Sample collection
During May and August 2020, four and five trunks (dbh 20-25 cm),
respectively, were sampled in the surroundings of Nižbor (Czechia,
49°59’09.9”N 13°56’47.5”E, 390 m.a.s.l.). The sampling site is situated
in a continuously forested area belonging to Protected Landscape Area
Křivoklátsko. The average annual temperature is 9 °C and the average
annual precipitation is 494.9 mm (Lány observatory, Czech
Hydrometeorological Institute). Spruce trees infested by I.
typographu s were randomly selected, felled, cut into logs and
transported to the Institute of Microbiology of the CAS, Prague. Logs
were incubated in the exterior in a shaded place simulating the original
forest site and successively sampled for various bark beetle life stages
regardless of the gender (parental adults, larvae, pupae, young
(teneral) adults, and infested pigmented and intact phloem as a control,
see sampling scheme, Fig. 1). We also sampled freshly laid eggs;
however, we were not able to ensure their proper surface sterilization.
Thus, the eggs were omitted from the analysis. Beetle species
determination was based on their macromorphology under a binocular
magnifier (Nikon SZ30, Minato, Japan) using determination literature
(Pfeffer, 1955). Beetle samples were taken out of the galleries and
surface sterilized by subsequent washing with 70% ethanol, 2% Tween 80
(Avantor, USA) and sterile distilled water. Randomly selected 5
galleries from each log were sampled and pooled into one representative
sample containing 10 individuals. Suspicious or parasitized galleries or
individuals were excluded from the study. In the summer season, the
parts of the infested phloem (2x5 mm) from active galleries and parts of
intact phloem (2x5 mm) were collected. The single sample consisted of
five phloem pieces collected on the same log, and pooled. Fresh intact
phloem was sampled at the beginning of the beetle development at least
10 cm from the young maternal gallery where no colonization by
microorganisms was visible. Infested phloem was sampled at the end of
the development of teneral adults and was distinct by the colorization
of plant tissues. Pooled samples were frozen at -80°C in Eppendorf tubes
till DNA/RNA extraction, if necessary.