We investigated behavioural responses of Phoracantha mastersi Pascoe—an irruptive species associated with sub-alpine Eucalyptus pauciflora woodland decline in southeast Australia—to a small suite of commercial semiochemicals. Drawing upon field-based observations of male sexual aggregation, we also evaluated responses of male beetles to conspecifics engaged in sustained mating and active copulation. Tests were conducted using a Y-maze within which treatment-related visual cues were eliminated.

Experimental population and conditions

We used field-captured adult P. mastersi, obtained during the austral summer (January-February) of 2023 and 2024. Beetles were captured during nocturnal beetle surveys of sub-alpine stands of Eucalyptus pauciflora ssp. Niphophila in three locations: Smiggin Holes (1700 m asl; -36.3921 S, 148.4276 E), Perisher Valley (1760 m asl; -36.4095, 148.4154), and Guthega (1650 m asl; -36.3817, 148.3723) on the Kosciuszko plateau in southeast Australia. Surveys comprised active searches during January-March over three consecutive summers (2021/23 to 2023/24). Captured insects were stored individually within 70 ml specimen jars. Species and sex identity were resolved consistent with Wang (1995). Following capture, all insects to be used in olfactometry trials were transferred to cool storage and held at 4-5 °C until required.

Response to semiochemicals

We conducted experiments of olfactory responses in beetles captured during both 2023 and 2024. Experiments comprised individual assays within a Y-maze olfactometer constructed of heat-moulded 100-mm diameter acrylic tubing. Air is drawn into the olfactometer by a vacuum pump (Rocker 300) via a chamber containing activated charcoal, through two humidifying borosilicate water bubblers, separate flow meters and treatment/control volatile chambers, and then into the arms of the Y-maze itself. Both the arms and entry tube are fitted with interchangeable heat-moulded end caps allowing flow from volatile chambers to be manually alternated and internal tubing to be cleaned between trials. Direct line-of-sight between the Y-maze and the volatile chambers is prevented by their placement behind an opaque plastic shield.

Preliminary tests of beetle behaviour indicated that geotaxis was strongly expressed in the laboratory. In response, we mounted the Y-maze at a 70° inclination and inserted a cylinder of fibreglass mesh into main tube to assist vertical movement. Consistent with results for cerambycids elsewhere (Allison et al., 2004), we tested for positive bias in response to the generic kairomones - (e.g., Miller, 2006; Miller et al., 2016; Morewood et al., 2002) and -pinene (e.g., Gonçalves et al., 2020) and ethanol (e.g., Gonçalves et al., 2020; Miller, 2006; Morewood et al., 2002), and semiochemicals 3-hydroxy-2-octanone (e.g., Molander et al., 2019), 3-hydroxy-2-hexanone (e.g., Imrei et al., 2019), monachamol (e.g., Andrade et al., 2024; Miller et al., 2016), fuscamol, fuscamol acetate and p-cymene (e.g., Collignon et al., 2019), as well as negative bias in response to D-limonene (e.g., Gonçalves et al., 2020) and verbenone. With exception to ethanol, all semiochemicals were supplied by Synergy Semiochemicals Corporation, Canada.

Immediately prior to conducting assays, we eliminated all light from the laboratory and restricted illumination to head-torches emitting only red light. Illuminated displays on laboratory equipment were also shielded. Candidate beetles, identified within randomly assigned sample schedule, were then removed from cool storage and acclimated to room temperature (~22.0 °C) for ~30 minutes and pipette-fed 0.15-0.20 mL of 10% v/v sucrose/water solution.  Assays focussed solely on the response of males because of their greater availability and more active behaviour both in the field and captivity.

Semiochemicals were mixed with paraffin oil at a rate of 100 mg/mL. Before introducing experimental subjects, a cotton pad infused with a diluted semiochemical was placed inside a fibreglass mesh cage and suspended at the inlet valve in an arm of the Y-maze, and a control pad, infused only with paraffin, in the opposing arm. Selection of treatment and control arms followed a randomised experimental schedule. Air flow was monitored during experiments to ensure a constant 4 L min^-1 to both arms of the Y-maze. An acclimated male was then introduced to the Y-maze via the base of the main tube, and the trial commenced. To eliminate all light sources and visual cues, head-torches were not illuminated during trials.

Assays were concluded when the subject had either arrived and remained at the mesh in the endcap of one arm of the Y-maze, or terminated if the subject did not express a definitive choice (i.e., remained stationary in the main tube, Y-junction, or part-way along an arm) after ten minutes. Whether the subject exhibited an apparent selection, and the direction (i.e., left or right) and source of the incoming air associated corresponding with that choice (i.e., semiochemical-paraffin mix or paraffin-only control) was recorded. We also conducted assays using a paraffin-only control in both arms, to test for systematic bias toward either of the Y-maze arms. The Y-maze was then cleaned with ethanol, allowed to fully dry and aerate, before a new candidate beetle was added for the next assay. Assays continued until a minimum of 50 concluded with a definitive choice. Beetles were returned to cool storage at the conclusion of assays for each semiochemical and a new, randomly selected cohort acclimated for the next set of tests.

Response to mating pairs

We conducted assays aimed at testing responses associated with olfactory stimuli generated during mating using beetles captured during both 2023 and 2024. Individual assays were conducted using the same Y-maze and animal husbandry, but a separate insect population, used in the semiochemical experiment.

To commence, two male/female pairs of acclimated beetles were randomly selected and introduced to the separate volatile chambers delivering air to the Y-maze arms. Physical contact within chambers housing non-mating pairs was prevented by the presence of fibre-glass mesh. Non-separating mesh was added to the floor of the chamber holding the mating pair. After their introduction to the volatile chamber, mating pairs were monitored until copulation commenced. Assays were conducted consistent with the protocol used for semiochemical experiments. We retained mating/non-mating pairs in volatile chambers for sequential assays until separation of the male/female genital bridge.

Preliminary analysis indicated that a positive bias was expressed by subject males toward mating pairs was principally confined to a short period after copulation commenced. In response, during 2024 we restricted assays to a single subject male immediately after copulation by a mating pair commenced. Assays were concluded or terminated consistent with the 2023 mating-pair experiment. Mating/non-mating pairs, chamber to which they were added and the identity of the male subject introduced to the Y-maze followed a predetermined experimental protocol.

Analysis

We first tested for left-right bias within the Y-maze using a two-sided χ² test. We then used one-sided binomial tests for selection tendencies in response to the presence of semiochemicals. Consistent with results for cerambycids elsewhere (Allison et al., 2004), we tested for positive bias in response - and -pinene and ethanol, and semiochemicals 3-hydroxy-2-octanone, 3-hydroxy-2-hexanone, monachamol, fuscamol, fuscamol acetate and p-cymene, and negative bias in response to the presence of D-limonene and verbenone. Because a common population was subsampled to conduct assays, and individual beetles were randomly included in assays for more than one semiochemical, we used a Bonferroni correction to adjust for the family-wise error rate across all results.

To test responses to mating pairs we separately analysed data from our 2023 and 2024 experiments. For 2023 data, we first used a one-sided binomial test for bias toward Y-maze arms drawing air from mating pairs for the entire duration of mating. Using data arising from our 2024 trials focussed on the response of males to immediately after the commencement of copulation, we tested for bias toward actively copulating pairs. Because they were based upon subjects independent of those used in semiochemical assays, and from each other, both tests applied a non-adjusted threshold significance level (i.e., α = 0.05). All analyses were conducted using JMP 17.1 (SAS Institute Inc. 2022)