Two experiments were carried out to screen for immune-related transcript changes. In the first, adult worker bees from a single local A. mellifera ligustica colony were removed, then injected abdominally with either dilute phosphate-buffered saline or saline solution containing 103 live cells of E. coli or 103 vegetative spores of the honey bee bacterial pathogen Paenibacillus larvae. These bees, along with uninjected controls, were maintained for 24 h at high humidity and 34 °C and then were immediately frozen at −70 °C prior to RNA extraction. To assess immune responses following natural infection, eight 1st-instar larval bees from the same stock were given per os challenges of P. larvae in their food [(50 spores/µl as described in Evans (2004) ], then maintained 24 h at 34 °C and high humidity. Parallel control larvae were given the same food without bacterial spores. All samples were frozen at −80 °C following incubation.