Thiotrophic mutualism - cooperation goes empirical

Evolutionary theory of interspecies cooperation depicts the initiation and maintenance between two mutualistic partners to be driven by pre- and postinfection processes such as partner choice, partner sanction, partner fidelity feedback, and byproduct benefits. Transmission, the mode of symbiont transfer into the next host generation through infection of a free-living population (horizontal) or through inheritance of the symbiont mostly from the mother (vertical) plays a key role in the evolution of mutualism. A mathematical model predicts that postinfection mechanisms, byproduct usage, the costs of transmission, and availability the symbiont population in the free-living environment determine whether transmission remains horizontal or can evolve into a vertical mode. However, empirical data are scarce and restricted to a few bacterial model systems. Thus this proposed research project aims at establishing the mutualism between the marine, colonial ciliate Zoothamnium niveum and its sulfur oxidizing, symbiotic bacterial partner Candidatus Thiobios zoothamnicoli as a thiotrophic model system for testing evolutionary concepts of interspecies cooperation. In contrast to most other thiotrophic associations, this system can be cultivated, grows fast and reproduces rapidly. Two important empirical aspects will be studied 1) symbiont population genetics and demography using multilocus sequence of several protein-encoding genes typing either based on universal conserved primers or single cell genomics and 2) autotrophy of symbiont and nourishment of host in context of host and symbiont fitness using pulse - chase experiments and tissue autoradiography and nano- scale secondary ion mass spectrometry, pulse labeling of host DNA synthesis and immunocytochemistry to measure host proliferation, counting of asexual produced swarmers to estimate host reproduction, and scanning electron microscopy to measure frequency of dividing symbionts. This will allow us gain insights into the symbiont heterogeneity on subspecies level, the total symbiont population size and demography, the autotrophic capabilities of the symbiont and the mode of host nutrition through translocation processes from symbiont to host as well as through free-living microbes. Moreover, by testing for host sanction and partner fidelity feedback, evaluating the costs of vertical transmission and the availability of the free-living symbiont, this empirical information will allow us to enlighten the interwoven dynamics between the mode of transmission and the mechanisms of maintenance of mutualism. These proposed studies will enhance our understanding on the diversity and functioning of bacterial mutualism and attempts to fertilize theoretical evolutionary biology with empirical achievements.

No one lives alone. Numerous microbes colonize the roots of plants and the gut of animals, including humans. To a large extent, these microbes are beneficial for the host. Occasionally pathogens attack and attempt to disturb this balanced equilibrium. Also when beneficial microbes turn into cheaters through mutations, the balance is disturbed and the persistence is threatened. Evolutionary game theory develops theoretical concepts for the evolution of mutualism. Some oft these concepts have been tested in this research project on 'Thiotrophic mutualism cooperation goes empirical' in the mutualistic association between a colonial ciliate host and its bacterial symbiont. They live in the sea on sunken wood, such as wooden boats and piers in harbors in the northern Adriatic Sea. As a first step we sequenced the genome of the symbiont and compared populations collected from the Caribbean Sea in Guadeloupe, Belize and Cuba, the Atlantic Sea in Madeira, and the Adriatic Sea in Slovenia. In experiments carried out in Piran, Slovenia we could show that the symbionts fix carbon, similar to algae in the sea and plants on land. Instead of sunlight as energy source, the chemical oxidation of hydrogen sulfide from the wood with oxygen from the seawater generated energy for carbon fixation. Part of the fixed carbon was concomitantly released to the host and taken up as food source. This release was automatic because the bacterial cell membrane was leaky. This type of partner support is called byproduct benefit, an important mechanism to maintain mutualism. The host, however, not only lives from the byproduct but also feeds on its symbionts directly, thus regulating the population density. Further other microbes from the surrounding seawater were also digested. These processes were shown using labeled inorganic carbon and autoradiography and nanoscale secondary ion mass spectrometry. In further experiments the density of free-living microbes in the seawater was manipulated and resulted in different growth rates of the host. Interestingly, also the symbionts adjusted its growth rate, although they live on chemicals and are not directly influenced by surrounding microbes. This mechanism of coupled fitness is called partner fidelity feedback, an important mechanism well known from other mutualistic relationships. Further, the host could be cultivated without its symbiont. In game theory this phenomenon is called loner strategy. Surprisingly, the morphology of the aposymbiotic phenotype was very different from the symbiotic phenotype. As of yet, this morphological plasticity, called polyphenism, was unknown among thiotrophic symbioses, but is well known from predator prey relationships. In conclusion, this project was extremely successful in testing several concepts of evolutionary game theory with empirical experiments in this promising model system of a colonial ciliate and its bacterial symbiont.