Immune stimulation of pupae using β-glucans

We injected β−1,3-glucans to test whether the changes in the chemical profile of infected pupae may be caused by an immune stimulation (Vilcinskas and Wedde, 1997; Unestam and Söderhäll, 1977; Gunnarsson, 1988). Soluble β−1,3-glucans were acquired by suspending 5 mg of Zymosan-A (Saccharomyces cerevisiae cell wall fragments [Sigma-Aldrich]) in 1 ml of sterile physiological ant saline (as described in [Aubert and Richard, 2008]). The Zymosan suspension was vortexed for 1 hr at 3200 rpm before being centrifuged at 10000 rcf for 5 min. The supernatant that contains the soluble β-glucans (Vilcinskas and Wedde, 1997) was then removed and stored at 4°C until use. As a control we used sterile ant physiological saline (Aubert and Richard, 2008). Pupae were artificially unpacked from their cocoons (as above) and placed gently into a sponge harness. Using fine glass capillaries (with spike to aid injection; inner diameter = 25 µm [BioMedical Instruments, Germany]), a microinjector (parameters: pi = 120 hPa, ti = 0.3 s, pc = 20 hPa [FemtoJet, Eppendorf, Germany]) and a micromanipulator (Luigs and Neumann, Germany), we injected 46 nl of the β-glucan solution or ant physiological saline through the pupae’s first tergite, into their haemocoel. We cleaned the capillaries between injections using 96% ethanol. Half of the pupae were frozen at – 80°C immediately after injection whilst the remainder were kept alone in individual plaster dishes for a further 48 hr, before then also being frozen. Frozen pupae were then used for molecular and chemical analyses (below).