Martin Kaltenpoth

Symbiotic Bacteria Protect Wasp Larvae from Fungal Infestation

Symbiotic associations between different organisms are of great importance for evolutionary and ecological processes [1-4]. Bacteria are particularly valuable symbiotic partners owing to their huge diversity of biochemical pathways that may open entirely new ecological niches for higher organisms [1-3]. Here, we report on a unique association between a new Streptomyces species and a solitary hunting wasp, the European beewolf (Philanthus triangulum, Hymenoptera, Crabronidae). Beewolf females cultivate the Streptomyces bacteria in specialized antennal glands and apply them to the brood cell prior to oviposition. The bacteria are taken up by the larva and occur on the walls of the cocoon. Bioassays indicate that the streptomycetes protect the cocoon from fungal infestation and significantly enhance the survival probability of the larva, possibly by producing antibiotics. Behavioral observations strongly suggest a vertical transmission of the bacteria. Two congeneric beewolf species harbor closely related streptomycetes in their antennae, indicating that the association with protective bacteria is widespread among philanthine wasps and might play an important role in other insects as well. This is the first report on the cultivation of bacteria in insect antennae and the first case of a symbiosis involving bacteria of the important antibiotic-producing genus Streptomyces.

Streptomyces as symbionts; an emerging and widespread theme?

Streptomyces bacteria are ubiquitous in soil, conferring the characteristic earthy smell, and they have an important ecological role in the turnover of organic material. More recently, a new picture has begun to emerge in which streptomycetes are not in all cases simply free-living soil bacteria but have also evolved to live in symbiosis with plants, fungi and animals. Furthermore, much of the chemical diversity of secondary metabolites produced by Streptomyces species has most likely evolved as a direct result of their interactions with other organisms. Here we review what is currently known about the role of streptomycetes as symbionts with fungi, plants and animals. These interactions can be parasitic, as is the case for scab-causing streptomycetes, which infect plants, and the Streptomyces species Streptomyces somaliensis and Streptomyces sudanensis that infect humans. However, in most cases they are beneficial and growth promoting, as is the case with many insects, plants and marine animals that use streptomycete-produced antibiotics to protect themselves against infection. This is an exciting and newly emerging field of research that will become increasingly important as the search for new antibiotics switches to unusual and under-explored environments.

Actinobacteria as mutualists: general healthcare for insects?

Mutualistic microorganisms are well known to play a key role in providing nutrients for successful growth and reproduction in many insects. Several recent studies indicate that they can be equally important for the protection of the host and its nutritional resources against pathogen attack. In particular, different actinobacteria have been found to defend ants, beetles and wasps against detrimental microorganisms by producing antibiotics. The extraordinary abilities of actinobacteria to exploit a wide variety of carbon and nitrogen sources and their extensive repertoire of secondary metabolites probably predispose this group to engage in protective symbioses. Defensive mutualisms with actinobacteria might constitute a general and widespread theme in the ecology and evolution of arthropods, and the study of the secondary metabolites involved promises to uncover novel drug candidates for human medicine.

Geographical and ecological stability of the symbiotic mid-gut microbiota in European firebugs, Pyrrhocoris apterus (Hemiptera, Pyrrhocoridae)

Symbiotic bacteria often play an essential nutritional role for insects, thereby allowing them to exploit novel food sources and expand into otherwise inaccessible ecological niches. Although many insects are inhabited by complex microbial communities, most studies on insect mutualists so far have focused on single endosymbionts and their interactions with the host. Here, we provide a comprehensive characterization of the gut microbiota of the red firebug (Pyrrhocoris apterus, Hemiptera, Pyrrhocoridae), a model organism for physiological and endocrinological research. A combination of several culture‐independent techniques (454 pyrosequencing, quantitative PCR and cloning/sequencing) revealed a diverse community of likely transient bacterial taxa in the mid‐gut regions M1, M2 and M4. However, the completely anoxic M3 region harboured a distinct microbiota consisting of facultative and obligate anaerobes including Actinobacteria (Coriobacterium glomerans and Gordonibacter sp.), Firmicutes (Clostri‐dium sp. and Lactococcus lactis) and Proteobacteria (Klebsiella sp. and a previously undescribed Rickettsiales bacterium). Characterization of the M3 microbiota in different life stages of P. apterus indicated that the symbiotic bacterial community is vertically transmitted and becomes well defined between the second and third nymphal instar, which coincides with the initiation of feeding. Comparing the mid‐gut M3 microbial communities of P. apterus individuals from five different populations and after feeding on three different diets revealed that the community composition is qualitatively and quantitatively very stable, with the six predominant taxa being consistently abundant. Our findings suggest that the firebug mid‐gut microbiota constitutes a functionally important and possibly coevolved symbiotic community.

Localization and transmission route of Coriobacterium glomerans, the endosymbiont of pyrrhocorid bugs.

Endosymbiotic gut bacteria play an essential role in the nutrition of many insects. Most of the nutritional interactions investigated so far involve gammaproteobacterial symbionts, whereas other groups have received comparatively little attention. Here, we report on the localization and the transmission route of the specific actinobacterial symbiont Coriobacterium glomerans from the gut of the red firebug, Pyrrhocoris apterus (Hemiptera: Pyrrhocoridae). The symbionts were detected by diagnostic PCRs and FISH in the midgut section M3, in the rectum and in feces of the bugs as well as in the hemolymph of some females. Furthermore, adult female bugs apply the symbionts to the surface of the eggs during oviposition, from where they are later taken up by the hatchlings. Surface sterilization of egg clutches generated aposymbiotic insects and thereby confirmed the vertical transmission route via the egg surface. However, symbionts were readily acquired horizontally when the nymphs were reared in the presence of symbiont-containing eggshells, feces, or adult bugs. Using diagnostic PCRs and partial sequencing of the 16S rRNA gene, closely related bacterial symbionts were detected in the cotton stainer bug Dysdercus fasciatus (Hemiptera: Pyrrhocoridae), suggesting that the symbiosis with Actinobacteria may be widespread among pyrrhocorid bugs.

An out-of-body experience: the extracellular dimension for the transmission of mutualistic bacteria in insects

Across animals and plants, numerous metabolic and defensive adaptations are a direct consequence of symbiotic associations with beneficial microbes. Explaining how these partnerships are maintained through evolutionary time remains one of the central challenges within the field of symbiosis research. While genome erosion and co-cladogenesis with the host are well-established features of symbionts exhibiting intracellular localization and transmission, the ecological and evolutionary consequences of an extracellular lifestyle have received little attention, despite a demonstrated prevalence and functional importance across many host taxa. Using insect–bacteria symbioses as a model, we highlight the diverse routes of extracellular symbiont transfer. Extracellular transmission routes are unified by the common ability of the bacterial partners to survive outside their hosts, thereby imposing different genomic, metabolic and morphological constraints than would be expected from a strictly intracellular lifestyle. We emphasize that the evolutionary implications of symbiont transmission routes (intracellular versus extracellular) do not necessarily correspond to those of the transmission mode (vertical versus horizontal), a distinction of vital significance when addressing the genomic and physiological consequences for both host and symbiont.

Actinobacteria as essential symbionts in firebugs and cotton stainers (Hemiptera, Pyrrhocoridae)

Actinobacteria engage in defensive symbioses with several insect taxa, but reports of nutritional contributions to their hosts have been exceptionally rare. Cotton stainers (Dysdercus fasciatus) and red firebugs (Pyrrhocoris apterus) (both Hemiptera, Pyrrhocoridae) harbour the actinobacterial symbionts Coriobacterium glomerans and Gordonibacter sp. as well as Firmicutes (Clostridium sp. and Lactococcus sp.) and Proteobacteria (Klebsiella sp. and a Rickettsiales bacterium) in the M3 region of their mid-gut. We combined experimental manipulation with community-level analyses to elucidate the function of the gut symbionts in both pyrrhocorid species. Elimination of symbionts by egg-surface sterilization resulted in significantly higher mortality and reduced growth rates, indicating that the microbial community plays an important role for host nutrition. Fitness of symbiont-deprived bugs could be completely restored by re-infection with the original microbiota, while reciprocal cross-infections of microbial communities across both pyrrhocorid species only partially rescued fitness, demonstrating a high degree of host-symbiont specificity. Community-level analyses by quantitative PCRs targeting the dominant bacterial strains allowed us to link the observed fitness effects to the abundance of the two actinobacterial symbionts. The nutritional mutualism with Actinobacteria may have enabled pyrrhocorid bugs to exploit Malvales seeds as a food source and thereby possibly allowed them to occupy and diversify in this ecological niche.

Defensive microbial symbionts in Hymenoptera

Summary In all stages of their life cycle, insects are threatened by a multitude of predators, parasites, parasitoids and pathogens. The lifestyles and feeding ecologies of some hymenopteran taxa render them especially vulnerable to pathogen infestation. Specifically, development in sub-terranean brood cells, mass provisioning of resources for the offspring and the life of social insects in large communities can enhance the risk of pathogen infestation and/or the spread of disease among conspecifics. To counteract these threats, insects have evolved mechanical, chemical and behavioural defences as well as a complex immune system. In addition to the hosts own defences, some Hymenoptera are associated with protective symbionts. Leaf-cutting ants, solitary digger wasps, bees and bumblebees engage in symbiotic interactions with bacteria that protect the adult host, the developing offspring or the food resources against microbial infections. In the well-studied cases of ants and wasps, the protective activity is mediated by the production of antimicrobial secondary metabolites. In other symbiotic interactions, however, competitive exclusion and immune priming may also play an important role in enhancing protection. Phylogenetic studies indicate that the defensive associations in Hymenoptera are generally more dynamic than the intimate nutritional mutualisms, with horizontal transfer or de novo uptake of the symbionts from the environment occurring frequently. Mutualistic micro-organisms can also significantly influence the outcome of host-parasitoid interactions. Some insects are protected by symbiont-produced toxins against parasitic wasps. Ichneumonid and braconid parasitoids, on the other hand, are associated with symbiotic viruses that are injected into the caterpillar host during oviposition and suppress its immune system to the advantage of the parasitoid. The increasing affordability of next-generation sequencing technologies will greatly facilitate the analysis of insect-associated microbial communities and undoubtedly uncover a plethora of as yet unknown protective symbioses. However, a detailed understanding of the hosts natural history is indispensable for elucidating the fitness benefits of the symbionts and the molecular basis of symbiont-conferred protection.

Partner choice and fidelity stabilize coevolution in a Cretaceous-age defensive symbiosis

Significance Symbiotic microbes are essential for the survival of many multicellular organisms, yet the factors promoting cooperative symbioses remain poorly understood. Three genera of solitary wasps cultivate antibiotic-producing Streptomyces bacteria for defense of their larvae against pathogens. Here we show that the wasp ancestor acquired the protective symbionts from the soil at least 68 million years ago. Although mother-to-offspring symbiont transmission dominates, exchange between unrelated individuals and uptake of opportunistic microorganisms from the environment occasionally occurs. However, experimental infections of female beewolves reveal that the wasps selectively block transmission of nonnative bacteria to their offspring. These findings suggest a previously unknown mechanism to maintain a specific symbiont over long evolutionary timescales and help to explain the persistence of bacterial mutualists in insects. Many insects rely on symbiotic microbes for survival, growth, or reproduction. Over evolutionary timescales, the association with intracellular symbionts is stabilized by partner fidelity through strictly vertical symbiont transmission, resulting in congruent host and symbiont phylogenies. However, little is known about how symbioses with extracellular symbionts, representing the majority of insect-associated microorganisms, evolve and remain stable despite opportunities for horizontal exchange and de novo acquisition of symbionts from the environment. Here we demonstrate that host control over symbiont transmission (partner choice) reinforces partner fidelity between solitary wasps and antibiotic-producing bacteria and thereby stabilizes this Cretaceous-age defensive mutualism. Phylogenetic analyses show that three genera of beewolf wasps (Philanthus, Trachypus, and Philanthinus) cultivate a distinct clade of Streptomyces bacteria for protection against pathogenic fungi. The symbionts were acquired from a soil-dwelling ancestor at least 68 million years ago, and vertical transmission via the brood cell and the cocoon surface resulted in host–symbiont codiversification. However, the external mode of transmission also provides opportunities for horizontal transfer, and beewolf species have indeed exchanged symbiont strains, possibly through predation or nest reuse. Experimental infection with nonnative bacteria reveals that—despite successful colonization of the antennal gland reservoirs—transmission to the cocoon is selectively blocked. Thus, partner choice can play an important role even in predominantly vertically transmitted symbioses by stabilizing the cooperative association over evolutionary timescales.

Unearthing carrion beetles' microbiome: characterization of bacterial and fungal hindgut communities across the Silphidae

Carrion beetles (Coleoptera, Silphidae) are well known for their behaviour of exploiting vertebrate carcasses for nutrition. While species in the subfamily Silphinae feed on large carcasses and on larvae of competing scavengers, the Nicrophorinae are unique in monopolizing, burying and defending small carrion, and providing extensive biparental care. As a first step towards investigating whether microbial symbionts may aid in carcass utilization or defence, we characterized the microbial hindgut communities of six Nicrophorinae (Nicrophorus spp.) and two Silphinae species (Oiceoptoma noveboracense and Necrophila americana) by deep ribosomal RNA amplicon sequencing. Across all species, bacteria in the family Xanthomonadaceae, related to Ignatzschineriao larvae, were consistently common, and several other taxa were present in lower abundance (Enterobacteriales, Burkholderiales, Bacilli, Clostridiales and Bacteroidales). Additionally, the Nicrophorinae showed high numbers of unusual Clostridiales, while the Silphinae were characterized by Flavobacteriales and Rhizobiales (Bartonella sp.). In addition to the complex community of bacterial symbionts, each species of carrion beetle harboured a diversity of ascomycetous yeasts closely related to Yarrowia lipolytica. Despite the high degree of consistency in microbial communities across the Silphidae—specifically within the Nicrophorinae—both the fungal symbiont phylogeny and distance‐based bacterial community clustering showed higher congruence with sampling locality than host phylogeny. Thus, despite the possibility for vertical transmission via anal secretions, the distinct hindgut microbiota of the Silphidae appears to be shaped by frequent horizontal exchange or environmental uptake of symbionts. The microbial community profiles, together with information on host ecology and the metabolic potential of related microorganisms, allow us to propose hypotheses on putative roles of the symbionts in carcass degradation, detoxification and defence.