Carpenter Bees (Xylocopa) Harbor a Distinctive Gut Microbiome Related to That of Honey Bees and Bumble Bees.

Eusocial corbiculate bees, including bumble bees and honey bees, maintain a socially transmitted core gut microbiome that contributes to digestion and pathogen defense. In contrast, solitary bees, which have fewer opportunities for direct interhost transmission, typically have less consistent microbiomes dominated by bacteria associated with pollen and food reserves. Carpenter bees (genus Xylocopa) are long-lived bees that are not eusocial but that often live in shared nesting sites. We characterized gut microbiomes for Xylocopa micans, X. mexicanorum, X. tabaniformis parkinsoniae, and X. virginica and for five solitary bee species from other genera (Andrena, Habropoda, Megachile, and Svastra), sampled in the same localities in central Texas. Unexpectedly, all four Xylocopa species had microbiomes dominated by bacterial lineages previously known only from social bees or other insect groups. Microbiomes were similar across three Xylocopa species and included lineages in the families Bifidobacteriaceae, Orbaceae, Lactobacillaceae, Pseudomonadaceae, and Enterobacteriaceae. In contrast, X. virginica had a distinct microbiome dominated by the genus Bombilactobacillus, a group abundant in guts of eusocial bees. Phylogenetic analyses support a past transfer of bacterial lineages into Xylocopa from bumble bees or honey bees. Gut microbiome compositions of Xylocopa species were distinct from those of other co-occurring solitary bees that had variable gut microbiomes dominated by bacteria from environmental sources. IMPORTANCE Gut microbiomes from social bees, such as honey bees and bumble bees, are conserved and consist of host-restricted bacteria that are transmitted among sterile female workers within a colony and that are important to the health of these key insect pollinators. In contrast, solitary bee species typically have more erratic, environmentally acquired microbiomes. Carpenter bees (genus Xylocopa) can be solitary as they lack a worker caste, and each female can excavate nests and raise offspring alone, although females are often social share nests at least in some species. This study showed that the gut microbiomes of four Xylocopa species have distinctive and consistent compositions and are dominated by bacterial lineages previously known from honey bees and bumble bees. Thus, eusociality is not required for bees to maintain a specialized, host-restricted gut microbiome. These findings suggest that gut bacteria are transmitted at shared nesting sites and that they play a role in host ecology.

The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila -colonizing Orbaceae.

Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli , a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying new host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species. IMPORTANCE: To fully understand how microbes have evolved to interact with their environments, one must be able to modify their genomes. However, it can be difficult and laborious to discover which genetic tools and approaches work for a new isolate. Bacteria from the recently described Orbaceae family are common in the microbiomes of insects. We developed the Pathfinder plasmid toolkit for testing the compatibility of different genetic parts with newly cultured bacteria. We demonstrate its utility by engineering Orbaceae strains isolated from flies to express fluorescent proteins and characterizing how they colonize the Drosophila melanogaster gut. Orbaceae are widespread in Drosophila in the wild but have not been included in laboratory studies examining how the gut microbiome affects fly nutrition, health, and longevity. Our work establishes a path for genetic studies aimed at understanding and altering interactions between these and other newly isolated bacteria and their hosts.

The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae.

Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli, a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying microbial ecology and host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species.