Host hydrocarbons protect symbiont transmission from a radical host defense.

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.

Streptomyces sp. M54: an actinobacteria associated with a neotropical social wasp with high potential for antibiotic production.

Streptomyces symbionts in insects have shown to be a valuable source of new antibiotics. Here, we report the genome sequence and the potential for antibiotic production of "Streptomyces sp. M54", an Actinobacteria associated with the eusocial wasp, Polybia plebeja. The Streptomyces sp. M54 genome is composed of a chromosome (7.96 Mb), and a plasmid (1.91 Kb) and harbors 30 biosynthetic gene clusters for secondary metabolites, of which only one third has been previously characterized. Growth inhibition bioassays show that this bacterium produces antimicrobial compounds that are active against Hirsutella citriformis, a natural fungal enemy of its host, and the human pathogens Staphylococcus aureus and Candida albicans. Analyses through TLC-bioautography, LC-MS/MS and NMR allowed the identification of five macrocyclic ionophore antibiotics, with previously reported antibacterial, antitumor and antiviral properties. Phylogenetic analyses placed Streptomyces sp. M54 in a clade of other host-associated strains taxonomically related to Streptomyces griseus. Pangenomic and ANI analyses confirm the identity of one of its closest relatives as Streptomyces sp. LaPpAH-199, a strain isolated from an ant-plant symbiosis in Africa. In summary, our results suggest an insect-microbe association in distant geographic areas and showcase the potential of Streptomyces sp. M54 and related strains for the discovery of novel antibiotics.

Phylogenetic analyses of antibiotic-producing Streptomyces sp. isolates obtained from the stingless-bee Tetragonisca angustula (Apidae: Meliponini).

Many insects have been associated with actinobacteria in protective symbiosis where antimicrobial metabolites inhibit host pathogens. However, the microbiota of neotropical insects such as the stingless-bee Tetragonisca angustula is poorly explored. T. angustula is a meliponid bee widely distributed in Latin America, its honey is traditionally exploited because of its ethno-pharmacological properties and its antimicrobial activity has been demonstrated. Also, the well-structured nest of this species allows exploration of the microbiota of its different components. Even though Streptomyces spp. have been cultured from stingless-bees, little is known about their role in this insect-microbe relationship. In this study, we examined the association between culturable actinobacteria and T. angustula, and evaluated the isolates' potential as antimicrobial producers. We isolated 51 actinobacteria from adult bees and different substrates of the hive of T. angustula (pollen and honey storage, garbage pellets and cerumen). We then performed a 16S rRNA phylogenetic analysis that clusters the bacteria to previously described lineages of host-associated Streptomyces. In addition, all the isolates were classified according to their antibacterial activity against human pathogens, measured by a growth inhibition test based on diffusion in agar. More than 50 % of our isolates exhibit antimicrobial activity, mainly to Gram-positive bacteria and fungi and only two against Gram-negative bacteria. Additionally, we obtained electron micrographs of adult bees with what appears to be patches of hyphae with Streptomyces-like cell morphology on their body surface. Our results suggest that T. angustula possibly uptakes and transfers actinobacteria from the environment, acting as vectors for these potentially beneficial organisms. This research provides new insights regarding the microbiota associated with T. angustula and justify future studies exploring the full diversity of the microbial community associated with the hive and the possible exchange of microbes with the crops they pollinate.

Secondary metabolites in a neotropical shrub: spatiotemporal allocation and role in fruit defense and dispersal.

Deciphering the ecological roles of plant secondary metabolites requires integrative studies that assess both the allocation patterns of compounds and their bioactivity in ecological interactions. Secondary metabolites have been primarily studied in leaves, but many are unique to fruits and can have numerous potential roles in interactions with both mutualists (seed dispersers) and antagonists (pathogens and predators). We described 10 alkenylphenol compounds from the plant species Piper sancti-felicis (Piperaceae), quantified their patterns of intraplant allocation across tissues and fruit development, and examined their ecological role in fruit interactions. We found that unripe and ripe fruit pulp had the highest concentrations and diversity of alkenylphenols, followed by flowers; leaves and seeds had only a few compounds at detectable concentrations. We observed a nonlinear pattern of alkenylphenol allocation across fruit development, increasing as flowers developed into unripe pulp then decreasing as pulp ripened. This pattern is consistent with the hypothesis that alkenylphenols function to defend fruits from pre-dispersal antagonists and are allocated based on the contribution of the tissue to the plant's fitness, but could also be explained by non-adaptive constraints. To assess the impacts of alkenylphenols in interactions with antagonists and mutualists, we performed fungal bioassays, field observations, and vertebrate feeding experiments. In fungal bioassays, we found that alkenylphenols had a negative effect on the growth of most fungal taxa. In field observations, nocturnal dispersers (bats) removed the majority of infructescences, and diurnal dispersers (birds) removed a larger proportion of unripe infructescences. In feeding experiments, bats exhibited an aversion to alkenylphenols, but birds did not. This observed behavior in bats, combined with our results showing a decrease in alkenylphenols during ripening, suggests that alkenylphenols in fruits represent a trade-off (defending against pathogens but reducing disperser preference). These results provide insight into the ecological significance of a little studied class of secondary metabolites in seed dispersal and fruit defense. More generally, documenting intraplant spatiotemporal allocation patterns in angiosperms and examining mechanisms behind these patterns with ecological experiments is likely to further our understanding of the evolutionary ecology of plant chemical traits.

Evidence for Widespread Associations between Neotropical Hymenopteran Insects and Actinobacteria.

The evolutionary success of hymenopteran insects has been associated with complex physiological and behavioral defense mechanisms against pathogens and parasites. Among these strategies are symbiotic associations between Hymenoptera and antibiotic-producing Actinobacteria, which provide protection to insect hosts. Herein, we examine associations between culturable Actinobacteria and 29 species of tropical hymenopteran insects that span five families, including Apidae (bees), Vespidae (wasps), and Formicidae (ants). In total, 197 Actinobacteria isolates were obtained from 22 of the 29 different insect species sampled. Through 16S rRNA gene sequences of 161 isolates, we show that 91% of the symbionts correspond to members of the genus Streptomyces with less common isolates belonging to Pseudonocardia and Amycolatopsis. Electron microscopy revealed the presence of filamentous bacteria with Streptomyces morphology in brood chambers of two different species of the eusocial wasps. Four fungal strains in the family Ophiocordycipitacea (Hypocreales) known to be specialized insect parasites were also isolated. Bioassay challenges between the Actinobacteria and their possible targeted pathogenic antagonist (both obtained from the same insect at the genus or species level) provide evidence that different Actinobacteria isolates produced antifungal activity, supporting the hypothesis of a defensive association between the insects and these microbe species. Finally, phylogenetic analysis of 16S rRNA and gyrB demonstrate the presence of five Streptomyces lineages associated with a broad range of insect species. Particularly our Clade I is of much interest as it is composed of one 16S rRNA phylotype repeatedly isolated from different insect groups in our sample. This phylotype corresponds to a previously described lineage of host-associated Streptomyces. These results suggest Streptomyces Clade I is a Hymenoptera host-associated lineage spanning several new insect taxa and ranging from the American temperate to the Neotropical region. Our work thus provides important insights into the widespread distribution of Actinobacteria and hymenopteran insects associations, while also pointing at novel resources that could be targeted for the discovery of active natural products with great potential in medical and biotechnological applications.