Immediately after collection larva were decapitated and gutted.
During collections, larvae were transported in a cryogenic vapour shipping tank (MVE Vapor Shipper, Minnesota Valley Engineering Inc., New Prague, MN, USA) held constant at −150°C until arrival at UBC where the samples are stored long-term at −80°C.
In order to avoid interaction with outside environment after collection, the larvae were maintained in the bee combs prior to their use in the assay.
Our collection of larvae was haphazard rather than strictly random but our collection methods were identical among our different treatments throughout our experiments ensuring that no biases were introduced to our findings.
The E-strain colony was established from a 2005 collection of larvae from maize stems by Smiljana Tomse (Agriculture and Forestry Institute, Novo Mesto, Slovenia).
The collection of larvae of coastal marine invertebrates at often great distances from the coast [ 3], and the often limited genetic structuring of widely separated populations of high-dispersal species [ 4, 5] reinforce this notion.
Panel shows numbers (see also Figure 1B,B′ ) of LTP positive neurons/brain hemisphere (red) and Repo positive glia/brain (blue) from staged First, second, early and mid third instar (3rd-72h = 72 hr and 3rd-96h = 96 hr after egg collection) wt larvae.
(D, E, G ) Plotted are percentages (Y-axis) of hatched embryos (D ; nYF = 198, nPF = 198; parental flies kept for three generations on respective food types before embryo collection), pupariated larvae (E ; nYF = 163 and nPF = 133; time of pupal development was unchanged) and of living mated females (G ; nYF = 40 and nPF = 35) over time (X-axis).
Two quality control steps ensured the accurate collection of diapausing larvae: Diapausing larvae were only collected after 100% of the previous batch of their mother's offspring (a given virgin female) had entered diapause.
