Gunilla B. Toth

INDUCIBLE CHEMICAL RESISTANCE TO HERBIVORY IN THE BROWN SEAWEED ASCOPHYLLUM NODOSUM

Physical damage caused by herbivores or artificial clipping is known to induce responses in secondary chemistry as well as increased resistance to further grazing in a large number of terrestrial vascular plants, but this has only rarely been described for marine algae. In the present study, it was found that a few weeks of grazing by the gastropod Littorina obtusata can induce substantially increased concentrations of phlorotannins in the intertidal brown seaweed Ascophyllum nodosum.In contrast, grazing by the isopod Idotea granulosa and simulations of herbivory through momentary and continuous clipping caused no significant changes in phlorotannin levels. This indicates a high degree of specificity in the elicitation of chemical responses to physical damage, something which previously has been shown for terrestrial vascular plants but not for marine algae. Ascophyllum plants that had been grazed by L. obtusata were also less susceptible than undamaged plants to further grazing by gastropods, but no such induced resistance was found in the experiments with I. granulosa. Feeding experiments with undamaged Ascophyllum plants and artificial food containing different levels of phlorotannins provided further support for the sensitivity of the feeding behavior of L. obtusata, and the insensitivity of I. granulosa, to intraspecific variation in the phlorotannin content of Ascophyllum. Since L. obtusata is specialized to live and feed on a few fucoid species, including A. nodosum, the results imply that phlo- rotannins have an important mediating role in the interactions between these macroalgae and L. obtusata. The experimentally induced increase of phlorotannins was consistent with results from phlorotannin analyses of Ascophyllum individuals from natural populations, where plants that had been heavily grazed by L. obtusata contained significantly higher levels of phlorotannins compared to undamaged plants. A field survey of the distribution and abundance of L. obtusata revealed that the density of the gastropod is highly variable at the same spatial scale as the phlorotannin content of Ascophyllum in the study area. These results suggest that grazing by L. obtusata can be an important factor in explaining natural phenotypic variation in the phlorotannin content of Ascophyllum. Together with the results from a few other studies on the interactions between meso- herbivores and marine algae, the results of this study support the previously proposed hypothesis that it is feeding by relatively small, less mobile herbivores that is most likely to cue for induced production of defense chemicals in seaweeds. More studies on such interactions may reveal that the apparent rarity of inducible chemical defenses in seaweeds is misleading.

Copepods induce paralytic shellfish toxin production in marine dinoflagellates

Among the thousands of unicellular phytoplankton species described in the sea, some frequently occurring and bloom-forming marine dinoflagellates are known to produce the potent neurotoxins causing paralytic shellfish poisoning. The natural function of these toxins is not clear, although they have been hypothesized to act as a chemical defence towards grazers. Here, we show that waterborne cues from the copepod Acartia tonsa induce paralytic shellfish toxin (PST) production in the harmful algal bloom-forming dinoflagellate Alexandrium minutum. Induced A. minutum contained up to 2.5 times more toxins than controls and was more resistant to further copepod grazing. Ingestion of non-toxic alternative prey was not affected by the presence of induced A. minutum. The ability of A. minutum to sense and respond to the presence of grazers by increased PST production and increased resistance to grazing may facilitate the formation of harmful algal blooms in the sea.

New trends in marine chemical ecology

This essay is the outcome of a colloquium convened in November 2005 at the Benthos Laboratory of the Stazione Zoologica Anton Dohrn in Ischia, Italy, on chemical ecology and the role of secondary metabolites in the structuring and functioning of marine biodiversity. The participants of the workshop are part of the European Network of Excellence MarBEF (Marine Biodiversity and Ecosystem Function), a consortium of 56 European marine institutes to integrate and disseminate knowledge and expertise on marine biodiversity. Here we review some of the new trends and emerging topics in marine chemical ecology. The first section deals with microbial chemical interactions. Microbes communicate with each other using diffusible molecules such as N-acylhomoserine lactones (AHL). These are regulators in cell-density-dependent gene regulation (quorum sensing) controlling microbial processes. In chemical interactions with higher organisms, microbes can act either as harmful pathogens that are repelled by the host’s chemical defense or as beneficial symbionts. These symbionts are sometimes the true producers of the host’s secondary metabolites that have defensive and protective functions for their hosts. We also describe how allelochemicals can shape phytoplankton communities by regulating competition for available resources, and also interactions among individuals of the same species. Compounds such as the diatom-derived unsaturated aldehydes have been demonstrated to act as info chemicals, and they possibly function as a diffusible bloom-termination signal that triggers an active cell death and bloom termination at sea. The same molecules have also been shown to interfere with the reproductive capacity of grazing animals deterring future generations of potential predators. Such compounds differ from those that act as feeding deterrents since they do not target the predator but its offspring. Many of the neurotoxins produced by dinoflagellates act as feeding deterrents, and laboratory experiments have shown that ingestion of these algae by some microzooplankton and macrozooplankton can cause acute, responses such as death, incapacitation, altered swimming behavior, and reduced fecundity and egg-hatching success. These effects may rarely occur in nature because of low individual grazing rates on dinoflagellate cells and grazing on other food sources such as microflagellates and diatoms. We also consider the nutritional component of marine plant-herbivore interactions, especially in the plankton, and the information available on the effects of growing conditions of algae on the production of toxic metabolites. Species producing saxitoxins seem to consistently produce the highest amounts of toxins (on a per cell basis) in the exponential phase of growth, and there is a decrease in their production under nitrogen, but not under phosphorus stress, where the production actually increases. We try to explain the circumstances under which organisms defend themselves chemically and argue that the most likely explanatory model for the production of secondary metabolites used for defense in planktonic organisms is the carbon nutrient balance hypothesis, which predicts that most algae produce their toxins mainly under conditions where carbon is in excess and nitrogen (or other nutrients) is limiting. We also discuss chemically mediated macroalgal-herbivore interactions in the benthos and the large variation in concentration of seaweed defense metabolites at different spatial and temporal scales. Seaweeds have been shown to produce a large variety of secondary metabolites with highly variable chemical structures such as terpenoids, acetogenins, amino acid derivates, and polyphenols. Many of these compounds probably have multiple simultaneous functions for the seaweeds and can act as allelopathic, antimicrobial, and antifouling or ultraviolet-screening agents, as well as herbivore deterrents. We also provide examples of interactions between marine benthic invertebrates, especially sponges, molluscs, and cnidarians, that are mediated by specific secondary metabolites and discuss the role of these in shaping benthic communities.

Formation of harmful algal blooms cannot be explained by allelopathic interactions

Many planktonic microalgae produce a range of toxins and may form harmful algal blooms. One hypothesis is that some toxins are allelopathic, suppressing the growth of competitors, and it has been suggested that allelopathy may be one important mechanism causing algal blooms. In a metaanalysis of recent experimental work, we looked for evidence that allelopathy may explain the initiation of algal blooms. With few exceptions, allelopathic effects were only significant at very high cell densities typical of blooms. We conclude that there is no experimental support for allelopathy at prebloom densities, throwing doubts on allelopathy as a mechanism in bloom formation. Most studies tested allelopathy using cell-free manipulations. With simple models we show that cell-free manipulations may underestimate allelopathy at low cell densities if effects are transmitted during cell–cell interactions. However, we suggest that the evolution of allelopathy under field conditions may be unlikely even if based on cell–cell interactions. The spatial dispersion of cells in turbulent flow will make it difficult for an allelopathic cell to receive an exclusive benefit, and a dispersion model shows that dividing cells are rapidly separated constraining clone selection. Instead, we propose that reported allelopathic effects may be nonadaptive side effects of predator–prey or casual parasitic cell–cell interactions.

OPTIMAL DEFENSE THEORY: ELASTICITY ANALYSIS AS A TOOL TO PREDICT INTRAPLANT VARIATION IN DEFENSES

Optimal defense theory (ODT) predicts that plants should have the highest defense levels in parts that have the highest value in terms of fitness. Assumptions about differences in fitness value among plant parts have previously been based on general reasoning, e.g., that reproductive tissue is more valuable than vegetative tissue since fitness ultimately depends on reproduction. Here we present a first example of how demographic elasticity (proportional sensitivity) analysis can be used as a tool to generate more objective and specific estimates of the fitness value of different plant parts, based on the entire life cycle. These estimates were further used to generate testable predictions about intraplant variation in defenses of the brown seaweed Ascophyllum nodosum (knotted wrack). The predictions were clearly supported by the results from both analyses of chemical defense levels (phlorotannins) and bioassays (feeding preference experiments), which showed that reproductive tissues had the lowest defense level of the three examined tissue types, in contrast to previous assumptions. We suggest that a stronger focus on variation in life history among species and populations, with demographic perturbation analysis as one important tool, could lead to a better understanding of intraplant variation in defense levels, in seaweeds as well as vascular plants and other modular organisms.

Influence of light and nitrogen on the phlorotannin content of the brown seaweeds Ascophyllum nodosum and Fucus vesiculosus

Phlorotannins, C-based defence compounds in brown seaweeds, show a high degree of spatial and temporal variation within seaweed species. One important model explaining this variation is the Carbon Nutrient Balance Model (CNBM), which states that the relative supply of carbon and limiting nutrients will determine the level of defence compounds in plants. Nitrogen is often considered to be the limiting nutrient for marine macroalgal growth and the CNBM thus predicts that when the carbon:nitrogen ratio is high, photosynthetically fixed carbon will be allocated to production of phlorotannins. In the present study, we evaluated the effects of light (i.e. carbon) and nitrogen on the phlorotannin content of two intertidal brown seaweeds, Ascophyllum nodosum and Fucus vesiculosus. This was done in an observational field study, as well as in a manipulative experiment where plants from habitats with different light regimes were subjected to different nitrogen and light treatments, and their phlorotannin content was measured after 14 days. The results showed that there was a negative relationship between tissue nitrogen and phlorotannin content in natural populations of F. vesiculosus, but not in A. nodosum. In the short term, the phlorotannin content in both algal species was not affected by changes in nitrogen availability. Exposure to sunlight had a positive effect on the phlorotannin content in natural populations of both algal species but, in the manipulative experiment, only F. vesiculosus showed a rapid response to changes in light intensities. Plants subjected to sunlight contained higher phlorotannin content than shaded plants. In conclusion, the results imply that nitrogen availability explains some of the natural variation in the phlorotannin content of F. vesiculosus, but the light environment has greater importance than nitrogen availability in predicting the phlorotannin content of each species.

INDUCIBLE AND CONSTITUTIVE DEFENSES OF VALUABLE SEAWEED TISSUES: CONSEQUENCES FOR HERBIVORE FITNESS

Optimal Defense Theory predicts that plants exposed to herbivory should allocate more resources to produce costly secondary metabolites in tissues with higher fitness values. To increase plant resistance, the secondary metabolites must have a negative impact on the preference and/or performance of herbivores. We tested the hypotheses that induction of secondary metabolites (phlorotannins) in a brown seaweed in response to grazing by herbivorous gastropods will differ between seaweed tissues with different fitness values (basal stipes and annual shoots), and that the subsequent change in food value will affect the fitness (growth and fecundity) of the gastropods. Induction of phlorotannins was significant in both tissue types but was more pronounced in basal stipes, which have a higher fitness value. Basal tissues also had significantly higher constitutive defense levels than did apical tissues. No effects of algal tissue type or grazing history on the growth rate of the gastropods were detected. However, the number of viable eggs was significantly lower for gastropods feeding on basal shoots, and there was a significantly lower proportion of viable eggs produced by gastropods that were offered previously grazed seaweed tissues. The results show that induced resistance, and its variation among different plant parts, can have significant negative effects on herbivore performance that may reduce future herbivore pressure and thus enhance plant fitness.

Removal of Dissolved Brown Algal Phlorotannins Using Insoluble Polyvinylpolypyrrolidone (PVPP)

Tannins, a large and diverse group of phenolic secondary metabolites, are common in terrestrial plants and marine brown algae. It is sometimes desirable to remove the tannins from plant or algal extracts, e.g., when isolating enzymes and nucleic acids, when using certain colorimetric methods to quantify the tannin content, or to create reliable controls when using tannins in experimental studies. Insoluble polyvinylpolypyrrolidone (PVPP) can be used to specifically remove tannins from solution. In the present study, we evaluated the effect of different factors (amount of PVPP, number of PVPP treatments, type of solvent, pH, and incubation time) on the PVPP removal of dissolved brown algal phlorotannins. Our results imply that there is a limited amount of phlorotannins that can bind to a given amount of PVPP, and that it is preferable to use low quantities of PVPP repeatedly, compared to using fewer treatments with a high amount of PVPP. Furthermore, we found no consistent effect on the removal of phlorotannins due to solvent type (acetone, methanol, distilled water or filtered seawater). There was a slight decrease in the amount of phlorotannins removed from extracts with increasing pH when repeatedly treated with PVPP. All phlorotannins were removed from extracts with pH ≤6.2, and 89% of the initial phlorotannin content was removed at pH 9.7. These results are compared with previous methodological studies on tannin removal with PVPP. Furthermore, the implications of phlorotannin removal in analytical and ecological investigations are discussed.

Marine dinoflagellates show induced life-history shifts to escape parasite infection in response to water-borne signals

Many dinoflagellate species form dormant resting cysts as a part of their life cycle, and in some freshwater species, hatching of these cysts can be delayed by the presence of water–borne signals from grazing zooplankton. Some marine dinoflagellates can form temporary cysts, which may function to resist unfavourable short–term environmental conditions. We investigated whether the marine dinoflagellate Alexandrium ostenfeldii is able to induce an increased resistance to the parasitic flagellate Parvilucifera infectans by forming temporary cysts. We performed several laboratory experiments where dinoflagellates were exposed either to direct contact with parasites or to filtered water from cultures of parasite–infected conspecifics (parasite–derived signals). Infection by P. infectans is lethal to motile A. ostenfeldii cells, but temporary cysts were more resistant to parasite infection. Furthermore, A. ostenfeldii induced a shift in life–history stage (from motile cells to temporary cysts) when exposed to parasite–derived water–borne signals. The response was relaxed within a couple of hours, indicating that A. ostenfeldii may use this behaviour as a short–term escape mechanism to avoid parasite infection. The results suggest that intraspecies chemical communication evoked by biotic interactions can be an important mechanism controlling life–history shifts in marine dinoflagellates, which may have implications for the development of toxic algal blooms.

Mesoherbivores reduce net growth and induce chemical resistance in natural seaweed populations

Herbivory on marine macroalgae (seaweeds) in temperate areas is often dominated by relatively small gastropods and crustaceans (mesoherbivores). The effects of these herbivores on the performance of adult seaweeds have so far been almost exclusively investigated under artificial laboratory conditions. Furthermore, several recent laboratory studies with mesoherbivores indicate that inducible chemical resistance may be as common in seaweeds as in vascular plants. However, in order to further explore and test the possible ecological significance of induced chemical resistance in temperate seaweeds, data are needed that address this issue in natural populations. We investigated the effect of grazing by littorinid herbivorous snails (Littorina spp.) on the individual net growth of the brown seaweed Ascophyllum nodosum in natural field populations. Furthermore, the capacity for induced resistance in the seaweeds was assessed by removing herbivores and assaying for relaxation of defences. We found that ambient densities of gastropod herbivores significantly reduced net growth by 45% in natural field populations of A. nodosum. Seaweeds previously exposed to grazing in the field were less consumed by gastropod herbivores in feeding bioassays. Furthermore, the concentration of phlorotannins (polyphenolics), which have been shown to deter gastropod herbivores, was higher in the seaweeds that were exposed to gastropod herbivores in the field. This field study corroborates earlier laboratory experiments and demonstrates that it is important to make sure that the lack of experimental field data on marine mesoherbivory does not lead to rash conclusions about the lack of significant effects of these herbivores on seaweed performance. The results strongly suggest that gastropods exert a significant selection pressure on the evolution of defensive traits in the seaweeds, and that brown seaweeds can respond to attacks by natural densities of these herbivores through increased chemical resistance to further grazing.