Oviposition and larval mycelia preference of the saproxylic European stag beetle

In the light of evidence-based conservation, we analysed the relation between the European stag beetle (Lucanus cervus) and a selection of ten common wood-decomposing fungi. We studied log preference and number of offspring for logs inoculated with different fungi during oviposition in a cafeteria experiment. A second experiment assessed survival and relative growth rates of larvae placed in substrates colonized by the different fungal species. The selected fungi used in both experiments are phylogenetically diverse and represent different rot types. Females laid no eggs in logs with brown-rot fungi and all larvae died in brown-rot mycelia within 33 days due to black spot. All white-rot fungi were accepted for oviposition, with no species-specific preferences. The larval survival rate in white-rot species was at least 57% and the relative growth rate of larvae was positive (except for the toxic Omphalotus illudens). At rot type level, we can confirm the optimal oviposition hypothesis, a match between oviposition and larval habitat. Our findings contradict the association of brown-rot fungal species with stag beetles, as reported in some literature, which is probably based on the presence of fruiting bodies. We conclude that the European stag beetle is a white-rot dependent species and accepts numerous species of white-rot fungi without a clear preference. This is relevant for evidence-based conservation when restoring its habitat or building artificial habitats (such as log piles) as well as for ex-situ breeding campaigns.

Methods

Ten fungal species were selected for the larval as well as the oviposition experiments (Table 1). All selected species have a wide distribution in Europe and are capable of colonising subterranean deciduous dead wood. Some of the selected species have been mentioned in the literature to be associated with the European stag beetle (Table 1). Furthermore, species were selected to represent different rot types (based on Funguild, Nguyen et al., 2016) and taxonomic groups (Basidiomycota and Ascomycota). Finally, Omphalotus illudens (French and Garrettson) (Schwein.) Bresinsky & Besl was included as it contains the toxin illudin, which is at least toxic to humans and rats (French & Garrettson, 1988; Tanaka et al., 1996).

Table 1. Selected fungus species in alphabetical order within rot type used in the two experiments with abbreviated name (Abbr.), rot type (Nguyen et al., 2016), division, reference for its association with L. cervus, and number of successful inoculated logs.

Abbr.

Scientific name

English name

Rot type

Division

Association with L. cervus

Successful log inoculations

Am

Armillaria mellea (Vahl) P. Kumm.

Honey fungus

White rot

Basidiomycota

6/10

Fv

Flammulina velutipes (Curtis) Singer

Velvet Shank

White rot

Basidiomycota

10/10

Gl

Ganoderma lucidum (Fr.) P. Karst.

White rot

Basidiomycota

Ganoderma applanatum (Pers.) Pat.; Rink, 2006; Rink & Sinsch, 2008

10/10

Gf

Grifola frondosa (Dicks.) Gray

Hen of the Woods

White rot

Basidiomycota

10/10

Km

Kuehneromyces mutabilis (Schaeff.) Singer & A.H.Sm.

Sheathed woodtuft

White rot

Basidiomycota

2/10

Oi

Omphalotus illudens (French and Garrettson) (Schwein.) Bresinsky & Besl

Jack o' Lantern

White rot

Basidiomycota

0/10

Tv

Trametes versicolor (L.) Lloyd

Turkeytail

White rot

Basidiomycota

10/10

Fp

Fomitopsis pinicola (Sw.) P. Karst.

Redbelted Bracket

Brown rot

Basidiomycota

Tochtermann, 1992

10/10

Ls

Laetiporus sulphureus (Bull.) Murrill

Chicken-of-the-woods

Brown rot

Basidiomycota

Scherf, 1985

8/10

Cg

Chaetomium globosum Kunze

Soft rot

Ascomycota

0/10

Oviposition experiment

A cafeteria experiment was set up to study the oviposition preference of females for the different selected fungi. For this, fresh beech logs (Fagus sylvatica Linnaeus, 1753) of 40 cm length and diameters between 10-15 cm were collected at Begijnenbos in Buizingen (Belgium, 50°45'N, 4°18'E) from a stand thinning at the end of January 2023. Hundred logs (with similar volume) were inoculated by placing 100 spawn dowels in each log, ten logs for each fungal species. The inoculation was performed by the company Mycelia (Deinze, Belgium) in a laminar flow chamber and subsequently logs were sealed in mushroom bags with aeration strips to avoid contamination. Logs were stored at 25 °C and 90% RH for four months to allow the mycelia to colonise the logs.

Colonization of some species was unsuccessful, resulting in only seven species remaining with a varying number of logs (2-10, Table 1). Secondly, species-specific decomposition rates resulted in a varying degree of log density (hardness). The only two logs of Kuehneromyces mutabilis (Schaeff.) Singer & A.H.Sm. were cut in half to gain a total of 68 logs used in the oviposition experiment. The length and diameter at both ends and weight of each log was measured before the start of the experiment in order to calculate its volume and density.

Ten plastic boxes measuring (l x w x h) 80 x 60 x 42 cm with 16 6 mm aeration holes at the side were subdivided into ten uniform compartments of 16 x 30 x 30 cm using plastic biplex sheets. Each compartment was filled randomly with either a vertically positioned log and covered with wood compost (gabco Kompostierung GmbH, Aachen, Germany), or solely with wood compost (n=32) so that each fungus species was used only once in each box (Table S1). The substrate height was 30 cm presenting a flat surface with the biplex sheets and only the logs protruding on top allowing accessibility for the adult females to all compartments by the 12 cm of free space up to the lid. The boxes were filled on June 13th, 2023, and placed in a climate chamber with a constant temperature of 17 °C.

Ten adult male and female stag beetles were collected from three nearby populations (Huizingen, Overijse, and Sint-Genesius-Rode) in Belgium between the 21st and 23rd of June 2023. These different populations belong to a once continuous population but are currently quite small, isolated, and genetically impoverished (Cox et al., 2020). When placing one couple in each of the designated boxes on June 30th, the male and female were selected from a different population as the final offspring will be used for restocking with genetic enriched individuals (Table S2). For this experiment, we will not consider the population genetics and fitness in different environments.

The beetles were fed with beetle jelly (beetlejelly.eu) and subjected to weekly monitoring. Dead adult beetles (parents) were removed and if required about 1 L of water was added to dry compartments.

On October 4th, 2023, the number of offspring was counted a first time by carefully emptying each compartment. The number of eggs and three larval instars (L1, L2, and L3) found in the substrate or on the surface of the wood was noted and removed from the oviposition boxes. Furthermore, the number of galleries in the logs containing most likely one or more larvae were also noted. The logs were placed back and the number of remaining larvae was counted again on May 3rd 2024 by breaking open all the logs with galleries. The total number of offspring as well as the log preference (logs with at least one egg or larva) was calculated by compiling both assessments. Both were analysed, log preference represents females acceptance of certain fungi while number of larvae might also account for the females' preference to lay more eggs or for higher survival rates of eggs and larvae in certain fungi. Furthermore, female stag beetles lay eggs in groups and groups become smaller with female age (Tochtermann, 1992) which might interfere with offspring size.

The mycelia of several species grew outside the log and colonised the surrounding wood compost. Especially the mycelia of the brown-rot fungi F. pinicola and L. sulphureus were found to grow strongly in the compost, even in adjacent compartments. These mycelia can be easily recognised by their wet, slimy texture and a strong sulphuric odour. The presence of brown-rot in other compartments was therefore noted on October 4th and the logs of these two species were removed from the boxes and further reared separately.

Larval experiment

To gain larvae, three stag beetle pairs were collected in Glons, Belgium (50°45'N, 5°32'E) in June 2022 and placed in breeding containers with white-rotten oak (Quercus spp.) wood buried in fermented beech sawdust. The population in Glons is part of the well-connected population along the Sambre-Meuse valley which is isolated (86 km) from the Brabant population where the adults for the oviposition experiment came from. The populations were selected for restocking and reintroduction goals and not specifically for the experiments. The adults were fed with beetle jelly and remained in the containers.

For the rearing substrate, two mushroom bags with 5 l of beech sawdust were inoculated for each of the ten selected fungal species (Table 1). The sawdust was intermixed with whole wheat grains, wheat bran, gypsum, and limestone powder as additives to optimise initial fungal growth. The substrates were produced by Mycelia (Deinze, Belgium) with their normal rearing standards. These mature mycelia substrates were used to rear the stag beetle larvae.

On October 5th, 2022, 69 larvae (58 L2, 11 L1) were collected from the rearing containers. Consequently, 69 1-litre transparent plastic containers (l x w x h) 15.5 x 10.5 x 7 cm with seven 3 mm-aeration holes just under the lid were filled with mycelia substrate, seven for each fungal species except for Chaetomium globosum Kunze with six replicates. The larvae were weighed, the larval instar determined, and randomly assigned to one of the containers.

The larvae were placed in a climate chamber at 16 °C and checked about weekly. On November 7th, 2022, i.e. on day 33, surviving larvae were weighed and the experiment ceased as some substrates were starting to get covered with green moulds while other mycelia were growing very vigorously resulting in encapsulation of the larvae (see results). Relative growth rate (RGR) was calculated from the initial (i) and final (f) weight as:

RGR = (Weight-f – Weight-i) / Weight-i.