The decreasing number of veteran trees in Europe threatens old-growth habitats and the fauna they support. This includes rare taxa, such as the violet click beetle, Limoniscus violaceus (Müller, 1821).

Samples of wood mould were taken from all beech trees in Windsor Forest previously confirmed to have contained L. violaceus larvae, and from trees where L. violaceus had not previously been detected, the latter categorised as having high, medium or low likelihood of containing the beetle during recent surveys. Habitat characteristics were measured, and volatile profiles determined using gas-chromatography mass-spectrometry.

Water content significantly differed between tree hollows of different violet click beetle status, high-potential habitats having higher and relatively stable water content compared with habitats with medium or low potential of beetle occupancy. Several volatile organic compounds (VOCs) were significantly associated with L. violaceus habitats. No differences in other characteristics were detected.

The distinction in water regime between habitats highlights that recording this quantitatively could improve habitat surveys. Several potential L. violaceus attractant VOCs were identified. These could potentially be integrated into existing monitoring strategies, such as through volatile-baited emergence traps or volatile-based surveying of habitats, for more efficient population monitoring of the beetle.

Study sites and sample collection

All samples and data were collected from Windsor Forest (51°26’02.5”N, 0°38’37.2”W). Windsor Forest contains many veteran trees with large hollows that afford habitat for many saproxylic invertebrates (JNCC 2019; Fowles 2020). Limoniscus violaceus was first recorded in Windsor Forest in 1937 and this location was thought to be the home of the largest UK population of the beetle at the time of this study. Windsor Forest is designated a Special Area of Conservation (SAC) under Annex II of the EC Habitats Directive.

The tree hollow microhabitats at Windsor Forest have all been assessed and each rated according to L. violaceus occupancy potential (i.e. habitat status): confirmed, and low, medium or high potential of beetle presence. Habitat statuses were determined in 2014-15 by entomological experts from Natural England and Buglife contracted to survey the site. Veteran beech trees were categorised as high potential if they had basal hollows, blown tops and evidence of heart rot comprising black wood mould within the hollow. Medium potential trees lacked obvious basal hollows but had their top blown off, trunk damage or early-stage basal hollows. Low potential trees were intact beech with no obvious or accessible hollows, or substrate too dry and exposed to be L. violaceus habitat. Breeding populations of L. violaceus were confirmed in five trees by presence of larvae, the surveys taking place 1 to 2 years before this study, thus very likely that beetles remain given their 1 to 2 year larval development.

The number of trees sampled in the present study was based around the number of confirmed L. violaceus habitats, all of which were sampled alongside a similar number from each other survey category. Non-confirmed trees were selected based upon proximity to confirmed trees, the volume and accessibility of wood mould, and to satisfy approximate equivalence to the number of confirmed trees. Wood mould was collected from each sampled Windsor Forest tree in April, May and June 2016, the period during which adult L. violaceus is active (pers. comm. Sarah Henshall, Buglife). Samples were taken from 16 trees at Windsor Forest, comprising five trees with confirmed activity of L. violaceus (all of the confirmed L. violaceus habitat trees present at the time of collection), and three, four and four trees considered to have, respectively, high, medium and low likelihood of violet click beetle presence. All sampling was carried out with a Natural England habitat disturbance license. Samples were taken from standing trees and a single fallen tree. Substrate samples of approximately 100 cm3 in volume were removed by gloved hand from basal hollows. One substrate sample (~15 cm3) was taken from each tree hollow in each month by taking wood mould from a few centimetres below the surface in a gloved hand and sealing it in a universal tube for analysis of volatile organic compounds (VOCs).  The tree diameter at 1.3 m from the ground, and hollow entrance dimensions halfway up and along each hole were measured. The substrate samples were transported to the laboratory at Cardiff University on the same day and stored at 4 °C.

Wood mould characterisation

Density (oven dry mass / fresh volume; g cm-3), water content (% oven dry mass) and water potential (MPa) were determined for each sample. Each of these is intimately associated with the decay process: density determines the physical resistance and affects moisture relations; water content is relevant both in terms of whether there is enough for physiological processes to occur or too much, which restricts aeration, but suffers from the drawback that its value varies not only depending on the amount of water present but also on the density of the material; and water potential indicates the availability of water, the latter known to be particularly important in facilitating fungal activity. Density and water content were determined by measuring fresh volume and mass, oven-drying for 5 days at 60 °C, and reweighing. Water potential of fresh wood mould samples was determined in a Decagon Devices WP4C Dew Point PotentiaMeter (METER Group Inc., Pullman, WA, USA) at 25 °C.

VOC analysis

The VOCs present in each wood mould sample were determined using thermal desorption gas-chromatography time-of-flight mass-spectrometry (TD-GC-TOF-MS) by tipping the wood mould into plastic food-grade roasting bags and sealing them for 30 min at room temperature (~25 °C), allowing the VOCs to diffuse into the bag’s headspace. Using an Easy-VOC manual hand pump (Markes International Ltd., Llantrisant, UK), 500 ml of the air from the headspace was extracted over a SafeLokTM thermal desorption tube (TenaxTA/Sulficarb, Markes International Ltd.). A control sample was prepared by pumping 500 ml of air from the laboratory atmosphere over a thermal desorption tube for each round of sampling. A retention standard was prepared by directly loading 1 µl of C8-C20 alkane standard solution (Sigma-Aldrich, St. Louis, MO, USA) into a thermal desorption tube.

The thermal desorption tubes were placed in a Markes International TD-100 Thermal Desorber (Markes International Ltd.) which desorbed the tubes at 100 °C for 5 min, followed by 280 °C for 5 min with a 40 ml min-1 trap flow. Trap desorption and transfer were carried out with a temperature increase of 20 °C sec-1, a maximum temperature of 300 °C for 3 min, with a split flow of 5 ml min-1. The VOCs were then separated in an Agilent 7890A GC system (Agilent Technologies, Santa Clara, CA, USA) with helium used as a carrier gas at 2 ml min-1 under constant flow conditions for 2 min at 40 °C. This process was followed by a temperature increase of 5 °C min-1 up to a maximum temperature of 240 °C, at which point the temperature remained constant for 5 min. The mass spectra of the separated VOCs were then recorded from m/z 30-350 in a time-of-flight ALMSCO BenchTOF-dx (Markes International Ltd.).

Data from GC-MS were processed using MSD ChemStation (Agilent Technologies Inc. 2005) and AMDIS, with a custom retention-indexed mass spectral library of compounds, a compiled list of compounds identified across all of these samples and others taken from wood mould that have been checked and verified against the NIST 2011 library. All VOCs scoring more than 80 % in forward and backward fit in the NIST 2011 library were included in the custom library of mass spectra. Data were normalised as proportions of each volatile profile and were square root transformed to prevent large values from biasing the results.