Electronic Supplementary Material
Sandra Steiger, Klaus Peschke, Wittko Francke & Josef K. Müller
Supplementary Methods
Analysis of cuticular compounds
Samples were quantified on a HP 6890 gas chromatograph with a split/spitless injector (300°C, automatic sampling, injection of 1μl). A fused silica column (DB-1, 30 m x 0.25 mm ID, 0.25 μm, J & W Scientific, Folsom, Canada) was used with a helium flow of 1ml/min. Oven temperature started at 35°C for 2 min, was then raised quickly to 100°C at a rate of 20°C/min, then more slowly to 300°C at 6°C/min, and remained at the final temperature for 25 min. The flame ionisation detector (FID) was run with 40 ml H2/min and 450 ml air/min. Chemical identification was performed on a coupled gas chromatograph-mass spectrometer system (HP 6890 series GC – HP 5973 MSD) at an electron impact ionisation of 70eV. The capillary column and temperature program were the same as those used for GC. We identified n-alkanes and methyl-branched alkanes by their mass spectra (Blomquist & Dillwith 1985; Doolittle et al. 1995; Jackson & Blomquist 1976; Nelson 1993; Nelson et al. 1972; Pomonis et al. 1980), and confirmed the identity of peaks with retention indices (Carlson et al. 1998; Francis & Veland 1981). The positions of double bonds in unsaturated hydrocarbons were determined by interpreting the mass spectra of the dimethyl disulfide derivatives (DMDS, Francis & Veland 1981). The position of the double bonds in olefins with more than two double bonds could not be elucidated by using DMDS adducts. However, from comparison of the mass spectra and retention indices of synthetic compounds (syntheses were carried out by W. F., to be published), structures of alkatetraenes and alkapentaenes could be assigned. The identification of the positions of the double bonds of alkatrienes and alkahexaenes was beyond the scope of our study. The preliminary characterisation of trienes and hexaenes were based on their mass spectra and on a comparison with the mass spectra of the existing synthetic alkatetraenes and alkapentaenes (for alkatrienes see also Millar 2000). Relative retention indices of branched and unsaturated hydrocarbons were calculated by using the retention times of the straight-chain hydrocarbons present in the extracts (Steinmetz et al. 2003; Van den Dool & Kratz 1963).
Contamination by oral and anal secretions
Beetles tended to release oral and anal secretions as they were handled and placed in the freezer. To analyse which substances in the extract did not derive from the cuticle, but originated from oral and anal secretions, secretions of living beetles were soaked up into small pieces of filter paper which were subsequently washed for 15 min in pentane. The respective extracts were analysed by GC-MS.
External sources of chemicals
To ascertain that differences found between the extracts of breeding and non-breeding individuals did not originate from other sources than the beetles themselves, control groups were established. Parental beetles might acquire chemicals through contact with the carcass during preparation or through contact with larvae when caring. To control for the former we rubbed living non-breeding females (n = 6) intensively with a carcass that had been treated by a pair of beetles for approximately 96 h. This procedure was repeated once a day for a period of four days. Shortly after the last time rubbing, beetles were killed for chemical analysis. To control for larvae as a source of chemicals, pairs (n = 6) of beetles were first subjected to the same protocol as the beetles in the parental group. However, instead of providing them with 10 larvae for caring, beetles were killed for chemical analysis when their first larvae hatched. Therefore, these beetles were in a parental state, but had never contact with larvae.
Supplementary Results
External sources of chemicals
The higher amounts of polyene hydrocarbons in parental beetles in comparison to non-parental ones could either be caused by a difference in polyene production of the beetles or by a transfer of chemicals originating from external sources. In the pentane extract of non-breeding females rubbed with prepared carrion, no increased amount of polyenes was detected. The extracts of such treated females were characterised by a significantly lower proportion of polyenes compared to the extracts of parental females (ANOVA, post hoc Bonferroni, p < 0.001; Fig. 3), but by amounts similar to that of non-breeding females (post hoc Bonferroni, p > 0.05). We were also able to exclude the possibility that polyenes are produced by larvae and transferred to the parents by showing that a high amount of polyenes was also found in parental beetles that never encountered their larvae (comparison with non-parental beetles: post hoc Bonferroni, p < 0.001; comparison with parental beetles with larvae: post hoc Bonferroni, p > 0.05; Fig. 3).
Supplementary Table
Table 2: Squared Mahalanobis distances between the group centroids (referring to DA of figure 1; MM, males fed mealworms; MF, females fed mealworms; CM, males fed vertebrate carrion; CF, females fed vertebrate carrion; PM, parental males; PF, parental females). Significance level for all distances: p < 0.0001.
group / MM / MF / CM / CF / PM / PFMM / 0,00 / 35,83 / 292,88 / 298,25 / 611,70 / 689,87
MF / 0,00 / 264,97 / 225,76 / 678,48 / 761,28
CM / 0,00 / 24,46 / 584,30 / 682,49
CF / 0,00 / 646,73 / 740,53
PM / 0,00 / 24,31
PF / 0,00
Supplementary Figure
Fig.3 Mean ± SE relative amount of polyenes with more than two double bonds in the extract of females from different treatments (with each n = 6; np = non-parental, npc = non-parental rubbed with vertebrate carcass, p = parental without larvae, pl = parental with larvae). Different letters above the bars indicate statistically significant differences (ANOVA, F = 46.28, p < 0.001).
Supplementary References
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