Predators, parasites and pathogens can significantly reduce the reproductive success of insects. Developmental stages are especially prone to pathogen infestation given their often limited mobility as well as trade-offs that lead to limited resources being allocated into growth instead of defense[1–4]. Additionally, many insect species rear their offspring in subterranean nesting sites where the progeny is continuously threatened by soil pathogens[5–7]. To counteract these threats, insects have evolved an intricate immune system as well as chemical and behavioral defenses, including brood care, nesting in habitats which are difficult to invade for predators or brood parasites, and the application of antimicrobial substances as defense against pathogen infestation[7–10].
Sociality has been documented to provide insects with collective defensive behaviors aimed at eliminating parasites from group members and thereby limiting the spread of infections within the colony[11–14]. Specifically, allogrooming can serve as an efficient strategy to mechanically remove fungal spores, nematodes, mites and other parasites[11, 15, 16]. By contrast, solitary species are usually lacking these behavioral defenses[14, 17–19], making other means of protection of their offspring against fungal and bacterial pathogens particularly important. Although nest hygienic behaviors have also been observed in solitary insects, there is often no or only limited contact between adult and developing individuals, so chemical mechanisms are likely to play a more important role for antimicrobial defense[7, 20–22]. Concordantly, the production and use of various defensive compounds for the protection of the developing offspring has been described for a number of solitary insect species[8, 23–28].
In addition to the insect’s own defenses, recent studies have demonstrated that several taxa team up with bacterial symbionts to protect the host, the offspring or its nutritional resources against pathogens, predators, parasites, or parasitoids[20, 21, 29–33]. This protection can be mediated by (i) competitive exclusion of pathogenic microorganisms[34–36], (ii) interaction with the host’s immune system to enhance resistance against pathogenic infestation[17, 37, 38], or (iii) the production of chemicals that harm and/or deter antagonists[29, 33, 39–41].
Interestingly, many of the mutualistic microorganisms involved in defensive partnerships with insects belong to the bacterial phylum Actinobacteria[42, 43]. Members of this group appear to be predisposed towards engaging in defensive symbioses by their widespread distribution in the soil, the ability to subsist in nutritionally deficient environments, and, notably, their capacity to produce a wide variety of secondary metabolites with antimicrobial properties[40, 42, 44, 45]. These compounds may provide an efficient way to defend the producer against pathogenic or competing microorganisms, making Actinobacteria particularly suitable partners for defensive mutualistic interactions with insect hosts. Especially species of the genus Streptomyces are efficient producers of antimicrobial substances, with over two-thirds of the clinically relevant natural product antibiotics originating from this genus[44, 46]. Despite the common perception of antibiotics as agents of chemical warfare among competing microorganisms, evidence for the natural roles of these compounds and their defensive activity under in vivo conditions is still scarce[47, 48]. In fact, recent studies suggest that their primary effect in maintaining microbial communities may be the modulation of gene transcription at low concentrations rather than the elimination of competitors.
Fungus-farming ants represent a prime example of protective symbioses. The ants cultivate symbiotic fungi as a food source for their colony. To protect their source of nourishment from fungal infestation by the pathogenic fungus Escovopsis, the ants engage in a protective symbiosis with actinomycete bacteria which have been reported to produce antibiotic substances that inhibit the growth of Escovopsis[30, 41, 50–52], thereby protecting the fungal gardens without affecting the cultivar itself. Similarly, the fungus growing bark beetle Dendroctonus frontalis is associated with bacteria of the genus Streptomyces. The bacteria, present in the fungal galleries as well as in the mycangia of the beetle, produce the antifungal substance mycangimycin, which can protect the beetle’s nutritional resources against the antagonistic fungus Ophiostoma minus[33, 39].
Solitary digger wasps in the tribe Philanthini (‘beewolves’, Hymenoptera: Crabronidae) engage in a defensive symbiosis with bacteria of the genus Streptomyces for the protection of the developing offspring[20, 40, 54, 55]. Female beewolves cultivate the symbiotic microorganisms in specialized antennal gland reservoirs and secrete them into the brood cells prior to laying an egg on one of the provisioned bees. After oviposition, the female seals the brood cell with sand and subsequently does not provide any further brood care. As beewolf development can take up to nine months, including a long period of larval diapause during hibernation in the humid underground brood cells, the beewolf’s offspring is continuously threatened by pathogenic bacteria and fungi that may invade the brood cells from the surrounding soil as well as from the remains of the provisioned honeybee prey. As an efficient broad-spectrum defense, the larvae incorporate the symbiotic streptomycetes into the silken walls of their cocoons, where the symbionts produce at least nine different antibiotic substances that serve as an “antimicrobial combination prophylaxis” against pathogenic bacteria and fungi during the long and vulnerable period of hibernation.
The mutualistic association of beewolves and Streptomyces provides a unique opportunity to study the natural role of antimicrobial compounds in a symbiotic context[20, 40, 54, 58]. Here we investigated the dynamics of population size and antibiotic production of the symbiotic Streptomyces on the beewolf cocoon in order to elucidate the mechanisms that allow for the long-term defense against pathogens. The results yield insights into an efficient strategy for offspring protection in a solitary insect and provide a unique case study on the long-term efficacy of bacterial secondary metabolites directly in the natural environment.