Microbial Dynamics within Shed Mucosal Secretions of Hirudo verbana, the European Medicinal Leech.

摘要

Microbes are widespread throughout our planet, residing in soils, oceans, and even beneath the arctic glaciers. Most interact with each other and other life forms, and there is no known complex organism that lacks an associated microbiome. These microbial symbionts are critical to the survival and proliferation of their host by assisting in nutrient provisioning, physiological development, immunological priming, providing protection from pathogens, contributing to predator evasion, etc. As such, the transmission of these symbionts is a crucial aspect of host biology. Despite a growing appreciation for the prevalence of mixed transmission (incorporating vertical, or from a parental route, and horizontal, or environmental, mechanisms) for establishing microbial symbioses, features that enable these infections are not well understood. My work investigated the mechanistic basis of symbiont acquisition by the European medicinal leech, Hirudo verbana. In the leech cocoon, only a portion of the albumenotrophic larvae obtain their beneficial gut symbiont, the Gammaproteobacterium Aeromonas veronii. However, by early adulthood, all leeches harbor this bacterium, indicating the complementation of incomplete vertical transmission by an unknown horizontal mechanism. Insight from a number of other organisms, aquatic and terrestrial, suggests that host-secreted mucus may provide a transmission vehicle. Through the use of genetically-tractable A. veronii, I demonstrated that cyclical host mucosal secretions are seeded by digestive tract symbionts. Using quantitative PCR, I verified that A. veronii are not only viable, but also proliferate within mucosal casts at a frequency synchronous to host shedding. Subsequent experiments using mucus inoculated with a gfp-expressing A. veronii demonstrates that mucosal contact is sufficient for symbiont transmission to novel hosts. Importantly, behavior assays show that leeches are attracted to these castings, providing an efficient mechanism for transmission of A. veronii between conspecifics. Additional behavioral assays show that host symbiont-state does not influence attraction towards mucus and that symbiont content of the mucus does not alter attraction, suggesting that A. veronii exploits a preexisting host physiological process.;Lastly, Illumina-based RNA-seq was used to identify how the A. veronii transcriptome, with regards to metabolism and information processing, responds to the lifestyle shift from mutualistic within the leech digestive tract to free-living within shed mucus. This dual mode of symbiont transmission may prove evolutionarily advantageous, as it not only ensures the infection of leeches by beneficial symbionts, but also provides accessibility to a higher genetic diversity of symbionts and biome lifestyle options to A. veronii beyond that of mutualism. Additionally, examination of the composition of the mucosal microbial community utilizing two separate culture-independent sequencing techniques (i.e. Sanger-sequenced 16S rRNA clone libraries and Illumina deep-sequencing of the V3-V4 hypervariable region of the 16S rRNA gene) revealed that a diverse microbiota resides within the mucus, consisting of both previously-described and potentially novel leech symbionts. Additional transcriptomic analyses indicate that this microbial community actively engages in cell-to-cell communication via quorum sensing and potential DNA transfer. In depth genomic analyses also prove that a novel microbial species, Pedobacter, is a major mucosal microbiota member worthy of future study. Understanding the features that enable mixed transmission, particularly those involving mixed species assemblages, may prove instrumental for the development of strategies to promote beneficial symbioses.