BioSample

Mountain pine beetle-attacked Lodgepole pine (Pinus contorta) trees were felled in Spring 2008 in Jackman Flats Provincial Park, BC, Canada (approx. N 52 55.958’ W 119 22.457’) and later placed in laboratory emergence cages at UNBC. Between Apr. 17 and May 10, 2008, 180 live mountain pine beetle (Dendroctonus ponderosae Hopkins) emerged adults were collected from the laboratory emergence cages and sent to UBC on moist filter paper. Between May 8 and May 22, beetles were immobilized on ice, antennectomized, treated topically on their abdominal venter with 10 micrograms of racemic juvenile hormone in 0.5 microlitres acetone and held in the dark in groups of 10 on moist KimWipes in 60 mL plastic Dixie cups for 1, 5 or 16 hr after which time their anterior midguts and adhering fatbody were dissected under water and immediately frozen in liquid nitrogen. At each time point, 60 insects with approximately an equal number of sexes were treated. Total RNA (482 micrograms) was extracted from frozen midgut/fat body tissue of 180 insects (weight undetermined) using the Qiagen RNeasy Mini Plant Kit. mRNA (8.2 micrograms) was purified from total RNA (366 micrograms) using the Oligotex mRNA Kit (Qiagen). First strand cDNA was prepared from 1 microgram of mRNA, SuperScript III reverse transcriptase (Invitrogen), CDS-3M primer (Evrogen), and the SMART IV Oligonucleotide (Clontech ). Second strand cDNA was prepared by LD-PCR with Phusion Hot Start DNA Polymerase (Finnzymes, Espoo, Finland). This cDNA was then normalized with duplex-specific nuclease and amplified following the TRIMMER-DIRECT (Evrogen) protocol. This cDNA is the same as used for Sanger EST sequencing with clone IDs beginning with DPO10 (dbEST accessions GT397484-GT419918).