Acquisition and activity of bacterial symbionts in marine invertebrates

Abstract

Chemosynthetic symbioses evolved multiple times in a wide diversity of host species and from many different bacterial lineages. The symbionts provide nutrition to the hosts by fixing CO2 into biomass using reduced inorganic compounds as energy sources. This gives the hosts a physiological advantage to colonize and thrive in nutrient poor habitats. Two key questions that have emerged in symbiosis research are 1) how do the hosts acquire their symbionts and 2) what reduced compounds can be used by the symbionts as energy source to fix CO2 into biomass. This PhD thesis consists of two parts that will each deal with one of these two fundamental questions. In the first part of this thesis, two manuscripts describe the symbiont colonization of host tissues in the deep-sea mussel Bathymodiolus from hydrothermal vents. Bathymodiolus harbors its chemosynthetic symbionts intracellularly in gill tissues and, as in all bivalves, the gills grow throughout the mussel's life. This raises the question how the newly developed gill tissues are colonized by symbionts. Symbiont colonization of newly formed gill tissues was investigated using fluorescence in situ hybridization with symbiont-specific probes on semi-thin sections of whole juveniles. In addition, posterior ends of adult gills were also analyzed, as new gill filament formation occurs here. In the smallest juveniles, symbionts had colonized a wide range of epithelial tissues, revealing a widespread distribution of symbionts in many different juvenile organs. In contrast, juveniles larger than 9 mm had symbionts only in their gills. These observations indicate an ontogenetic shift in symbiont colonization from an indiscriminate infection of almost all epithelia in early life stages to spatially restricted colonization of gills in later developmental stages of Bathymodiolus. Analyses of the posterior end of both juvenile and adult gill tissues further showed that all gill filaments except the first most recently formed 7 to 9 filaments harbored symbionts. Newly formed gill tissues of Bathymodiolus are thus initially symbiont free and only later become infected with symbionts as they extend and differentiate, suggesting a life long de novo colonization by the endosymbionts of aposymbiotic host cells. In the second part of this thesis I investigated the physiological capabilities of the symbionts of Olavius algarvensis. This marine worm lacks both a digestive and excretory system. Instead it relies on a symbiotic community of two gammaproteobacterial sulfur oxidizers, two deltaproteobacterial sulfate reducers, and a spirochete for nutrition and waste recycling. External energy sources for the symbiotic association have remained enigmatic because of extremely low concentrations of reduced sulfur compounds and organic substrates in the worms habitat. Using a metaproteomic approach and incubation experiments I showed that hydrogen (H2) and carbon monoxide (CO) are additional energy sources for the symbiosis of O. algarvensis. The finding of elevated CO and H2 concentrations in the worm s habitat further confirmed the ecological importance of both substrates for the worm symbiosis. One of the sulfur-oxidizing symbionts incorporated high amounts of CO2 into its biomass in the presence of CO, which was determined using 13C-labeled bicarbonate in the incubation medium and subsequent nanoSIMS analyses. The metaproteomic study further revealed a high expression of proteins involved in highly efficient pathways and high-affinity uptake transporters for the recycling and conservation of energy, nitrogen, and carbon sources. This indicates that the nutrient-poor nature of the worm s habitat exerted a strong selective pressure in shaping this association.