Julia Kubanek

Seaweed resistance to microbial attack: A targeted chemical defense against marine fungi

Pathogenic microbes can devastate populations of marine plants and animals. Yet, many sessile organisms such as seaweeds and sponges suffer remarkably low levels of microbial infection, despite lacking cell-based immune systems. Antimicrobial defenses of marine organisms are largely uncharacterized, although from a small number of studies it appears that chemical defenses may improve host resistance. In this study, we asked whether the common seaweed Lobophora variegata is chemically defended against potentially deleterious microorganisms. Using bioassay-guided fractionation, we isolated and characterized a 22-membered cyclic lactone, lobophorolide (1), of presumed polyketide origin, with sub-μM activity against pathogenic and saprophytic marine fungi. Deterrent concentrations of 1 were found in 46 of 51 samples collected from 10 locations in the Bahamas over a 4-year period. Lobophorolide (1) is structurally unprecedented, yet parts of the molecule are related to tolytoxin, the scytophycins, and the swinholides, macrolides previously isolated from terrestrial cyanobacteria and from marine sponges and gastropods. Until now, compounds of this structural class have not been associated with marine macrophytes. Our findings suggest that seaweeds use targeted antimicrobial chemical defense strategies and that secondary metabolites important in the ecological interactions between marine macroorganisms and microorganisms could be a promising source of novel bioactive compounds.

Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed

Organism surfaces represent signaling sites for attraction of allies and defense against enemies. However, our understanding of these signals has been impeded by methodological limitations that have precluded direct fine-scale evaluation of compounds on native surfaces. Here, we asked whether natural products from the red macroalga Callophycus serratus act in surface-mediated defense against pathogenic microbes. Bromophycolides and callophycoic acids from algal extracts inhibited growth of Lindra thalassiae, a marine fungal pathogen, and represent the largest group of algal antifungal chemical defenses reported to date. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging revealed that surface-associated bromophycolides were found exclusively in association with distinct surface patches at concentrations sufficient for fungal inhibition; DESI-MS also indicated the presence of bromophycolides within internal algal tissue. This is among the first examples of natural product imaging on biological surfaces, suggesting the importance of secondary metabolites in localized ecological interactions, and illustrating the potential of DESI-MS in understanding chemically-mediated biological processes.

Macroalgal terpenes function as allelopathic agents against reef corals

During recent decades, many tropical reefs have transitioned from coral to macroalgal dominance. These community shifts increase the frequency of algal–coral interactions and may suppress coral recovery following both anthropogenic and natural disturbance. However, the extent to which macroalgae damage corals directly, the mechanisms involved, and the species specificity of algal–coral interactions remain uncertain. Here, we conducted field experiments demonstrating that numerous macroalgae directly damage corals by transfer of hydrophobic allelochemicals present on algal surfaces. These hydrophobic compounds caused bleaching, decreased photosynthesis, and occasionally death of corals in 79% of the 24 interactions assayed (three corals and eight algae). Coral damage generally was limited to sites of algal contact, but algae were unaffected by contact with corals. Artificial mimics for shading and abrasion produced no impact on corals, and effects of hydrophobic surface extracts from macroalgae paralleled effects of whole algae; both findings suggest that local effects are generated by allelochemical rather than physical mechanisms. Rankings of macroalgae from most to least allelopathic were similar across the three coral genera tested. However, corals varied markedly in susceptibility to allelopathic algae, with globally declining corals such as Acropora more strongly affected. Bioassay-guided fractionation of extracts from two allelopathic algae led to identification of two loliolide derivatives from the red alga Galaxaura filamentosa and two acetylated diterpenes from the green alga Chlorodesmis fastigiata as potent allelochemicals. Our results highlight a newly demonstrated but potentially widespread competitive mechanism to help explain the lack of coral recovery on many present-day reefs.

Chemically mediated competition between microbes and animals: microbes as consumers in food webs.

Microbes are known to affect ecosystems and communities as decomposers, pathogens, and mutualists. However, they also may function as classic consumers and competitors with animals if they chemically deter larger consumers from using rich food-falls such as carrion, fruits, and seeds that can represent critical windfalls to both microbes and animals. Microbes often use chemicals (i.e., antibiotics) to compete against other microbes. Thus using chemicals against larger competitors might be expected and could redirect significant energy subsidies from upper trophic levels to the detrital pathway. When we baited traps in a coastal marine ecosystem with fresh vs. microbe-laden fish carrion, fresh carrion attracted 2.6 times as many animals per trap as microbe-laden carrion. This resulted from fresh carrion being found more frequently and from attracting more animals when found. Microbe-laden carrion was four times more likely to be uncolonized by large consumers than was fresh carrion. In the lab, the most common animal found in our traps (the stone crab Menippe mercenaria) ate fresh carrion 2.4 times more frequently than microbe-laden carrion. Bacteria-removal experiments and feeding bioassays using organic extracts of microbe-laden carrion showed that bacteria produced noxious chemicals that deterred animal consumers. Thus bacteria compete with large animal scavengers by rendering carcasses chemically repugnant. Because food-fall resources such as carrion are major food subsidies in many ecosystems, chemically mediated competition between microbes and animals could be an important, common, but underappreciated interaction within many communities.

Community and ecosystem level consequences of chemical cues in the plankton

Aquatic organisms produce compounds that deter consumers, alter prey behavior, suppress or kill target and nontarget species, and dramatically affect food-web structure, community composition, and the rates and pathways of biogeochemical cycles. Toxins from marine and freshwater phytoplankton create health hazards for both aquatic and terrestrial species and can significantly affect human activities and the economic vitality of local communities. A reasonable case can be made that phytoplankton metabolites such as dimethyl sulfide (DMS) link interaction webs that span hundreds to thousands of kilometers and connect production from oceanic phytoplankton to desert cacti and coyotes via zooplankton, fishes, and sea birds. The possible role of DMS in global heat budgets expands this effect even further. The ecosystem-wide and potentially global consequences of aquatic chemical cues is an underappreciated topic that warrants additional attention.

Fitness consequences for copepods feeding on a red tide dinoflagellate: deciphering the effects of nutritional value, toxicity, and feeding behavior

Phytoplankton exhibit a diversity of morphologies, nutritional values, and potential chemical defenses that could affect the feeding and fitness of zooplankton consumers. However, how phytoplankton traits shape plant–herbivore interactions in the marine plankton is not as well understood as for terrestrial or marine macrophytes and their grazers. The occurrence of blooms of marine dinoflagellates such as Karenia brevis suggests that, for uncertain reasons, grazers are unable to capitalize on, or control, this phytoplankton growth—making these systems appealing for testing mechanisms of grazing deterrence. Using the sympatric copepod Acartia tonsa, we conducted a mixed diet feeding experiment to test whether K. brevis is beneficial, toxic, nutritionally inadequate, or behaviorally rejected as food relative to the palatable and nutritionally adequate phytoplankter Rhodomonas lens. On diets rich in K. brevis, copepods experienced decreased survivorship and decreased egg production per female, but the percentage of eggs that hatched was unaffected. Although copepods showed a 6–17% preference for R. lens over K. brevis on some mixed diets, overall high ingestion rates eliminated the possibility that reduced copepod fitness was caused by copepods avoiding K. brevis, leaving nutritional inadequacy and toxicity as remaining hypotheses. Because egg production was dependent on the amount of R. lens consumed regardless of the amount of K. brevis eaten, there was no evidence that fitness costs were caused by K. brevis toxicity. Copepods limited to K. brevis ate 480% as much as those fed only R. lens, suggesting that copepods attempted to compensate for low food quality with increased quantity ingested. Our results indicate that K. brevis is a poor food for A. tonsa, probably due to nutritional inadequacy rather than toxicity, which could affect bloom dynamics in the Gulf of Mexico where these species co-occur.

Cytotoxic alkaloids from the flatworm Prostheceraeus villatus and its tunicate prey Clavelina lepadiformis

Abstract Four novel alkaloids, lepadins B ( 2 ) and C ( 3 ) and villatamines A ( 4 ) and B ( 5 ), have been isolated from the predatory flatworm Prostheceraeus villatus and its tunicate prey Clavelina lepadiformis. Lepadins A ( 1 ) and B ( 2 ) and villatamine B ( 5 ) exhibit significant in vitro cytotoxicity against human cancer cell lines.

Intraspecific variation in palatability and defensive chemistry of brown seaweeds: effects on herbivore fitness

When offered a choice between brown seaweeds (Phaeophyta) from shallow inshore populations versus deeper offshore populations along the mid-Atlantic coast of the United States of America, the herbivorous amphipod Ampithoe longimana consistently preferred plants from the inshore populations. This was the case for three species (Dictyota menstrualis, Spatoglossum schroederi, and Sargassum filipendula) collected from each of a single inshore and offshore site, and for one species (D. menstrualis) collected from each of three inshore and three offshore sites. Bioassay-guided fractionation of chemical crude extracts from D. menstrualis suggested that the relative unpalatability of the offshore plants was due to the lipid-soluble secondary metabolites 4β-hydroxydictyodial A and 18,O-dihydro-4β-hydroxydictyodial A 18-acetate, along with minor compounds that were not fully identified. The inshore-offshore pattern did not appear to result from induction of defenses due to herbivory by mesograzers, as mesograzer densities were higher on the more palatable inshore plants. Herbivore feeding preferences for inshore versus offshore seaweeds matched the effects of those seaweeds on their fitness. When juvenile amphipods were raised on inshore versus offshore tissues of D. menstrualis, amphipod survivorship, growth, and ovulation were significantly suppressed on the offshore compared to the inshore tissues. Few previous investigations have studied intraspecific variance in seaweed palatability. We extend these by showing that between-population differences in palatability can persist for several years and by demonstrating that this variance is chemically based and has dramatic effects on herbivore fitness.

Metabolomics and proteomics reveal impacts of chemically mediated competition on marine plankton

Significance Microscopic marine algae (phytoplankton) are responsible for much of Earths photosynthesis, serving as the base of a massive food web supporting fisheries. Phytoplankton compete for limiting resources, with some species producing noxious compounds that kill competitors or inhibit their growth. The red-tide dinoflagellate Karenia brevis is one such allelopathic species, causing growth suppression of other phytoplankton and negatively impacting coastal ecosystems. Metabolomic and proteomic approaches were used to characterize the sublethal physiological impacts of K. brevis allelopathy on two competing phytoplankton, providing insights into the physiological mechanisms by which allelopathy occurs and the metabolic pathways that enable resistance in co-occurring competitors. Competition is a major force structuring marine planktonic communities. The release of compounds that inhibit competitors, a process known as allelopathy, may play a role in the maintenance of large blooms of the red-tide dinoflagellate Karenia brevis, which produces potent neurotoxins that negatively impact coastal marine ecosystems. K. brevis is variably allelopathic to multiple competitors, typically causing sublethal suppression of growth. We used metabolomic and proteomic analyses to investigate the role of chemically mediated ecological interactions between K. brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. The impact of K. brevis allelopathy on competitor physiology was reflected in the metabolomes and expressed proteomes of both diatoms, although the diatom that co-occurs with K. brevis blooms (A. glacialis) exhibited more robust metabolism in response to K. brevis. The observed partial resistance of A. glacialis to allelopathy may be a result of its frequent exposure to K. brevis blooms in the Gulf of Mexico. For the more sensitive diatom, T. pseudonana, which may not have had opportunity to evolve resistance to K. brevis, allelopathy disrupted energy metabolism and impeded cellular protection mechanisms including altered cell membrane components, inhibited osmoregulation, and increased oxidative stress. Allelopathic compounds appear to target multiple physiological pathways in sensitive competitors, demonstrating that chemical cues in the plankton have the potential to alter large-scale ecosystem processes including primary production and nutrient cycling.

High content live cell imaging for the discovery of new antimalarial marine natural products

BackgroundThe human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum.MethodsA high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope.ResultsImage analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasites phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts.ConclusionCollectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials.