DOI: 10.5061/dryad.f7m0cfz7h
title: Data from: Attraction of <em>Phoracantha mastersi</em> (Coleoptera: Cerambycidae) to copulation-associated olfactory stimuli

description: 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.
description: <strong>Experimental population and
 conditions</strong> We used field-captured adult
 <em>P. mastersi</em>, obtained during the austral summer
 (January-February) of 2023 and 2024. Beetles were captured during
 nocturnal beetle surveys of sub-alpine stands of <em>Eucalyptus
 pauciflora</em> ssp. <em>Niphophila</em> 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. <strong>Response to
 semiochemicals</strong> 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<sup>-1</sup> 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
 (<em>i.e.</em>, 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
 (<em>i.e.,</em> left or right) and source of the incoming air
 associated corresponding with that choice (<em>i.e.,</em>
 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.
 <strong>Response to mating pairs</strong>
 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.
 <strong>Analysis</strong> We first
 tested for left-right bias within the Y-maze using a two-sided
 χ<sup>2</sup> 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 <em>p</em>-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
 (<em>i.e.</em>, α = 0.05). All analyses were conducted using
 JMP 17.1 (SAS Institute Inc. 2022)
description: # Data from: Attraction of *Phoracantha mastersi* (Coleoptera:
 Cerambycidae) to copulation-associated olfactory stimuli Dataset DOI:
 [10.5061/dryad.f7m0cfz7h](10.5061/dryad.f7m0cfz7h) ## Description of the
 data and file structure ### Files and variables #### File:
 PMAS_SEMIOCHEMICAL.csv **Variable description:**  * DATE: Calendar date
 assay conducted MM/DD/YYYY * SEMIOCHEMICAL: Semiochemical used in the
 assay * POSITION: Y-maze arm with assayed semiochemical (Left/Right) *
 OUTCOME: Y-maze arm selected by subject (Left/Right). 'None' if
 no selection made #### File: PMAS_COPULATION.csv **Variable
 description:**  * DATE: Calendar date assay conducted MM/DD/YYYYMATING_F:
 Field-assigned code for mating female * MATING_M: Field-assigned code for
 mating male * MATING_F: Field-assigned code for mating female *
 NON-MATING_F: Field-assigned code for non-mating female * NON-MATING_M:
 Field-assigned code for non-mating male * POSITION: Y-maze arm with mating
 male and female * OUTCOME: Y-maze arm selected by subject (Left/Right).
 'None' if no selection made #### File: PMAS_MATING.csv
 **Variable description:**  * DATE: Calendar date assay conducted as
 MM/DD/YYYY * TIME: 24-hour time at which assay commenced AS HH:MM: SS *
 MATING_F: Field-assigned code for mating female * MATING_M: Field-assigned
 code for mating male * MATING_TIME: Duration of mating forthe  assayed
 mating male and female in minutes * NON-MATING_F: Field-assigned code for
 non-mating female * NON-MATING_M: Field-assigned code for non-mating male
 * POSITION: Y-maze arm with mating male and female * OUTCOME: Y-maze arm
 selected by subject (Left/Right). 'None' if no selection made