Host hydrocarbons protect symbiont transmission from a radical host defense

Abstract

Symbioses with microbes play a pivotal role in the evolutionary success of insects, and can lead to intimate host-symbiont associations. However, how the host maintains a stable symbiosis with its beneficial partners while keeping antagonistic microbes in check remains incompletely understood. Here, we uncover a mechanism by which a host protects its symbiont from the host's own broad-range antimicrobial defense during transmission. Beewolves, a group of solitary digger wasps (Hymenoptera: Crabronidae), provide their brood cells with symbiotic Streptomyces bacteria that are later transferred to the cocoon and protect the offspring from opportunistic pathogens by producing antibiotics. In the brood cell, however, the symbiont-containing secretion is exposed to a toxic burst of nitric oxide (NO) released by the beewolf egg, which effectively kills antagonistic microorganisms. How the symbiont survives this lethal NO burst remained unknown. Here, we report that upon NO exposure in vitro, the symbionts mount a global stress response, but this is insufficient to ensure survival at brood cell-level NO concentrations. Instead, in vivo bioassays demonstrate that the host's antennal gland secretion (AGS) surrounding the symbionts in the brood cell provides an effective diffusion barrier against NO. This physicochemical protection can be reconstituted in vitro by beewolf hydrocarbon extracts and synthetic hydrocarbons, indicating that the host-derived long-chain alkenes and alkanes in the AGS are responsible for shielding the symbionts from NO. Our results reveal how host adaptations can protect a symbiont from host-generated oxidative and nitrosative stress during transmission, thereby efficiently balancing pathogen defense and mutualism maintenance.

Keywords: Streptomyces; defensive symbiosis; immune system; mutualism; nitric oxide.

Fig. 1.

Life cycle of the European Beewolf (P. triangulum). (A) Beewolf females hunt Apis mellifera workers and paralyze them by injecting their venom into the bee’s thorax (51). They provide their subterranean brood cells with one to several bees, embalmed in a secretion from their postpharyngeal gland (41). This secretion contains high amounts of long-chain saturated and unsaturated hydrocarbons that prevent infection of the provisions by reducing water condensation (–44). The female deposits an egg on top of the provisions (52). Before sealing the brood cell, the female applies an AGS rich in linear unsaturated and saturated hydrocarbons (53) and containing the defensive symbiont Streptomyces philanthi to the brood cell ceiling (46, 54). (B) The beewolf egg sanitizes the brood cell by releasing high amounts of toxic nitric oxide (NO), with NO emission peaking at ~14 to 16 h after oviposition (39). While NO effectively kills microbial opportunists in the brood cell (39), S. philanthi withstands NO via a previously unknown mechanism. (C) After hatching and feeding on the provisioned bees for several days, the larva integrates S. philanthi into its cocoon (46). On the cocoon surface, the symbionts produce an antibiotic cocktail (47, 55) that provides protection from microbial infestation (46, 47). (D) After 4 to 6 wk or in the following summer, the larva undergoes metamorphosis, and the adult ecloses from the cocoon (52, 56).

Fig. 2.

Changes in gene expression in (A) free-living S. coelicolor (indicated by the symbol of filamentous bacteria) and (B) symbiotic S. philanthi (indicated by the beewolf head) 6 h after in vitro exposure to NO in comparison to exposure to N₂ (indicated by the petri dish), and in (C) symbiotic S. philanthi in the AGS within beewolf brood cells in the presence vs. the absence of a beewolf egg emitting NO (indicated by the beewolf brood cell). Significant gene expression differences are highlighted in color (adjusted P < 0.05 and twofold to fourfold differential expression in blue, and more than fourfold change in red). Log-transformed values of some extremely highly expressed genes were set to 5 to improve readability and are indicated with triangles instead of circles.

Fig. 3.

Symbionts display a drastic change in the expression of general stress response genes, as well as oxidative and nitrosative stress–specific responses in vitro, but not in vivo (i.e., within the beewolf brood cell). Treatment-specific expression of genes associated with (A) the response to nitrosative and oxidative stress, (B) DNA interaction, (C) protein protection, and (D) redox state controlling. Scoe = free-living S. coelicolor (also indicated by the symbol of filamentous bacteria), Sphi = symbiotic S. philanthi (also indicated by the beewolf head), AGS = antennal gland secretion. The petri dish indicates gene expression in vitro, the beewolf brood cell symbolizes gene expression within the AGS. The P denotes genes for which the respective protein was detected in the proteomic analysis of the AGS.

Fig. 4.

The beewolf female’s AGS blocks diffusion of NO both in vivo and in vitro. (A) Beewolf brood cell in an observation cage, with paralyzed bees at the bottom of the brood cell and the beewolf egg on top of one of the bees. The AGS is visible as small white specks at the ceiling of the brood cell, in this case a transparent plastic sheet. (B) Enlarged region of the brood cell from A, showing the AGS on the brood cell ceiling. (C) Representative filter paper prepared with NO indicator that turned brown after NO exposure in the brood cell, with a clear zone where the AGS is localized, enlarged in (D). (E and F) Same areas as in C and (D) under fluorescent light, corroborating the position of the AGS by its autofluorescence. [Scale bars: (A, C, and E): 4 mm; (B, D, and F): 1 mm.] (G) Quantification of indicator reaction upon NO exposure in beewolf brood cells. Comparison of normalized mean gray values between zones of filter paper treated with AGS vs. the surrounding area exposed to NO in the brood cell, and a control without NO exposure (artificial brood cell without beewolf egg that was excavated next to the target brood cell). Different letters above the boxes indicate significant differences (Tukey’s HSD, P < 0.05). (H) Quantification of indicator reaction upon NO exposure in solutions with a cover of beewolf CHCs vs. a control of hexane (Wilcoxon rank sum exact test, W = 0, P = 0.002, N = 6). The Inset shows the dark blue indicator solution in control tubes after NO exposure (Left) as compared to the transparent solutions covered by beewolf CHCs that prevent NO diffusion (Right).