Mimetic host shifts in a social parasite of ants: Analytical data of surface semio-chemicals on Maculinea rebeli larvae

We tracked the changing chemical profiles of caterpillars from each region when reared with each ant, from uncontaminated pre-adoption final instars, to individuals 6 weeks after adoption, and finally to the latter after isolation from ants for 5 days, which allows acquired semio-chemicals to dissipate and prompts the hungry caterpillars to release their own secretions. Hexane extracts of surface chemicals were obtained from: (i) unparasitized Myrmica schencki and M. sabuleti on our study sites in the Pyrenees, Poland, and the naïve ants in France that had not experienced M. rebeli (n = 5 workers from 5 nests of each ant per locality); (ii) 8 batches per region of pre-adoption final instar M. rebeli larvae sampled after leaving G. cruciata before contact with ants (n = 5 larvae per batch, in total 40 individuals for each type of M. rebeli); (iii) M. rebeli larvae after living 6 weeks with naïve French ants, n = 5 (Spanish+schencki and Polish+sabuleti), n = 3 (Spanish+sabuleti and Polish+schencki); and (iv) M. rebeli larvae reared as in (iii), then isolated from ants and kept unfed and singly in sterile conditions for 5 days; n = 5 (Spanish+schencki), n = 4 (Polish+schencki), n = 3 (Spanish+sabuleti and Polish+sabuleti). The chemical and statistical analyses of extracts followed an established protocol. Maculinea extracts were concentrated to 20ml, ant workers to 50ml, and 2ml of every sample were analyzed by gas chromatography with mass spectrometric detection (GC-MSD) using a HP 5890II GC and HP 5971A MSD, and ultra-high purity helium as the carrier gas with 10 psi column head pressure. Mass spectral data were acquired in full scan mode over 40 - 600 m/z. Mass chromatograms were initially screened for hydrocarbons by examining the selected ion chromatogram of m/z = 57. The chromatogram was integrated at a threshold value of 12 (HP integrator) to obtain the areas under the peaks measuring the total ion count. With each sequence of samples we also analysed alkane standards (n-C20 - n-C36), and the position of each peak within that range in a sample was calculated as an Equivalent Chain Length (ECL). Mass chromatograms were inspected to ensure that they were free of gross interferences and that peaks of interest such as branched and straight alkanes and alkenes, were chromatographically distinct and symmetrical. We excluded peaks that were column bleed, siloxanes, or phthalate plasticizers as indicated by a characteristic abundant ion at m/z 149. Peaks of interest were tentatively identified by a combination of ECL number and inspections of their full scan mass spectra and matching with the NIST-97/08 mass spectral database. For statistical analysis, the area under each peak was expressed as the proportion of the sum of all peaks in the chromatogram. Samples were compared using multivariate and non-parametric multidimensional scaling (MDS) on the ranks of the Bray-Curtis similarities. The extent of a final lack of fit was assessed by a STRESS statistic before pair-wise differences between species and treatments were assessed using an analysis of similarities (ANOSIM) in Primer-e v6. We used the average pairwise distance between groups, and assessed two averages with a two-sample t-test, to compare differences in the shift of similarities between groups.