Protection of a defensive symbiont does not constrain the composition of the multifunctional hydrocarbon profile in digger wasps

Abstract

Hydrocarbons (HCs) fulfil indispensable functions in insects, protecting against desiccation and serving chemical communication. However, the link between composition and function, and the selection pressures shaping HC profiles remain poorly understood. Beewolf digger wasps (Hymenoptera: Crabronidae) use an antennal gland secretion rich in linear unsaturated HCs to form a hydrophobic barrier around their defensive bacterial symbiont, protecting it from brood cell fumigation by toxic egg-produced nitric oxide (NO). Virtually identical HC compositions mediate desiccation protection and prey preservation from moulding in underground beewolf brood cells. It is unknown whether this composition presents an optimized adaptation to all functions, or a compromise due to conflicting selection pressures. Here, we reconstitute the NO barrier with single and binary combinations of synthetic linear saturated and unsaturated HCs, corresponding to HCs found in beewolves. The results show that pure alkanes as well as 3 : 1 mixtures of alkanes and alkenes resembling the composition of beewolf HCs form efficient protective barriers against NO, indicating that protection can be achieved by different mixtures of HCs. Since in vitro assays with symbiont cultures from different beewolf hosts indicate widespread NO sensitivity, HC-mediated protection from NO is likely important across Philanthini wasps. We conclude that HC-mediated protection of the symbiont from NO does not exert a conflicting selection pressure on the multifunctional HC profile of beewolves.

Keywords: Philanthus; Streptomyces; hydrocarbon; mutualism; nitric oxide; symbiosis.

Figure 1.

Nitric oxide (NO) sensitivity of ‘S. philanthi’ biovariations and free-living Streptomyces at different NO concentrations. Symbionts (purple) were more sensitive to NO than free-living Streptomyces (off-white) (ANOVA, χ² = 103.5, d.f. = 2, p < 2.2 × 10⁻¹⁶), and growth inhibition increased with NO concentration (ANOVA, χ² = 200.3, d.f. = 2, p < 2.2 × 10⁻¹⁶). In addition, the strains varied in their NO sensitivity (ANOVA, χ² = 155.4, d.f. = 40, p = 1.618 × 10⁻¹⁵). The sizes of the circles indicate the number of replicates in the different growth categories. See electronic supplementary material, table S2 for strain designations.

Figure 2.

Reconstitution of the nitric oxide (NO) barrier effect with single and binary combinations of synthetic hydrocarbons (HCs). A total of 100 µg of HCs was applied for all treatments. Effectivity of HCs in blocking NO was measured as the change in coloration in an NO indicator solution, with higher OD₅₄₀ values indicating stronger oxidation and thus less protection against NO by the HC layer (see representative images on the top; for all images see electronic supplementary material, figure S4). Beewolf CHC extracts served as positive, and hexane as negative control. All HCs and the alkene–alkane ratio of binary combinations are found in P. triangulum AGS and HC extracts (electronic supplementary material, table S1, figure S1). Letters indicate significant differences (Tukey's HSD, n = 8, p < 0.05).