Computational protocol: A switch from constitutive chemical defence to inducible innate immune responses in the invasive ladybird Harmonia axyridis

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Protocol publication

[…] The antimicrobial activity of the haemolymph was determined using the common agar diffusion assay on E. coli D31 and M. luteus DSM 20030 plates as described previously []. Wells (3 mm diameter) were punched from the agar plates, and loaded with 3 µl of the haemolymph samples from untreated larvae/beetles and challenged beetles of each ladybird species. Eggs from both species were squeezed directly onto the agar plates. After incubation at 37°C for 24 h, the diameters of the inhibition zones were recorded. Different concentrations of gentamycin sulfate (Roth) and lysozyme (Fluka) were used as standards for calibration. The antibacterial activity of the haemolymph was presented as µg ml−1 gentamycin sulfate or lysozyme equivalents. For statistical calculations, one-way ANOVA and the Holm–Sidak test were performed by using SigmaPlot. A p < 0.05 was considered as statistically significant (see the electronic supplementary material). [...] Haemolymph samples were fractionated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 16.5 per cent tricine gradient gels (Bio-Rad), to determine the effect of bacterial challenge on haemolymph protein composition. The haemolymph from 10 adult beetles of each species (untreated and challenged) was collected into ice-cold phosphate-buffered saline (final dilution 1 : 2), centrifuged at 1500 rcf for 10 min at 4°C, and the supernatant was stored at –20°C. The separated proteins were stained using EZBlue (Sigma) and the bands were analysed using Quantity One 1-D analysis software (Bio-Rad). Lane comparison data were plotted with SigmaPlot v. 8.0. Bio-Rad polypeptide molecular-weight standards were used as size markers. The identification of the induced, low-molecular-weight protein bands by liquid chromatography mass spectrometry in elevated energy mode (LC-MSE) has been described previously []. […]

Pipeline specifications

Software tools SigmaPlot, Quantity One 1-D
Applications Miscellaneous, DNA fingerprinting
Diseases Gilbert Disease