Zostera marina and epibionts: the dynamics of chemical defenses. Dissertation for the Academic Degree Dr. rer. Nat. at the Faculty of Mathematics and Nature Sciences of the Christian-Albrechts-University Kiel, Helmholtz Centre for Ocean Research GEOMAR Submitted by Chi Guan Kiel, 2017 Zostera marina and epibionts: the dynamics of chemical defenses. Dissertation for the Academic Degree Dr. rer. Nat. at the Faculty of Mathematics and Nature Sciences of the Christian-Albrechts-University Kiel, Helmholtz Centre for Ocean Research GEOMAR Submitted by Chi Guan Kiel, 2017 1. Gutachter / Referee: PD Dr. Florian Weinberger 2. Gutachter / Referee: Prof. Dr. Deniz Tasdemir Tag der mündlichen Prüfung / Date of Disputation: 21.06.2017 Zum Druck genehmigt / Approved of Print: 27.06.2017 gez. Dekan Prof. Dr. Natascha Oppelt Dedicated to my love Nancy Contents Summary............................................................................................................................................ 9 Zusammenfassung...........................................................................................................................12 General Introduction...................................................................................................................... 15 Epibiosis.................................................................................................................................... 15 The consequences of epibiosis.................................................................................................. 16 The role of bacteria in epibiosis................................................................................................18 Responses of the hosts...............................................................................................................19 The importance of surface extracts...........................................................................................20 Regulation of antifouling defense............................................................................................. 20 Induced chemical defense......................................................................................................... 22 The seasonality of antifouling defense......................................................................................23 Eelgrass.....................................................................................................................................23 Thesis outline.............................................................................................................................24 Study hypotheses....................................................................................................................... 26 References..................................................................................................................................27 Publications and Contributions of Authors..................................................................................35 Chapters I. The role of rosmarinic acid and sulfated flavonoids in the chemical defense of the eelgrass Zostera marina against microfouling 36 II. Heat waves accelerate the chemical defense of Zostera marina against microfoulers 72 III. Seasonal variations in the anti-bacterial defense activity of Zostera marina L. correlate with seasonal variations in fouling pressure and density of microepibionts..........107 General Discussion........................................................................................................................ 142 7 Chemical antifouling defense of eelgrass....................................................................... 142 Phenolic compounds on eelgrass....................................................................................144 Regulation of antifouling defense................................................................................... 147 Reference......................................................................................................................... 154 Acknowledgement......................................................................................................................... 161 Erklärung.......................................................................................................................................163 8 Summary Marine ecosystems and terrestrial ecosystems differ in many aspects and one of these differences is in biofouling, which is considerably more important under water. The role of bacteria in biofouling is essential, as they precondition living and non-living surfaces for the settlement of other marine organisms. In most cases micro- and macro-epiphytes have harmful or even detrimental effects on their hosts, as they compete for resources, reduce the uptake of nutrients or solar energy and in some cases microepiphytes can even be pathogens. The bacterial density in the marine environment is rather high, with, for instance, 0.7 to 2.24 × 106 cells/ml in the western Baltic Sea. However most of the marine plants, such as the habitat forming species Zostera marina L., remain largely free of fouling although they are constantly exposed to those microbes. Such observations suggest that Z. marina may be capable of an antifouling defense. It was the primary goal of this work to identify relevant defense compounds and environmental factors that regulate the dynamics of their production. The chemical defense of Z. marina has been studied since the 1930s and various primary and secondary metabolites - especially phenolic compounds – have been identified as potentially defensive compounds that could defend plants from fouling. However, almost all of those compounds were isolated from the tissue of Z. marina and not from plant surfaces, where fouling actually occurs and the defensive effect would be required. In addition, only few studies determined minimum active concentrations of compounds that are required to deter ecologically relevant settlers and compared them to the concentrations of those compounds on host surfaces, which is important to identify ecologically relevant compounds that drive the natural antifouling defense of Z. marina. In this dissertation, ecologically relevant non-toxic repellents of bacterial microfoulers that are present in extracts from eelgrass surfaces were identified. In subsequent bioassay-guided fractionation steps, rosmarinic acid and two sulphated flavonoids (luteolin-7-sulphate and diosmetin /chrysoeriol-7-sulphate) were identified as bioactive components. All three compounds were dose-dependent in terms of antifouling defense, but rosmarinic acid turned out to be the most dominant and hence the most ecologically relevant of the three compounds. Concentrations of the two flavonoids and also of the known deterrent compound 9 zosteric acid were rather low in surface extracts but high in tissue extracts, which illustrates the importance to study surface extracts if ecologically relevant compounds are to be identified (Chapter I). To study the responses of Z. marina to temperature increase with the respect of chemical antifouling defense I incubated Z. marina several months in the Kiel Outdoor Benthocosm facility under a simulated natural temperature regime with or without application of periodic or single heat waves (+5 °C within one week) compared to the seasonal environmental change heat waves increased the antifouling defense relatively moderately, and the increase was more obvious in the last month of incubation, when the seasonal temperature increase was at its maximum. The surface concentration of rosmarinic acid was not altered by heat wave treatments but by the seasonal environmental change. At the same time it correlated to some degree with the abundance of potential bacterial foulers, suggesting that a demand-driven regulation of antifouling defense possibly exists in Z. marina. Given that heat waves had no reducing effect on the deterrent capacity of Z. Marina, global warming in the future will possibly not decrease the resistance of eelgrass against bacterial foulers (Chapter II). To further study the effects of seasonal changes on the antifouling defense activity of Z. marina in the natural Baltic Sea environment, seagrass surface extracts were collected monthly over one vegetation period. The peak of defense activity appeared in summer, when the abundance of potential bacterial foulers was also higher than in all other seasons, which provided additional evidence of a demand-driven antifouling defense strategy in Z. marina. Also the seasonality of rosmarinic acid surface concentrations correlated with the bacterial fouling pressure and the density of bacteria that settled on eelgrass plants, which was also observed in the benthocosm experiment described in Chapter II. Besides of rosmarinic acid, additional surface compounds must be relevant for the defense activity of eelgrass against fouling, since both measures correlated to some degree only. In addition, abiotic factors such as temperature and nitrogen supply seem to play important roles in the regulation of antifouling defenses and hence the seasonal fluctuation of this activity. However, the influence of temperature is perhaps indirect, as it drives an increased fouling pressure in summer (Chapter III). 10 In summary, this thesis demonstrates that a chemical defense of Z. marina against fouling exists and it identifies the ecologically most relevant surface compounds. Abiotic and possibly also biotic factors that influence