Refining the Roots of the Beewolf-Streptomyces Symbiosis: Antennal Symbionts in the Rare Genus Philanthinus (Hymenoptera, Crabronidae)

INTRODUCTION

Insects engage in a great diversity of symbiotic interactions with microorganisms (5). Bacterial symbionts in the gut or gut-associated organs that contribute to host nutrition are especially widespread and common (3, 8). Recently, however, an increasing number of mutualistic associations for the defense of the host or its nutritional resources against predators, pathogens, or parasitoids have been described (4, 13). Several such symbioses involve actinobacteria that are well known for the production of a large number of antibiotics, many of which are of outstanding clinical relevance (13). Notably, actinobacteria are an important first line of defense against pathogenic or commensal fungi in the fungus gardens of leaf-cutting ants (7, 11, 22, 25), as well as in the fungal galleries of Southern pine beetles (26).

Solitary digger wasps of the genera Philanthus and Trachypus (Hymenoptera, Crabronidae), the so-called beewolves, have been shown to engage in a highly specialized symbiotic association with actinobacteria of the genus Streptomyces (“Candidatus Streptomyces philanthi”) (14, 15, 17). The symbionts are cultivated in unique antennal gland reservoirs that constitute invaginations of the outer antennal cuticle (9). In the reservoirs, the bacteria are presumably provided with nutrients from the host via the associated gland cells or from the hemolymph (18). The bacteria are vertically transmitted by an unusual mechanism of posthatching transfer (16). Each brood cell is supplied with the symbiotic bacteria from the antennal gland reservoirs of the female beewolf. Later, the larva transfers the symbionts to its cocoon, where they provide an effective defense against detrimental microorganisms during the long period of hibernation in the brood cell (15). In Philanthus triangulum (Fabricius, 1775), this protective activity is mediated by at least nine different antibiotic substances that are produced by the symbionts on the cocoon (19). The complex cocktail serves as an effective defense against a broad spectrum of potentially pathogenic microorganisms, a strategy that is comparable to the combination prophylaxis used in human medicine (19). In addition to having this protective function, the bacterium-containing antennal gland secretion also guides the emergence of the beewolf progeny (28).

The antennal Streptomyces symbionts have been described for the genera Philanthus (14, 15) and Trachypus (17), while other genera of the subfamily Philanthinae (Cerceris, Aphilanthops, and Clypeadon) apparently lack the symbionts (14). The rare genera Philanthinus and Pseudoscolia, however, have not yet been investigated. While Pseudoscolia is closely related to Cerceris (1) and is therefore unlikely to harbor antennal symbionts, Philanthinus is the genus most closely related to Philanthus and Trachypus. Here, we used a combination of molecular and histological techniques to investigate Philanthinus quattuordecimpunctatus (F. Morawitz, 1888) for the presence of “Ca. Streptomyces philanthi” in order to shed light on the origin and distribution of defensive antennal symbionts in philanthine wasps.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Diagnostic PCR assays revealed the presence of Streptomyces bacteria in the antennae of all three P. quattuordecimpunctatus females. Although the gyrA gene has previously been found to be quite variable among “Ca. Streptomyces philanthi” strains from different host species (M. Kaltenpoth and E. Strohm, unpublished data), the 16S rRNA sequences and the gyrA sequences were identical for the three individuals, suggesting a specific and stable association between P. quattuordecimpunctatus and its “Ca. Streptomyces philanthi” strain. The 16S rRNA sequence exhibited a high degree of similarity with sequences of “Ca. Streptomyces philanthi” from other host species (98.6 to 100%) and clustered within the monophyletic symbiont clade (Fig. 1). Since Philanthinus is phylogenetically the most basal of the three genera that are associated with “Ca. Streptomyces philanthi,” a basal placement of the symbionts would have been expected under the assumption of an ancient infection event and subsequent codiversification of hosts and symbionts. Since this is not the case, the observed phylogenetic pattern is indicative of occasional horizontal transfer of symbionts as has been suggested earlier for the symbionts of Trachypus (17). It has to be noted, however, that the high similarities in 16S rRNA sequences among the symbionts severely limit the ability to draw conclusions on the evolutionary history of the symbiosis. More variable gene sequences are needed to resolve the phylogenetic relationships on these low taxonomic levels. GyrA appears to be a promising candidate, as symbiont gyrA sequences from different host species show much higher variability than 16S rRNA sequences (M. Kaltenpoth and E. Strohm, unpublished data).

Morphological sections of an antenna from a female P. quattuordecimpunctatus and subsequent 3D reconstruction revealed the presence of complex glands in six antennomeres (A4 through A9, Fig. 2A). This contrasts with findings obtained with both Philanthus (9) and Trachypus (17), which exhibit such glands in only five segments (A4 through A8). However, as in Philanthus and Trachypus, each gland reservoir in the P. quattuordecimpunctatus antenna consists of an invagination of the cuticle from the dorsal/proximal part of the antennomere that expands dorsally and distally. The reservoirs lack the lateral lobes that are more or less pronounced in different Philanthus and Trachypus species (9; E. Strohm, W. Goettler, and M. Kaltenpoth, unpublished data). The outer wall of the reservoir consists of a reinforced cuticle, while the inner wall appears thin and flexible. The reservoir is associated with a large number (>20) of complex gland cells (Fig. 2A and B). Each of these so-called class 3 gland units (according to reference 21) consists of the actual secretory cell where the secretion is synthesized and a corresponding canal cell that forms the ductule (Fig. 2C) through which the secretion reaches the gland lumen. The gland cells are located primarily at the proximal and distal ends of the antennomere; accordingly, the canals discharge mainly at the proximal and distal sides of the reservoir (Fig. 2A). The cytoplasm of the gland cells is rich in vesicles of various sizes, and the nuclei show abundant nucleoli, both indicators of a high secretory activity of these cells. The size of the gland reservoirs seems to decrease somewhat from the proximal to the distal antennomeres. The gland reservoirs are filled with dense clusters of filamentous bacteria. Fluorescence in situ hybridization with cross sections of a female P. quattuordecimpunctatus antenna confirmed the presence of symbiotic bacteria, since the symbionts were clearly stained with the “Ca. Streptomyces philanthi”-specific probe SPT177, as well as with the general eubacterial probes Cy3-EUB338 and FAM-EUB784 (Fig. 3).

The results of our histological and molecular analyses revealed the presence of complex antennal glands and symbiotic “Ca. Streptomyces philanthi” bacteria in the rare crabronid genus Philanthinus. Since antennal symbionts were previously found to be absent from Cerceris, Aphilanthops, and Clypeadon (14) but present in Philanthus and Trachypus (14, 17), the symbiosis with “Ca. Streptomyces philanthi” appears to be restricted to around 172 species of crabronid wasps in the genera Philanthus, Trachypus, and Philanthinus. Thus, it likely evolved somewhere along the branch leading to the tribe Philanthini (Fig. 4).

The overall shape of the gland reservoirs of Philanthinus is less complex than described for Philanthus and Trachypus, since the lateral lobe protruding from the main reservoir body is lacking. Assuming a phylogenetically basal position of Philanthinus, the structural complexity of the reservoirs may have increased during evolution, possibly as an adaptation to retain a significant proportion of symbiont cells in the lateral lobe of the reservoir after secretion of the main reservoir content into the brood cell. This increase in the residual population of symbionts may allow faster growth and replenishment of the bacteria in the gland reservoir, which in turn might reduce the time between successive brood cells. Interestingly, in Philanthinus the gland reservoirs are present in six antennomeres as opposed to only five in Philanthus and Trachypus. It remains unclear whether Philanthinus has gained or the ancestor of Philanthus and Trachypus has lost a reservoir, but the transition was apparently a simple step in the evolution of the symbiont cultivation organs in beewolf antennae.

We previously hypothesized that the glands originally evolved to provide a directional cue to the beewolf larva (28) and were only secondarily invaded by the symbiotic bacteria (9). Alternatively, the evolutionary origin of the gland structure and the symbiosis may be tightly linked, with soil streptomycetes initiating the evolution of the glands by colonizing the antennal cuticle. A similar scenario is likely for leaf-cutter ants that cultivate symbiotic Pseudonocardia bacteria on gland-assisted regions of their cuticle (6). It remains unclear, however, why only beewolves evolved these unique antennal glands that serve both to provide a directional cue to the larva and to cultivate the protective symbionts. Each of these functions would be expected to also provide a selective advantage to other ground-nesting members of Hymenoptera. Detailed behavioral and molecular studies are needed to understand how members of related taxa prevent pathogen infestation and direct offspring emergence without the elaborate antennal glands found in beewolves.