Description: Terrestrial plants are known to “garden” the microbiota of their rhizosphere via released metabolites (that can attract beneficial microbes and deter pathogenic microbes). Such a “gardening” capacity is also known to be dynamic in plants. Although microbial “gardening” has been recently demonstrated for seaweeds, we do not know whether this capacity is a dynamic property in any aquatic flora like in terrestrial plants. Here, we tested the dynamic microbial “gardening” capacity of seaweeds using the model invasive red seaweed Agarophyton vermiculophyllum. Following an initial extraction of surface-associated metabolites (immediately after field collection), we conducted a long-term mesocosm experiment for 5 months to test the effect of two different salinities (low = 8.5 and medium = 16.5) on the microbial “gardening” capacity of the alga over time. We tested “gardening” capacity of A. vermiculophyllum originating from two different salinity levels (after 5 months treatments) in settlement assays against three disease causing pathogenic bacteria and seven protective bacteria. We also compared the capacity of the alga with field-collected samples. Abiotic factors like low salinity significantly increased the capacity of the alga to deter colonization by pathogenic bacteria while medium salinity significantly decreased the capacity of the alga over time when compared to field-collected samples. However, capacity to attract beneficial bacteria significantly decreased at both tested salinity levels when compared to field-collected samples. Dynamic microbial “gardening” capacity of a seaweed to attract beneficial bacteria and deter pathogenic bacteria is demonstrated for the first time. Such a dynamic capacity as found in the current study could also be applicable to other aquatic host–microbe interactions. Our results may provide an attractive direction of research towards manipulation of salinity and other abiotic factors leading to better defended A. vermiculophyllum towards pathogenic bacteria thereby enhancing sustained production of healthy A. vermiculophyllum in farms

Description: In marine environments bacterial microfoulers are an important determinant for the settlement of algal and animal macrofoulers. At the same time fouling is usually subject to seasonal fluctuation. Additionally, the seagrass Zostera marina is prone to microfouling, although this marine spermatophyte is known to be chemically defended against bacterial settlers. Spermatophytes are often capable of induced or activated defences against biological enemies such as pathogens or herbivores, but it is still unknown whether they can fine-tune their antifouling-defence according to settlement pressure. We therefore assessed the seasonality of bacterial settlement pressure, defence against microsettlers and concentrations of a previously identified defence compound, rosmarinic acid, on surfaces of Z. marina. All examined variables peaked in summer, while they tended to be lower in spring and autumn. The seasonality of defence activity and rosmarinic acid surface concentration was positively correlated with the seasonal fluctuation of fouling pressure, which suggests that Z. marina can adjust its defence level to the relatively high bacterial fouling pressure in summer. Besides of biotic factors the seasonal change of environmental factors, such as nitrogen supply, and in particular temperature, also affected the defence level, either directly or through indirect effects on the microbial settlers.

Description: Epibacterial communities on seaweeds are affected by several abiotic factors such as temperature and acidification. Due to global warming, surface seawater temperatures are expected to increase by 0.5–5 °C in the next century. However, how epibacterial communities associated with seaweeds will respond to global warming remains unknown. In this study, we investigated the response of epibacterial communities associated with the invasive Gracilaria vermiculophylla exposed to 3 °C above ambient temperature for 4 months using a benthocosm system in Kiel, Germany, and 16S rRNA gene amplicon sequencing. The results showed that elevated temperature affected the beta-diversity of the epibacterial communities. Some potential seaweed pathogens such as Pseudoalteromonas, Vibrio, Thalassotalea, and Acinetobacter were identified as indicator genera at the elevated temperature level. Thirteen core raw amplicon sequence variants in the elevated temperature group were the same as the populations distributed over a wide geographical range, indicating that these core ASVs may play an important role in the invasive G. vermicullophylla. Overall, this study not only contributes to a better understanding of how epibacterial communities associated with G. vermiculophylla may adapt to ocean warming, but also lays the foundation for further exploration of the interactions between G. vermiculophylla and its epimicrobiota.

Description: In comparison with terrestrial plants the mechanistic knowledge of chemical defences is poor for marine macroalgae. This restricts our understanding in the chemically mediated interactions that take place between algae and other organisms. Technical advances such as metabolomics, however, enable new approaches towards the characterisation of the chemically mediated interactions of organisms with their environment. We address defence responses in the red alga Gracilaria vermiculophylla using mass spectrometry based metabolomics in combination with bioassays. Being invasive in the north Atlantic this alga is likely to possess chemical defences according to the prediction that well-defended exotics are most likely to become successful invaders in systems dominated by generalist grazers, such as marine macroalgal communities. We investigated the effect of intense herbivore feeding and simulated herbivory by mechanical wounding of the algae. Both processes led to similar changes in the metabolic profile. Feeding experiments with the generalist isopod grazer Idotea baltica showed that mechanical wounding caused a significant increase in grazer resistance. Structure elucidation of the metabolites of which some were up-regulated more than 100 times in the wounded tissue, revealed known and novel eicosanoids as major components. Among these were prostaglandins, hydroxylated fatty acids and arachidonic acid derived conjugated lactones. Bioassays with pure metabolites showed that these eicosanoids are part of the innate defence system of macroalgae, similarly to animal systems. In accordance with an induced defence mechanism application of extracts from wounded tissue caused a significant increase in grazer resistance and the up-regulation of other pathways than in the activated defence. Thus, this study suggests that G. vermiculophylla chemically deters herbivory by two lines of defence, a rapid wound-activated process followed by a slower inducible defence. By unravelling involved pathways using metabolomics this work contributes significantly to the understanding of activated and inducible defences for marine macroalgae.

Description: Mechanisms related to the induction of phlorotannin biosynthesis in marine brown algae remain poorly known. Several studies undertaken on fucoid species have shown that phlorotannins accumulate in the algae for several days or weeks after being exposed to grazing, and this is measured by direct quantification of soluble phenolic compounds. In order to investigate earlier inducible responses involved in phlorotannin metabolism, Fucus vesiculosus was studied between 6 and 72 h of grazing by the sea snail Littorina littorea. In this study, the quantification of soluble phenolic compounds was complemented by a Quantitative real-time PCR (qRT-PCR) approach applied on genes that are potentially involved in either the phlorotannin metabolism or stress responses. Soluble phlorotannin levels remained stable during the kinetics and increased significantly only after 12 h in the presence of grazers, compared to the control, before decreasing to the initial steady state for the rest of the kinetics. Under grazing conditions, the expression of vbpo, cyp450 and ast6 genes was upregulated, respectively, at 6 h, 12 h and 24 h, and cyp450 gene was downregulated after 72 h. Interestingly, the pksIII gene involved in the synthesis of phloroglucinol was overexpressed under grazing conditions after 24 h and 72 h. This study supports the hypothesis that phlorotannins are able to provide an inducible chemical defense under grazing activity, which is regulated at different stages of the stress response.

Description: The goals of this study were (1) to investigate whether Fucus vesiculosus regulates the production of its antifouling defence chemicals against epibacteria in response to light limitation and temperature shifts and (2) to investigate if different surface concentrations of defence compounds shape epibacterial communities. F. vesiculosus was incubated in indoor mesocosms at five different temperature conditions (5 to 25°C) and in outdoor mesocosms under six differently reduced sunlight conditions (0 to 100%), respectively. Algal surface concentrations of previously identified antifouling compounds - dimethylsulphopropionate (DMSP), fucoxanthin and proline – were determined and the bacterial community composition was characterized by in-depth sequencing of the 16S-rRNA gene. Altogether, the effect of different treatment levels upon defence compound concentrations was limited. Under all conditions DMSP alone appeared to be sufficiently concentrated to warrant for at least a partial inhibitory action against epibiotic bacteria of F. vesiculosus. In contrast, proline and fucoxanthin rarely reached the necessary concentration ranges for self-contained inhibition. Nonetheless, in both experiments along with the direct influence of temperature and light, all three compounds apparently affected the overall bacterial community composition associated with F. vesiculosus since tendencies for insensitivity towards all three compounds were observed among bacterial taxa that typically dominate those communities. Given that the concentrations of at least one of the compounds (in most cases DMSP) were always high enough to inhibit bacterial settlement, we conclude that the capacity of F. vesiculosus for such defence will hardly be compromised by shading or warming to temperatures up to 25°C.

Description: To evaluate the importance of anti-herbivore resistance for algal invasion success we compared resistance traits among specimens of the red macroalga Gracilaria vermiculophylla from six native populations in Korea and China and eight invasive populations in Europe and Mexico that were maintained under identical conditions in the laboratory. Herbivorous snails both from the native range (Littorina brevicula) and from the invaded range (Littorina littorea) consumed significantly less of seaweed specimens originating from non-native populations. Metabolome profiling revealed that this preference was correlated with an increased woundactivated production of deterring prostaglandins and hydroxyeicosatetraenoic acids. Thus, invasive populations of G. vermiculophylla are more strongly defended against challenge by herbivores and other biological enemies that cause local tissue or cell disruption and activate oxylipin production. Anthropogenic distribution of genotypes adapted to resist elevated feeding pressure probably contributed to the invasion success of this species.

Description: Grazing by the isopod Idotea baltica induces chemical defenses in the brown seaweed Fucus vesiculosus. A combination of a 33 day induction experiment, feeding choice assays and functional genomic analyses was used to investigate temporal defense patterns and to correlate changes in palatability to changes in gene expression. Despite permanent grazing, seaweed palatability varied over time. Controls were significantly more consumed than grazed pieces only after 18 and 27 days of grazing. Relative to controls, 562/402 genes were up-/down-regulated in seaweed pieces that were grazed for 18 days, i.e. when defense induction was detected. Reprogramming of the regulative expression orchestra (translation, transcription), up-regulation of genes involved in lipid and carbohydrate metabolism, intracellular trafficking, defense and stress response, as well as downregulation of photosynthesis was found in grazed seaweed. These findings indicate short-term temporal variation in defenses and that modified gene expression patterns arise at the same time when grazed seaweed pieces show reduced palatability. Several genes with putative defensive functions and cellular processes potentially involved in defence, such as reallocation of resources from primary to secondary metabolism, were revealed

Description: The genus Ulva is has broadly negative connotations because of its ability to form harmful “green tides” and the problems it causes with precise species identification, due to its morphological plasticity. During recent years, tides of unattached Ulva compressa U. Linneus 1753 with an atypical sheet-like morphology were for the first time observed in the German Baltic. Here we report that this nuisance alga is conspecific with the type strain of U. mutabilis Föyn 1958 from Faro in Portugal, an important model organism to study morphogenesis, morphogenetics and mutualistic interactions. Different approaches were used to examine conspecificity: (1) Comparisons on vegetative and reproductive features of cultured material of Ulva mutabilis and German Ulva compressa resulted in congruent results proving that a certain morphogenetic mutation pattern is shared. Spontaneous mutations of “slender-like” thalli are appearing whilst the common form exhibits a “leaf-like” wildtype morphology. (2) Interbreeding experiments of gametes of Ulva compressa and Ulva mutabilis were successful and showed a fertile first-generation offspring exhibiting the typical wildtype morphology similar to the phenotype of the parental generation. (3) Phylogenetic and species delimitation analyses were carried out on 128 tufA sequences of Ulva compressa specimens sampled in 2014–2016 in Germany and on tufA sequences of two clones of the strains Ulva mutabilis (sl-G[mt+]) and Ulva mutabilis (wt-[mt-]) to identify Molecular Operational Taxonomic Units (MOTUs). The Generalized Mixed Yule-Coalescent (GMYC) method comprises one major MOTU containing all included sequences of Ulva compressa and Ulva mutabilis, while reference sequences included in the analysis clustered outside this MOTU. This highly supports the monophyly of Ulva compressa and Ulva mutabilis, which can be treated as the same species. As a consequence, U. mutabilis is also a suitable model for future studies of green tides and their molecular and morphogenetic basis in the Baltic Sea.

Description: Fouling is a stressor that might determine the fate of seaweeds, but reports of algal adaptation to epibiosis are scarce. Previous comparisons have shown resistance to epibionts can be higher in non-native than in resident seaweed species, but we do not know whether it is an intrinsic trait of the non-natives or it has been acquired during the invasion process. We here compared native and non-native populations of the same algal species to elucidate this question. Resistance against two groups of epiphytes was assessed in living thalli and in artificial substrata coated with surface extracts, both gained from four Asian (native) and four European (non-native) populations of the red alga Gracilaria vermiculophylla. Two diatom species and two filamentous macroalgae were used as micro- and macro-epiphytes, and one of each type was collected in Asia, while the other came from Europe. Laboratory assays were done in both distributional ranges of G. vermiculophylla and in different seasons. We used a fully crossed design with the factors (i) ‘Origin of Gracilaria’, (ii) ‘Origin of epiphytes’, (iii) ‘Season’ and (iv) ‘Solvent used for extraction’. Both groups of epiphytes, regardless of their origin, attached less to living thalli and to surface extracts from non-native G. vermiculophylla. Fewer diatoms attached to hexane-based extracts, while fewer Ceramium filaments settled on extracts gained with dichloromethane. Our results show for the first time that non-native individuals of a seaweed are better defended against epiphytes than native conspecifics. Furthermore, we found evidence that at least a part of the defence is based on extractable secondary metabolites. We suggest that an enhanced defence against epiphytes after introduction is one reason for G. vermiculophylla’s invasion success. Our observation may also apply to other basibiont–epibiont interactions and could be a key feature of seaweed bioinvasions.