Description: DNA barcoding analysis, using tufA, revealed considerable differences between the expected and observed species inventory of Ulva sensu lato in the Baltic and North Sea areas of the German state of Schleswig-Holstein. Of 20 observed genetic entities, at least four (U. australis, U. californica, U. gigantea and Umbraulva dangeardii) had been introduced recently, whereas three others (one Ulva sp. and two Blidingia spp.) could not be identified at the species level and could also represent recently introduced species. In addition, the observed distributions of Kornmannia leptoderma and U. rigida were much more extensive than indicated by historical records, whereas Blidingia minima and Gayralia oxysperma were absent or much less common than expected. Barcoding analysis also revealed that both U. tenera (type material) and U. pseudocurvata (historical vouchers) from Helgoland, an off-shore island in the North Sea, actually belong to U. lactuca, a species that appears to be restricted to this island. Furthermore, past morphological descriptions of U. intestinalis and U. compressa have apparently been too restrictive and have been responsible for numerous misidentifications. The same is true for U. linza, which, in northern Germany, clusters into two genetically closely related but morphologically indistinguishable entities. One of these entities is present on Helgoland, while the second is present on North Sea and Baltic Sea mainland coasts.

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: A bioassay-guided approach was used to identify defense compounds that are present on the surface of Zostera marina and which inhibit settlement of microfoulers at natural concentrations. Moderately polar eelgrass surface extracts inhibited the settlement of seven marine bacteria and one yeast that originated from non-living substrata. In contrast, five other bacterial strains that had been directly isolated from eelgrass surfaces were all insensitive, which suggested a selective effect of surface metabolites on the microbial communities present on eelgrass. Bioassay-guided isolation of active compounds from the extracts in combination with UPLC-MS and 1H-NMR spectroscopy resulted in the identification of rosmarinic acid, luteolin-7-sulfate and diosmetin-7-sulfate or its isomer chrysoeriol-7-sulfate. All three compounds are nontoxic repellents, as they did not inhibit bacterial growth, but prevented bacterial settlement in a dose-dependent manner. Between 15.6 and 106.8 μg ml−1 of rosmarinic acid were present on the eelgrass surface, enough for half maximal settlement inhibition of bacteria.

Description: This study aimed to reveal the dynamics of inducible anti-herbivory traits in the brown seaweed Fucus vesiculosus, in response to grazing by the isopod Idotea baltica. As the molecular basis of defence induction in seaweeds is poorly understood, a transcriptomic approach was used to gain insight into cellular processes underlying defence induction and thus promote the mechanistic understanding of anti-herbivory responses in seaweeds. In a 27 day feeding-assayed induction experiment, temporal patterns of the induced anti-herbivory resistance of F. vesiculosus were assessed under laboratory conditions. Feeding assays were performed at three day intervals, using fresh and reconstituted food. Microarray hybridizations investigating the expression of genes 3 days before, as well as at the same time as, feeding assays revealed a decreased palatability of previously grazed F. vesiculosus pieces compared with non-grazed control pieces. Despite permanent exposure to grazers, F. vesiculosus palatability varied over time. Non-grazed F. vesiculosus pieces were significantly preferred to grazed pieces after 18 and again after 27 days of previous grazing, while their relative palatability for isopods was comparable at all other times during the experiment. Relative to controls, 562/402 genes were ≥ 1.5-fold up-/down-regulated in seaweed pieces that were grazed for 18 days, i.e. when induction of defences was detected in feeding assays. Massive reprogramming of the regulatory expression orchestra (translation, transcription) as well as up-regulation of genes involved in lipid and carbohydrate metabolism, intracellular trafficking, defence and stress response was found. At the same time, down-regulation of photosynthesis was observed in grazed seaweed pieces. This study suggests a high level of temporal variability in induced anti-herbivory traits of F. vesiculosus and reveals increased expression of genes with putative defensive functions in conjunction with the reallocation of resources from primary to secondary metabolism.

Description: Highlights: • Wound activated prostaglandin production was quantified in Gracilaria vermiculophylla. • Specimens from non-native populations contained higher prostaglandin concentrations. • Prostaglandins and related compounds deter animal consumers. • Activated defense through Prostaglandins was selected during Gracilaria's invasion history. • As a consequence specimens from invasive populations are not safe for human consumption. Abstract: The capacity of the East Asian seaweed Gracilaria vermiculophylla (“Ogonori”) for production of prostaglandin E2 from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. Wounding of non-native specimens resulted on average in 390% more 15-keto-PGE2, in 90% more PGE2, in 37% more PGA2 and in 96% more 7,8-di-hydroxy-eicosatetraenoic acid than wounding of native specimens. Not only PGE2, but also PGA2 and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.

Description: It was demonstrated previously that polar and non-polar surface extracts of the brown alga Fucus vesiculosus collected during winter from the Kiel Bight (Germany) inhibited bacterial attachment at natural concentrations. The present study describes the bioassay-guided identification of the active metabolites from the polar fraction. Chromatographic separation on a size-exclusion liquid chromatography column and bioassays identified an active fraction that was further investigated using nuclear magnetic resonance spectroscopy and mass spectrometry. This fraction contained the metabolites dimethylsulphopropionate (DMSP), proline and alanine. DMSP and proline caused the anti-attachment activity. The metabolites were further quantified on the algal surface together with its associated boundary layer. DMSP and proline were detected in the range 0.12–1.08 ng cm−2 and 0.09–0.59 ng cm−2, respectively. These metabolites were tested in the concentration range from 0.1 to 1000 ng cm−2 against the attachment of five bacterial strains isolated from algae and sediment co-occurring with F. vesiculosus. The surface concentrations for 50% inhibition of attachment of these strains were always 〈0.38 ng cm−2 for DMSP and in four cases 〈0.1 ng cm−2 for proline, while one strain required 1.66 ng cm−2 of proline for 50% inhibition. Two further bacterial strains that had been directly isolated from F. vesiculosus were also tested, but proved to be the least sensitive. This study shows that DMSP and proline have an ecologically relevant role as surface inhibitors against bacterial attachment on F. vesiculosus.

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.

Description: The chemical defence against microfouling in the brown seaweed Fucus vesiculosus was investigated and an inhibitor of bacterial settlement was isolated by bioassay-guided fractionation of non-polar surface extracts. UV-vis and mass spectrometry were used to identify the compound as the carotenoid fucoxanthin. The metabolite was tested at the natural concentration (in a surface volume based assay) against the settlement of four bacterial strains isolated from F. vesiculosus and 11 strains isolated from co-occurring algae and marine sediment. Surface concentrations between 1.4 and 6 μg cm-2 resulted in 50% inhibition of four of these isolates, which were studied in more detail using a surface area-based assay, while a fifth isolate proved to be less sensitive. The presence of fucoxanthin on the surface of F. vesiculosus was demonstrated with two different surface extraction methods. Fucoxanthin was detected at concentrations between 0.7 and 9 μg cm-2 on the algal surface. Fucoxanthin was still present at the algal surface after removal of associated diatoms through mechanical cleaning and germanium dioxide treatment and was thus mainly produced by F. vesiculosus rather than by diatoms. Thus, the photosynthetic pigment fucoxanthin appears to be ecologically relevant as a surface-associated antimicrobial agent, acting against the settlement of bacteria on the surface of the macroalga F. vesiculosus.