Controlled crosses
We collected mountain pine beetle larvae (F0 generation) from infected
lodgepole pine trees from Smokey River Lowlands near Grande Prairie
(Alberta, Canada; 54°21.376’ N, 118°19.112’ W) and BURNCO Quarry near
Canmore (Alberta, Canada; 51°04.026’ N, 115°17.237’ W) in October of
2014 (Supp. Fig. 1). These sites represent the two genetically distinct
sub-populations previously identified within Canada (Batista et al.,
2016; Janes et al., 2014; Samarasekera et al., 2012) and are referred to
in this study as North and South, respectively. Collected larvae
continued development in their natal bolts at 4°C until spring 2015.
Next, we placed bolts in opaque emergence boxes at room temperature
(approximately 22°C) and mature mountain pine beetles were collected
daily for a maximum of 15 days. Freshly emerged mountain pine beetles
were sexed using the auditory method described by Rosenberger et al.
(2016) to minimize physical damage. We temporarily stored the emerged
adults at 4°C until a sufficient number had been collected to begin
crosses (maximum five days). Further details are provided in Trevoy et
al. (2019).
We reared North x South crosses of the F0 generation in 55 cm bolts from
three lodgepole pine, Pinus contorta Douglas ex Louden (Pinales:
Pinaceae), trees felled in May 2015 at Nojack, Alberta (53°36.103’ N,
115°35.239’ W). Females were placed in 1.5 ml microcentrifuge tubes
attached to bolts to encourage gallery construction within a specific
bolt. Males from the opposite population were placed in the tube 48 h
later. Females or males that failed to enter the bolt within 24 h were
replaced with alternates from the same site of origin. We stored F0
crosses in a sealed room at approximately 22°C for six weeks, at which
point we transferred them to emergence boxes. F1 progeny were collected
over 15 days and physically sexed using the non-destructive auditory
method. Mountain pine beetle adults have been known to establish
secondary galleries in different trees (Janes et al., 2016). To ensure
that F1 beetles were assigned to the correct gallery, and therefore the
correct cross, each bolt was stripped and visually inspected. Any
progeny from a bolt containing multiple galleries was discarded from
further analysis.
We crossed F1 beetles with gallery siblings to produce highly related F2
individuals. Males tend to guard the entrance of a gallery and pack it
with frass (Reid, 1962). Thus, after seven days, we peeled back the wood
around each gallery to a depth of 5 cm and the male was removed for
genotyping. In accordance with Amman (1972), females were expected to
complete gallery construction within three weeks. Thus, we extracted
females at a later date in a similar fashion to males. Bolts were placed
in emergence boxes at approximately 22°C until additional F2 progeny
were no longer recovered. All F2 individuals were stored at –20°C prior
to DNA extraction after physical sexing, using the dimorphic seventh
tergite (Safranyik & Carroll, 2006). Sexing using the dimorphic seventh
tergite was reserved for F2 individuals because, while more accurate
than the auditory method, it typically causes damage to the specimen.
The crossing design and family summaries are illustrated in Supp. Fig.
1. Specimens have been vouchered at the E. H. Strickland Entomological
Museum at the University of Alberta with UASM numbers 391988-391991.