature was held at 60 °C for 1 min, then set to 220 °C at 10 °C/min. The carrier gas was helium (4.0 ml/min).
Head and antennae of male and female P. aubei were prepared between two glass electrodes filled with Ringer solution and attached to silver/silver chloride electrodes, which were connected to an IDAC 2 amplifier (SYNTECH, Ockenfels SYNTECH GmbH, Kirchzarten, Germany).
For analysing the extracts of T. occidentalis 'Brabant' and T. plicata 'Excelsa', the same column and the GC settings were used as above, but instead of splitless mode, injections were made in on-column mode.
As synthetic standard, 15 ng n-decyl acetate (S10Ac) in n-hexane solvent (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) was co-injected with the volatile collections, but also injected alone as a control (the FID peak of standard is not shown in the figures).
Electroantennographic analyses were made with both female and male antennae.

Analysis of volatile collections by gas chromatograph coupled to mass spectrometry
Chemical identification of components that evoked antennal responses from P. aubei, was based on calculation of Kováts Retention Indices and comparison of spectra to the NIST Webbook database (https:// webbo ok. nist. gov/ chemi stry/). Synthetic samples were injected (source: camphene and fenchene are supplied from Thermo Fisher Scientific Inc., Waltham, MA USA. The others are supplied from Sigma Aldrich, Merck KGaA, Darmstadt, Germany) and the retention times of the EAD active compounds were compared to verify the results. Methodology and instrumentation were based on our related, previous MS identifications (Bozsik et al. 2016(Bozsik et al. , 2022. Briefly, an Agilent 6890 GC (Agilent Technologies Inc.) coupled to an Agilent 5973 mass selective detector was used for GC-MS. The GC was equipped with a HP-5 MS UI (30 m × 0.32 mm × 0.25 μm, J&W, Agilent) column.
Each of the above-mentioned components was also present in the volatiles of cut T. occidentalis 'Smaragd' logs, in similar ratios ( Fig. 1B and Table 1). Likewise, the pattern of antennal responses to this subset of components was also similar. However, some additional components with strong antennal responses were also detected early, eluting just after the solvent peak. Notably α-pinene and α-thujene (compounds 1 and 2), which eluted right after n-hexane, produced overlapping FID-peaks, which evoked a noticeable pair of antennal responses (Fig. 1B). Their presence was further supported by GC-MS analysis. These two compounds, which together totaled 236 ng/injection (Table 1B'), accounted for the bulk of the volatiles from this sample. Their abundance was followed by that of camphene (compound 3), fenchene (compound 4), β-pinene (compound 5), myrcene (compound 6), limonene (compound 7), and p-cymene (compound 8) (Fig. 1B, Table 1B. Replicated in Supplementary Materials, Fig. 4B/2). Each of these compounds appeared in moderate amounts (Table 1B'), which were, abundant enough for evoking antennal responses. These compounds could be detected by the flame ionisation detector (FID) in the collections from healthy trees as well. However, these were present only in trace amounts and did not evoke antennal responses.
In comparison to healthy trees, a rather different volatile profile was emitted from T. occidentalis 'Smaragd' trunks housing P. aubei females in nuptial chambers.
One of the most striking differences was that α-pinene and α-thujene were the major components (1976 ng/ injection combined), which impressively overshadowed Fig. 1 Electroantennographic responses of male P. aubei in GC-EAD measurements to volatiles of from T. occidentalis 'Smaragd' samples of various types and health statuses as follows including. A healthy leaves and twigs, B declining, semi-dry trunk, C female P. aubei infested trunk, D the fresh frass of P. aubei females Table 1 Identified components of volatile collections related to Fig. 1 Volatiles collected from the trunks of T. occidentalis 'Smaragd' that are (A) healthy, (B) drought-afflicted, or (C) producing frass from infestations of P. aubei in fresh nuptial chambers on. An "x" denotes that the compound was found, and an "-" that it was below the limit of the antennal detector. Compounds are uniformly numbered across figures and tables. Amounts of compounds were calculated by integrating the area of the GC peaks, and comparing to 15 ng of S10Ac, the synthetic standard (pA*s) *Injected volume of samples was 2 μl   Fig. 4C/2). Fenchone, α-thujone, 1-octen-3-ol and β-thujone were also present, however in low amounts only (6.9, 9.8, 0.7 and 0.4 ng/injection, respectively) ( Table 1C, C'). Nevertheless, they evoked relatively strong antennal responses. However, camphor (compound 13) did not evoke a response (Fig. 1C). A much later eluting trace compound 15, with a strong antennal response, also was evident (Fig. 1C). This compound was absent from noninfested and drought-suffering T. occidentalis collections described above. Evidence that this is a beetle-specific compound will be published elsewhere. The profile of volatile components collected from the lumen of female-occupied nuptial chambers was similar to that collected from the external headspace. However, the amounts collected internally were much lower (Fig. 4E in Supplementary Material).
Volatiles collected from the frass of newly carved nuptial chambers also showed a very similar profile to the collections taken externally from the newly infested T. occidentalis trunks (Fig. 1D and Table 1D, D').
Volatiles of healthy T. occidentalis 'Brabant' trunks contained the same major, antennally active components, in similar ratios ( Fig. 2A and Table 2), as the healthy T. occidentalis 'Smaragd'. The composition of healthy T. plicata 'Excelsa' (Fig. 2B and Table 2) looked almost identical as well but did not contain fenchone. EAD responses of male and female P. aubei antennae did not differ for these collections.
The ratios of the antennally active components in the volatiles collected from T. occidentalis in the intact, drought-stressed or infested treatments, along with the direct collection from frass, are displayed in the Fig. 3 and  (Table 3). The dominant component in healthy tree volatiles was α-thujone, whilst stressed trees emitted mostly α-pinene and α-thujene.

Discussion
In this study, the GC-EAD profiles of volatiles collected from healthy, draught-induced and P. aubei infested T. occidentalis trunks were compared to each other by standard methodology. The aim was to search for differences between the patterns of those components which might be perceived by P. aubei, rather than pinpointing and identifying as many trace components as possible, irrespective to their role. This was a complementary approach to that followed in earlier publications, where the composition of volatiles of either draught-induced (Bozsik et al. 2016) or intact trees (Bozsik et al. 2022) had been studied separately. The main emphasis in the present study was to focus on components appearing in the volatiles of the host trees that are changed either by drought stress, or by infestation of P. aubei females. This is, to our knowledge, a first step along a new approach for describing T. occidentalis-P. aubei semiochemical interactions.
It should be mentioned, however, that a cut log is just a rough imitation of naturally draught afflicted tree in field conditions. Nevertheless, very similarly prepared, unpruned cut logs of C. sempervirens have been effectively used for field trapping of P. aubei (Mendel 1983). Another issue for sampling is that volatiles collected from Fig. 3 Relative area of the identified compounds from A healthy leaves and twigs, B declining, semi-dry trunk, C female P. aubei infested trunk, D fresh frass of P. aubei females Table 2 Identified components of volatile collections related to Fig. 2 For more explanation see Table 1 No. newly female-infested trees represent a blend of the tree-and beetle-specific compounds. Nevertheless, identifying compounds which are specific to all such samples, and yet also perceived by the specialised pest may help us to recognise the key stimuli. In turn the effort may assist in developing suitable blends for later use in monitoring systems. The abundance of some highly volatile, early eluting components, particularly α-pinene and α-thujene drastically increased in volatiles of stressed trees when compared to samples collected from healthy trees. This pattern was clearly observed also in drought-stressed trees, free from P. aubei and fresh galleries, indicating that these compounds are of plant origin and stress-related. Although they overlap in the FID-trace, distinguishable, dual antennal responses were detected, showing that each of them is perceived by P. aubei. The relative abundances of camphene, fenchene, β-pinene, myrcene, limonene and p-cymene also increased in volatiles of stressed trees, as compared to samples collected from healthy trees. Amongst them, β-pinene, myrcene and limonene separated distinctly in samples of trunks with female nuptial chambers suggesting a possible roll of the insects in their product that could be further investigated. However, there was no supporting evidence for such a roll in our more recent studies focussing on the female-produced pheromone (results will be reported elsewhere).
At the same time, the amounts of fenchone, α-thujone and β-thujone, which dominated the samples taken from healthy trees, significantly decreased in stressed trees. These shifts were especially spectacular in the cases of the P. aubei infested trunk and frass samples, but also could clearly be observed in less pronounced manner also in draught-stressed trees. This indicates that the decrease of these compounds can be attributed to a general tree response to stress. Fluctuations in abundancies of compounds strongly correlated to amplitudes of antennal responses of P. aubei in GC-EAD measurements. This pattern suggests that P. aubei can distinguish between healthy and stressed host trees, at least at the level of peripheral perception.
The relative overall low amounts of collected volatiles from the inner headspace (lumen) of nuptial chambers may have been a result of the short time of the collection procedure (see Supplementary Materials). In general, it cannot be ruled out as a less effective way of sampling and could be improved.
Amongst the highly volatile compounds, the joint occurrence and response to α-pinene and α-thujene in volatiles of stressed T. occidentalis was observed in a co-eluting emission peak that was matched by two separate antennal responses from P. aubei. It was shown for the first time in the present study that either drought stress or fresh infestation significantly increases the emission of these compounds, as compared to the emission of healthy trees. Nevertheless, α-pinene, as well as β-pinene, limonene and fenchone were chemically identified and electrophysiological responses confirmed earlier, albeit in smaller amounts, from intact T. occidentalis (Bozsik et al. 2022). Concerning the less volatile compounds, such as fenchone, α-thujone, β-thujone and camphor, they were discovered earlier by similar methods in volatiles of isolated T. occidentalis twigs (Bozsik et al. 2016).
It is worth mentioning that α-thujene, camphene, fenchene, β-pinene, myrcene, limonene and p-cymene were found for the first time to elicit antennal response from P. aubei in this study.
Significant biological activity of the compounds we identified from T. occidentalis volatiles has been reported in other bark beetle species. For example, α-pinene has been described in many contexts, such as influencing the response of Ips avulsus Eichhoff to its aggregation pheromone (Sullivan 2023), evoking antennal responses from I. typographus L. (Dickens 1981) and contributing to colonisation of Douglas fir by Dendroctonus pseudotsugae Hopkins (Knopf and Pitman 1972). However, α-thujene has rarely been reported in connexion to insect behaviour (for an overview see: Schiestl 2010), despite being identified from a number of plant species.
Myrcene, as a host tree compound, is a precursor of pheromone biosynthesis of Ips species (Hughes 1974;Byers and Birgersson 2012).
In some cases, limonene was found to be a repellent host monoterpene of some bark beetle species (Raffa et al. 1985;Cook and Hain 1988;Romón et al. 2017). In contrast, Miller (2007) described that limonene is an attractant kairomone of male and female white pinecone beetle, Conophthorus coniperda (Schwarz) (Coleoptera: Scolytidae).p-Cymene, along other terpenes, was found in large quantities in volatiles of pedunculate oak, Quercus robur L. logs, suffering from the activity of European oak bark beetle, Scolytus intricatus Ratzeburg, building maternal galleries (Vracková et al. 2000). Similarly, p-cymene and 1,8-cineole, induced volatiles of infested spruce trees were found to be equally strong inhibitors for I. typographus, resulting in shut down of the response to the pheromone (Andersson et al. 2010). Hou et al. (2021) described an odorant receptor for p-cymene in I. typographus and surmised that even if p-cymene is an induced defence compound, released by spruce trees as a response to infestation, and hence should be a toxic for the beetle, it still may serve as a kairomonal cue for the beetle to estimate density of colonising population and thus taken as a signal of host suitability (Hou et al. 2021). The other compounds from this study have been found in numerous cases in the essential oils of various plants and reported as semiochemicals (cf. Pherobase).
This study was limited to antennally active volatile constituents (potential kairomones for P. aubei) of T. occidentalis volatiles. However, it should be mentioned that the set of components found in the samples corroborated the results of earlier publications, which reported phytochemical analyses of the volatile constituents of T. occidentalis and related scale-leafed conifer trees (Szolyga et al. 2014;Lis et al. 2019;Ni et al. 2019).
Also, further studies are needed to reveal the behavioural relevance of the antennally active components, specific to declining host trees. Seasonality of behavioural response of beetles should also be studied in this respect. It has already been reported earlier that the preference for decaying trees is expressed only in the breeding season, when female beetles look for suitable host trees for preparing breeding galleries (Bozsik and Szocs 2017). Notably, in the breeding season, which occurs in spring and early summer in the continental climate, females have been recently activated after overwintering and are looking for the trunks of stressed trees for boring nuptial chambers to start preparing breeding galleries. However, in autumn, freshly emerged adults search for heathy trees for maturation feeding tunnels in tip of twigs as they prepare for overwintering (Bozsik and Szőcs 2017). The formation of this latter type of tunnel (single-armed, short overwintering gallery) is probably a resulted of an adaption process to the continental climate. Once these other factors are understood, this current study may better serve as a contribution to get closer to reveal the olfactory cues of P. aubei, and thus allow for developing monitoring traps for tracking the spread of this pest.