Marine chemical ecology in benthic environments

Melany P. Puglisi *a, Jennifer M. Sneed b, Raphael Ritson-Williams c and Ryan Young d
aChicago State University, Department of Pharmaceutical Sciences, Chicago, IL, USA. E-mail: mpuglisi@csu.edu
bSmithsonian Marine Station at Fort Pierce, Fort Pierce, FL, USA
cUniversity of Hawaii at Manoa, Biology Department, Honolulu, HI, USA
dNational University of Ireland, Galway, Ireland

Received 9th July 2018

First published on 28th September 2018


Covering: Most of 2013 up to the end of 2015

This review highlights the 2013–2015 marine chemical ecology literature for benthic bacteria and cyanobacteria, macroalgae, sponges, cnidarians, molluscs, other benthic invertebrates, and fish.


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Melany P. Puglisi

Melany P. Puglisi is an associate professor at Chicago State University. She received her BS from Southampton College, LIU in 1991 in chemistry, her MS in biology from the University of Guam in 1995, and her PhD in pharmacognosy from the University of Mississippi. Melany's postdoctoral research at Scripps Institution of Oceanography and the Smithsonian Marine Station at Fort Pierce focused on marine microbial chemical ecology. Chicago State University is a small teaching institution that provides opportunities to underserved populations. In 2016, she joined the Editorial Board of the Journal of Natural Products as the Book Review Editor. She is a co-editor of the book Chemical Ecology: The Ecological Impacts of Marine Natural Products, CRC Press. Her research interests involve the chemical interactions between benthic marine organisms and microorganisms.

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Jennifer M. Sneed

Jennifer Sneed is a research biologist at the Smithsonian Marine Station at Fort Pierce studying chemically mediated interactions between organisms in the marine environment. She specializes in understanding the roles that marine microbes play in the ecological interactions of other organisms. She has a MS in biology from the University of South Florida and a PhD in analytical chemistry from the Friedrich Schiller University in Jena, Germany. Jennifer began working at the Smithsonian Marine Station as a Postdoctoral Fellow in 2011 and was hired on as a full time research biologist in the fall of 2012.

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Raphael Ritson-Williams

Raphael is a Ph.D student in Zoology at the University of Hawai'i at Manoa, with a specialization in ecology, evolution and conservation biology. In 2002 he completed a M.S. degree from the University of Guam with a focus on marine chemical ecology. He worked for 10 years at the Smithsonian Marine Station at Fort Pierce studying a variety of organisms to better understand marine chemical ecology and coral reef recovery. He is currently researching the characteristics of marine habitats that confer resilience.

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Ryan Young

Ryan Young studied chemistry at Rhodes University, South Africa where he received his BSc (Hons) in 2007. He remained there for his PhD under the supervision of Prof Michael Davies-Coleman and received his PhD in 2011 for his work on antiplasmodial and antibiotic natural products. In 2012, Ryan moved to the University of South Florida to work with Prof. Bill Baker on chemical ecology of Antarctic marine organisms and disease vectors in Latin America and West Africa. Currently Ryan in employed at the National University of Ireland, Galway researching marine biodiscovery of Irish deep-sea marine organisms with Dr Louise Allcock. Ryan's research interests include chemical ecology of marine invertebrates, isolation, structure elucidation of new natural compounds in addition to semi-synthesis, med-chem and SAR studies.


1 Introduction

In this report, we review the literature of the chemical ecology of microorganisms, algae, marine invertebrates, and fish from benthic communities for the years between 2013 and 2015. Since our last review,1 the field has continued to expand with the discovery of novel metabolites2 and the development of new technologies.3–6 In this time period, a number of reviews were published. Pawlik et al.3 provided a historical perspective of marine chemical ecology and the impact of Scuba and new marine technologies this field. Kuhlisch and Pohnert4 included a section in their review to highlight the application of comparative metabolomics in marine chemical ecology. Chandramouli et al.5 reviewed the application of proteomics in the understanding of signaling pathways that affect larval attachment and metamorphosis. Heuschele and Selander6 covered the new technologies in development for the identification of molecules involved in chemosensing in copepods. For a comprehensive review of the chemical ecology of Antarctic benthic communities see Núñez-Pons and Avila.7 Schwartz et al.8 reviewed the chemical ecology of the marine plankton. Dobretsov et al.9 covered the literature on biofouling inhibition by heterotrophic microorganisms for the period 2006–2012. Egan et al.10 included a section on the effect of secondary metabolites from macroalgae on the colonization of epiphytic bacteria in their review of seaweed–bacteria interactions. Lopanik11 and Flórez et al.12 covered symbiotic microbial chemical defenses in marine invertebrates while Schmidt13 specifically focused on the symbiotic chemical defenses of ascidians. Additional reviews in this time period are more detailed covering a narrow area in marine chemical ecology. The chemical defenses and biosynthesis of secondary metabolite in cephalopods and the gastropod Dicathais orbita have been reviewed by Derby14 and Benkendorff,15 respectively. Saxitoxin and other marine neurotoxins were reviewed by Cusick and Sayler,16 da Gama et al.17 review mechanisms of antifouling by macroalgae.

The September 2015 issue of the Journal of Integrative and Comparative Biology included papers from the symposium “Chemicals that organize ecology: Toward a greater integration of chemoreception, organismal biology, and chemical ecology”. Two symposia concerning marine chemical ecology address the release of the allelopathic compounds dimethylsulfoniopropionate, dopamine and reactive oxygen species by green tides18 and the pharmacology of taste receptors and recognition of chemical defenses by consumers.19

2 Microorganisms

2.1 Bacteria

Biofilm bacteria produce chemical cues that induce settlement in a variety of invertebrate larvae. Two recent studies identified marine bacteria that induce settlement of Caribbean coral larvae. Sharp et al.20 tested three marine bacterial strains isolated from Symbiodinium spp. cultures for their ability to induce coral larval settlement. One strain, Roseivivax sp. 46E8, significantly increased settlement of Porites astreoides larvae during the logarithmic phase of the bacterium's growth. Roseivivax sp. belongs to a group of α-proteobacteria that are consistently found associated with corals. Cell-free filtrates of log phase cultures of Roseivivax sp. also induced settlement indicating the presence of an extracellular cue exuded by the bacterium. However, organic and aqueous extracts of the bacterial cells and cell-free filtrates of Roseovivax sp. had no effect on larval settlement. While the exact nature of the settlement cue has yet to be identified, the authors suggest that small nucleic acid-containing particles found in Roseivivax sp. cell-free filtrates may play a role in the settlement process.
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Another study focused on the role of bacteria in the settlement behavior of Caribbean corals demonstrated that Porites astreoides larvae will settle in response to natural biofilms.21 This response was eliminated when biofilms were treated with broad-spectrum antibiotics indicating that the production of an active settlement cue by a biofilm bacterium. To determine the source of the settlement cue, sixteen bacterial strains were isolated from the surfaces of crustose coralline algae and tested for their effects on P. astreoides settlement. Only one strain (Pseudoalteromonas sp. PS5) was found to induce settlement. Crude organic extracts of Pseudoalteromonas sp. cells also induced settlement and through bioassay-guided fractionation, a single compound, tetrabromopyrrole (TBP) 1, was identified as the active metabolite. TBP also induced settlement in two other Caribbean coral species, Orbicella franksi and Acropora palmata. The three coral species tested represent a diverse array of coral taxa and life history strategies. That the bacterially produced compound TBP induces complete settlement in all of the Caribbean coral species tested suggests a potentially important role for this compound in a wide variety of Caribbean corals.

The marine bacterium Bacillus amyloliquefaciens isolated from the octocoral Muriceopsis bayeriana chemically inhibited the growth of 7 of 8 terrestrial fungal strains tested and 4 of 5 marine fungal strains tested by secreting metabolites in the presence of the fungal strains.22 Using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), the authors were able to visualize the chemical interactions between the living bacterium and fungus on agar plates. They observed differential distribution patterns of compounds produced by the fungal colonies with some being present near the center of the colony and others secreted into the media. Detection of fungal metabolites was reduced at the margin of interaction with the bacterium indicating a possible consumption or secretion inhibition at the interaction site. A set of ions with m/z 1066, 1080, 1094 and 1108 were consistently detected surrounding the B. amyloliquefaciens colonies and were also found in the n-butanol extract of the bacterium. Combining the MALDI-IMS with MS/MS network analysis the ions were identified as belonging to the iturin family of compounds previously reported to exhibit antifungal activity. Based on bioassay guided fractionation, iturin (C11) 2, iturin (C12) 3, and iturin (C13) 4 were identified as the active antifungal agents in this interaction. This direct visualization of the compounds surrounding the bacterium and the fungus independently and in co-culture, in combination with MS/MS networking is a unique tool for investigating chemical interactions between culturable microbes.

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2.2 Cyanobacteria


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Soares et al.23 report the isolation and characterization of a novel metabolite and a known compound with antimicrobial activities from a mixed assemblage of filamentous cyanobacteria collected from Carrie Bow Cay, Belize. DNA analysis of the cyanobacterial mat revealed that the mat was composed of two strains, BCBC12-12.1 with phenotype similarities to Lyngbya majuscula and BCBC12-12.2 with phenotype similarities to the genus Hormoscilla. Bioassay-guided fractionation using the three fungal strains Dendryphiella salina, Fusarium sp. and Lindra thalassiae yielded carriebowlinol (5) and lyngbic acid (6). Both compounds inhibited the growth of all three fungal strains well below natural concentration; compound 5 (IC50 = 0.2–0.5 μM, natural concentration = 1.0 μM) was significantly more active than compound 6 (IC50 = 2.1–2.9 μM, natural concentration = 4.0 μM) in these assays. 5 also exhibited broad antibacterial activity against marine bacteria. The authors suggest that 5 and 6 serve as antimicrobial defenses in the cyanobacterial assemblage.

2.3 Fungi

Only a few studies of the chemical ecology of marine fungi have been published since our last review. In 2014, Martín-Rodríguez et al.24 published the first report of quorum-sensing (QS) inhibition by marine endophytic fungi isolated from a sponge and corals. The reporter strain Chromobacterium violaceum CVO26 was employed to screen from pure endophytic fungi cultures. Four active strains were identified: Sarocladium sp. (LAEE06) isolated from the sponge Agelas sp.; Fusarium sp. (LAEE13) isolated from the coral Pseudodiploria (previously Diploria) strigosa; Epicoccum sp. (LAEE14) also isolated from D. strigosa; and Khuskia sp. (LAEE21) isolated from the coral Plexaura flexuosa. Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) analysis showed the presence of known metabolites and potentially new compounds within the extracts. The major compounds in the chemical profiles were fusaric acid, trichosetin, beauvericin, and verrucarin B. The authors suggest that the complex mixture of fungal metabolites and fatty acids may contribute to the activity of the extracts.
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The fungus Penicillium sp. SCSGAF associated with the gorgonian coral Dichotella gemmacea was reported to significantly inhibit two bacterial strains and larval settlement of Balanus amphitrite.25 Further investigation yielded two new polyketides, 6,8,5′6′-tetrahydroxy-3′-methylflavone (7) and paecilin C (8) and six known analogs: secalonic acid D (9), secalonic acid B (10), penicillixanthone A (11), emodin (12), citreorosein (13) and isorhodoptilometrin (14) with different biological activities. Secalonic acid D (6), the major metabolite in the extract, and compounds 10 and 11 inhibited the growth of Micrococcus luteus UST950701-006, a larval settlement inducing strain, and the marine pathogen Pseudoalteromonas nigrifaciens UST010620-005. Metabolites 7, 12–14 significantly inhibited settlement of Balanus amphitrite with EC50 values of 6.7, 6.1, 17.9 and 13.7 μg mL−1.

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2.4 Microalgae

Using a metabolomics approach, Gillard et al.26 characterized the first known diatom sex pheromone in the benthic pennate diatom Seminavis robusta. Migrating mating type cell are designated as MT+. These motile cells move toward a group of attracting cells designated as MT during reproduction. This activity increased in the presence of medium from mating cultures of S. robusta. The presence of an extractable pheromone within the mating culture media was confirmed by passing the media over hydrophilic/lipophilic-balanced solid-phase extraction (HLB-SPE) cartridges and exposing the absorbent beads to MT+ cells. In order to identify the pheromone responsible for attracting the MT+ cells, the MT cultures exposed to MT+ media were compared to that of MT cultures not exposed to MT+ media using a metabolomics approach. One metabolite was significantly up-regulated in attractive MT media and was identified as L-diproline (15). When loaded onto SPE beads, L-diproline caused significant attraction of MT+ cells at concentrations ranging from 2 pmol mg−1 beads to 200 nmol mg−1 beads. The enantiomer D-diproline exhibited similar activity to 12 but is not naturally produced by the diatom.
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3 Macroalgae

3.1 Chemical defenses of macroalgae


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Chemical defenses of macroalgae against herbivores continues to be an active area of interest. Crude extracts and solvent partition fractions from the brown alga Sargassum tenerrimum were tested in laboratory and field feeding assays with fish from the same region as the algae collections.27 In both sets of assays, fish preferred the control over the crude extract, hexane partition and ethyl acetate partition. Chemical fractionation of the hexane and ethyl acetate partition yielded fucosterol, stigmast-5,24-dien-3-ol (16) as the major metabolite. In additional laboratory and field assays 16 deterred feeding at natural concentration at rates similar to the hexane partition.

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Phlorotannins from the brown alga Eisenia bicyclis and bromophenols, major components of extracellular secretions, from Eisenia and Ecklonia species, were investigated for their feeding deterrence activity against Turbo cornutus in laboratory experiments.28 Phloroglucinol (17), eckol (18), fucofuroeckol A (19), phlorofucofuroeckol A (20), dieckol (21), and 8,8′-bieckol (22) were incorporated into an artificial food composed of dextran and agar at natural concentrations. Compounds 19 and 20 significantly reduced feeding by T. cornutus at the concentration of 0.1 mM, however, 17 and 22 did not deter feeding behavior. Surprisingly, 18 and 21 stimulated the feeding activity at the concentration of 0.1 mM. Commercially obtained 2,4-dibromophenol and 2,4,6-tribromophenol were also investigated for their effect on feeding by T. cornutus at concentrations ranging from 1 μM to 1 pM. At 1 μM 2,4-dibromophenol significantly inhibited feeding while low concentrations stimulated feeding. 2,4,6-tribromophenol did not have an effect on feeding by T. cornutus.

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In previous studies of Ulvaria obscura from the North Pacific, algal exudates have been shown to exhibit a variety of biological activities against marine invertebrate and algae. These exudates contain high concentrations of dopamine (23) at 500 nM or higher.29 A recent study of 23 sought to determine if the biological activities observed for the U. obscura exudates against co-occurring macroalgae, crab larvae and juveniles were caused by the high concentrations of 23 in the exudate. A diverse set of assays were conducted with U. lactuca, the zygotes of Fucus distichus and the larvae and juveniles of the crabs Metacarcinus magister and Cancer oregonensis. Metabolite 23 effectively inhibited germination of F. distichus and U. lactuca growth at 5 nM and 50 nM, respectively. The survival rate of M. zoeae dropped significantly when exposed to 168 nM concentrations of 23, however, C. megalopae juveniles or megalopae were not affected. The authors propose that dopamine released into the sea water around U. obscura will regulate the co-occurring species around larger blooms.

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Several recent studies addressed the geographic variability of chemical defenses against herbivores. The marine amphipod Ampithoe longimana, a mesograzer, has been shown to prefer algae in the genus Dictyota over other possible food sources. In a recent study, A. longimana from subtropical Florida, warm-temperate North Carolina and cold-temperate New England regions were collected three times over a one-year period to determine the abundance of the amphipod in common species of algae at each site and determine if there was a preferred host for A. longimana.30A. longimana was abundant in the algae collected in the summer months at all locations, however populations were greatest in early summer in the North Carolina and Florida and late summer in Rhode Island. Lyophilized samples of Acanthophora, Ectocarpus, Gracilaria and Hincksia/Feldmannia spp. did not inhibit feeding nor were they preferred by amphipods, however geographic variation in feeding preference was reported for D. menstrualis, D. ciliolata, F. distichus, C. crispus and P. gymnospora. Populations of amphipods that live in a chemically defended seaweed preferred that species over others. In assays with the lipophilic extracts from D. ciliolate, local population of amphipods consumed more of the lipophilic extracts compared to foreign populations. The authors propose that the results from this study demonstrate that amphipod populations, known to be generalist herbivores, can locally adapt to their hosts and evolve a preference for algae with chemical defenses.

The palatability of crude extracts from common, chemically defended macroalgae from the Caribbean was tested against sea urchins collected from the sub-tropical coast of Florida, and temperate coasts of Massachusetts and Santa Barbara to determine if tropical herbivores develop a higher tolerance to chemical defenses found in tropical algae.31 Lipophilic extracts from Caulerpa sertularioides, Halimeda tuna, H. discoidea, Penicillus dumetosus, Dictyota ciliolata, D. pulchella, Spatoglossum schroederi, Stypopodium zonale and Palisada poiteaui were offered to four subtropical and three cold-temperate sea urchins in two sets of feeding assays. Feeding resistance patterns were generated using the Herbivore Resistance Scale. Extracts from C. sertularioides, D. ciliolate and D. pulchella deterred feeding by all sea urchins. Subtropical species were more likely to consume algal extracts compared to urchins collected from the temperate regions. Arbacia punctulata collected from Florida were less deterred by the extracts from Dictyota spp. and S. zonale extracts compared to A. punctulata collected from Massachusetts suggesting that exposure to the algae has allowed the sub-tropical population to adapt to the chemical defenses. The authors suggest that the evolutionary history of algae–herbivore interactions appear to shape the feeding patterns of generalist herbivores.

A study of Plocamium brasiliense addressed geographic variation of chemical defenses against the sea urchin Lytechinus variegatus and the crab Acanthonyx scutiformis.32 Samples of Plocamium brasiliense were collected from two sites approximately 30 km apart, Enseada do Forno and Praia Rasa, Búzios, Rio de Janeiro, Brazil. Crude extracts from both collections were compared by gas chromatography coupled with mass spectrometry (HRGCMS). The GCMS profile from P. brasiliense collected in Praia Rasa contained one major compound representing about 59% of the total extract content. Extracts from both sites were rich in halogenated monoterpenes. In paired assays with control foods Lytechinus variegatus avoided the crude extracts from both sites. However, when the crude extract from Enseada do Forno was tested in paired assays with the crude extract from Praia Rasa, L. variegatus consumed the 45% of the Enseada do Forno extract and only about 10% of the Praia Rasa extract. The authors hypothesize that the difference in feeding activity by the sea urchin is due to the presence of the major metabolite found in the Praia Rasa extract.

Another study of the chemical defenses produced by algae in the genus Plocamium investigated the relationship between the amphipod Paradexamine fissicauda and the red algae P. cartilagineum and Picconiella plumosa from Antartica.33P. fissicauda and five other species of amphipods were fed a diet of P. cartilagineum in no choice assays. Only P. fissicauda consumed measurable quantities of P. cartilagineum. In no choice feeding assays, the amphipod preferred Palmaria decipiens, Plocamium cartilagineum and Picconiella plumosa over the other species in all three trials. In the four choice assays that followed, the amphipod preferred P. cartilagineum and P. plumosa over the others. To test if P. fissicauda could sequester chemical defenses from P. cartilagineum, the fish Nototenia fissicauda was offered amphipods fed on a diet of P. cartilagineum against those fed on a diet of the undefended red alga P. plumosa, or Bovallia gigantia (control). Amphipods that were fed P. cartilagineum for 2 months were unpalatable to the fish while B. gigantia and amphipods fed on P. plumosa were readily consumed. Gas chromatography mass spectrometry analysis (GCMS) of the P. fissicauda extracts showed the presence of halogenated secondary metabolites produced by P. cartilagineum that were absent in the extract prepared from the amphipods that consumed P. plumosa. The authors conclude that P. fissicauda is sequestering chemical defenses from P. cartilagineum and may use them for their own defense.

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Herbivore specialists are rare in marine ecosystems. Elysia tuca is a specialist sea slug that lives on several species of algae including Halimeda incrassata.34 Field observation and laboratory colonization experiments with live algal or seagrass pieces demonstrated that E. tuca colonizes H. incrassata at a significantly higher rate than other Halimeda species. Additional experiments using cotton balls conditioned with the artificial seawater soaked with seaweeds demonstrated that E. tuca colonized cotton balls treated with H. incrassata at a significantly higher rate than other Halimeda species. Bioassay-guided fractionation yielded 4-hydroxybenzoic acid (24) as the foraging cue for E. tuca to its prey H. incrassata. Paired field experiments with 6 × 6 cloth squares soaked in either 24 or the solvent control in stands of H. incrassata for 24 hours showed that E. tuca were able to track 24 and preferentially settled on the adjacent algae. Observations in the field showed that Elysia were more abundant on reproductive Halimeda. Bioassay-guided fractionation of reproductive specimens yielded halimedatetraacetate (25), a known deterrent sequestered by E. tuca, as the chemical cue used by the sea slug to find the reproductive seaweed. Further field experiments showed that Halimeda growth was reduce by ∼50% during periods of heavy feeding by the sea slug. The authors also observed that Halimeda drops branches with Elysia. They propose that this is a defense that not only removes the herbivore but may also serve to reduce the possibility of fungal infection.

Another study of green algae in the genus Halimeda addressed the effect of ocean acidification on the production of chemical defenses against generalist herbivores by exposing the alga to different levels of the partial pressure of CO2 (pCO2) (300 to 2400 μatm) in laboratory experiments.35 Of the three species, only the net CaCO3 content (% dry wt) of H. incrassata decreased with increasing pCO2 levels. There was no measurable effect on the other two species. The concentrations of the major terpene halimedatetraacetate in the new growth of H. opuntia, H. incrassata and H. simulans at the end of the experiment were not changed by different pCO2 concentrations. Feedings assays incorporating CaCO3 and secondary metabolites at measured concentrations demonstrated that feeding by the common sea urchins Lytechinus variegatus and Diadema antillarum increased on diets with lower concentrations of CaCO3 and secondary metabolites. The authors suggest that changes in CaCO3 and secondary metabolite composition in some algal species affected by ocean acidification have the potential to weaken herbivore defenses.

3.2 Inducible defenses

Rasher and Hay36 reported the first experimental evidence of the induction of allelochemicals in a seaweed. This study investigated the induction of allelopathic defenses of the palatable seaweed Sargassum polycystum and the unpalatable seaweed Galaxaura filamentosum in the presence of its coral competitor Porites cylindrica and the effects of the interaction with algae and the coral on the growth and production of chemical defenses of the seaweed. Harvested corals were placed in contact with individual S. polycystum or G. filamentosum and deployed into the field. One half of the harvested coral branches were bleached for prior to attachment to the metal rack as controls. Prior to chemical extraction, four branches of each control and treated alga were removed, suspended on ropes and offered to natural assemblages of fishes. Hydrophobic extracts were prepared from the control and treated specimens and incorporated into Phytagel for allelopathy assays. These were attached to healthy P. cylindrica branches. Extracts were also coated on Padina boryana and Amphiroa crassa for feeding assays. Results of this sophisticated set of experiments demonstrated that allelochemicals produced by G. filamentosa were induced over the 8 days of the experiment, causing damage to the corals at twice the rate than the controls, at the cost of reduced growth and a reduction of chemical defenses to herbivores. These same effects or changes were not observed for S. polycystum.

3.3 Antimicrobial and antifouling defenses of marine algae

Antifouling defenses continue to be of interest for macroalgae in shallow water benthic communities. Rickert et al.37 conducted a year long, seasonal study of macrofouling and the role of chemical defense for the brown algae Fucus vesiculosus and F. serratus using a novel field assay. Epiphyte coverage was estimated each month by (1) screening a single thallus from each Fucus seaweed and a control, rough-surfaced PVC pipe exposed at the same site, for small fouling organisms under the microscope and (2) recording the settlement of common fouling organisms Amphibalanus improvisus and Mytilus edulis on these surfaces. Surface extracts of clean, non-fouled Fucus spp. were incorporated into a cellulose filter paper and covered with a polycarbonate filter membrane to better approximate the surface conditions of the alga. Once assembled, extracts were deployed into the field for five days. Seasonal variability was reported for micro- and macro-fouling patterns and the antifouling activity of surface extracts from both Fucus species. F. vesiculosus extracts prepared from samples collected in the spring and summer months deterred settlement of A. improvises, while F. serratus extracts did not exhibit anti-fouling activity against A. improvisus or M. edulis. The authors conclude that the variability observed is likely mediated by other metabolites or mechanical properties of Fucus spp.

To better define the roles of algal metabolites several studies examined the activity and composition of surface extracts. Using 454 pyrosequencing, Lachnit et al.38 determined that surface extracts from Fucus vesiculosus significantly reduced the attachment of Pseudomonas sp. isolated from the marine environment. The nonpolar fraction of the surface extracts from F. vesiculosus was separated into 8 fractions for antibacterial assays against Pseudomonas carregenovora, Pseudomonas sp., Zooshkiella sp., Bacillus aquimaris and B. subtilis. Two of these 8 fractions were then examined for activity in bacterial attachment assays with the same panel of microorganisms under laboratory conditions, followed by a 3 day field experiment to assess these same fractions, along with the aqueous crude extract partition for antibacterial properties. Vibrionales made up the largest proportion of microorganisms on the surface treated with the polar fraction. Surfaces coated with the nonpolar fraction had an 80% reduction of surface colonizing bacteria. Fractions containing the xanthophyll pigment fucoxanthin and other unidentified nonpolar surface compounds caused a significant 80% reduction of surface colonizing bacteria. Bioassay-guided fractionation of the active surface fraction with Pseudomonas sp. yielded the pigment fucoxanthin. The authors conclude that the composition of the surface bacteria is mediated by surface associated metabolites.

Bonnemaisonia hamifera, an invasive species in Scandinavian waters, has been shown to produce the herbivore deterrent compound 1,1,3,3-tetrabromo-2-heptanone. A study by Svensson et al.39 continued the investigation of the whole alga and this ecologically active metabolite to determine their allelopathic effects on native seaweeds and the benthic diatom Cylindrotheca fusiformis in a series of laboratory assays. Whole-seaweed assays were conducted by tying B. hamifera with one of the five seaweeds, Ceramium virgatum, Cystoclonium purpureum, Polysiphonia stricta, Pilayella littoralis, and Sphacelaria cirrosa, and placing the pairs in individual containers for 13 days. Controls consisted of identical pairs of algae tied together. Settlement assays were conducted with 1,1,3,3-tetrabromo-2-heptanone against C. fusiformis, spores of C. virgatum and P. stricta, and gametes of Ulva lactuca at eight concentrations (the highest at the natural concentration). A set of “coating experiments” by placing B. hamifera 1.5 cm away from thalli of native species on a plastic strip and submerging them in laboratory tanks or placing them in the field for one week to determine if 1,1,3,3-tetrabromo-2-heptanone was released from the invasive alga and transferred to the native species. Results from the co-culture experiments did not show an effect of the invasive species. However, 1,1,3,3-tetrabromo-2-heptanone inhibited the settlement of macroalgal propagules and microalgae at ecologically relevant concentrations. Further chemical investigation of hexanes extracts following the coating experiments demonstrated that the active compound was transferred from B. hamifera to the surface of the native algae in both laboratory and field experiments. The authors purport that this is the first demonstration of the transfer of an active metabolite from an invasive species and this is likely to contribute to the success of B. hamifera in Scandinavian waters.

3.4 Algal-coral interactions

To date, studies concerning the production of coral larval chemical cues from crustose coralline algae (CCA) and their associated bacteria are usually limited to only a few species of corals. In 2015, Tebben et al.40 reported the findings of a comprehensive study of larval settlement experiments conducted with 11 hard coral species from Australia, Guam, Singapore and Japan. They sought to determine the role of chemical cues from surface associated bacteria as well as CCA. In standard laboratory settlement assays, coral larvae did not settle on the bacterial biofilms associated with CCA. However, metamorphosis without attachment was induced in coral larvae by Pseudoalteromonas spp. in corals from all geographical regions. Bioassay-guided fractionation of the bacterial extracts yielded the known metabolite, tetrabromopyrrole (1), as the active metabolite. In contrast, experiments with live CCA and their crude extracts induced larval settlement. Glycoglycerolipids and polysaccharides were identified as the major classes of compounds in active fractions. These settlement cues appear to be of global significance in the settlement and metamorphosis of coral larvae.

4 Seagrass

Labyrinthula, the cause of wasting disease outbreaks in temperate and tropical seagrass beds, continues to be a concern. Trevathan-Tackett et al.41 conducted in vitro assays with the known phenolic compounds, vanillin (26), p-coumaric acid (27), 4-hydroxybenzoic acid (24), and 3,4-dihydroxybenzoic acid (28), and the flavone glycoside thalassiolin B (29) from Thalassia testudinum. Compounds were assayed individually against Labyrinthula at six concentrations to determine an IC50. Subsequent assays were conducted with a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 ratio of two compounds, each at the IC50 determined in the previous assays. Of the four phenolic compounds, 27 exhibited the most potent antimicrobial activity and 26 was the least potent, however all four were active below natural concentrations. The flavone glycoside 29 was reported to be significantly more active than the other four compounds in the study. Mild synergism was observed when combining 24 with 27. Bioassay-guided fractionation of T. testudinum yielded one active fraction containing an unrelated compound. The authors suggest that T. testudinum produces metabolites that may prevent infection by Labyrinthula.
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Building on previous studies that showed phenolic acid and condensed tannin in seagrasses deter herbivores, Steele and Valentine42 exposed wounded seagrass plants to grazing by Crepidula ustulatulina and Paracerceis caudata in laboratory induction assays followed by a second set of experiments to test for feeding deterrence in which the mesograzers were offered powdered seagrass tissue in agar in paired choice experiments. Gallic acid, ferulic acid and compounds 24, 26–28 were found in T. testudinum and H. wrightii extracts. H. wrightii also sporadically contained gentistic acid. After 15 days, overall phenolic compound concentrations decrease by 40–50% and tannin concentrations decrease by 20% in T. testudinum exposed the gastropods and isopods. However, the concentrations of 24 and 27 increased (25 to 85%) in tissues damaged by C. ustulatulina and P. caudata. In contrast, isopod feeding increased the concentrations of some shoalgrass phenolic acids by ∼30 to 50%, while gastropod grazing led to ∼25 to 50% higher concentrations of condensed tannins in shoalgrass leaves, suggesting that grazer identity and seagrass species play important roles in seagrass deterrent production. Amphipods (Batea catharinensis) consistently preferred agar food made from seagrass leaves with low phenolic concentrations in choice feeding experiments.

Iyapparaj et al.43 investigated the effects of extracts from Syringodium isoetifolium and Cymodocea serrulata for antibacterial, anti-microalgal, and antimacrofouling (including mollusc foot adhering, mussel, and crustacean) activities. Methanolic extracts of S. isoetifolium and C. serrulata possessed inhibitory activity against biofilm forming bacteria and microalgal strains at concentrations between 1.0 to 10 mg mL−1. The methanolic extracts also inhibited fouling by the limpet Patella vulgata at 6.0 mg mL−1. In contrast, the methanolic extracts were active at μg concentrations against the mussel Perna indica and the crustaecean Artemia salina. The authors suggest that the potential for development of antifouling agents using these seagrasses is growing and is opening promising and eco-friendly opportunities.

The role of volatile organic compounds in chemoreception by associated invertebrates and the effects of pH changes caused by ocean acidification in the concentration of these compounds was investigated for the seagrass Posidonia oceanica.44 Experiments were conducted at normal pH 8.1 and an acidified pH 7.7 relative to environmental pH of 8.1. While the majority of invertebrates recognized the volatile organic compounds signal at pH 8.1, there were changes in reactions in 54% of invertebrates in an acidified environment. The authors purport that this data suggests that changes in environmental CO2 does, in fact, skew the choices of invertebrates and other organisms coexisting with seagrass populations.

5 Sponges

Chemical defenses have been well characterized for sponges in the Caribbean and offer the opportunity to explore larger ecological questions. A recent study of the effects of overfishing on the assemblages of the sponges on coral reefs from 12 Caribbean countries in 2008 to 2012 was conducted to catalog sponge species as well as the type and abundance of spongivores.45 A total of 109 sponges were identified in the transects. More than 50% of the sponge cover was composed of 10 common species. Sponges known to produce secondary metabolites made up 57.5% of the sponges in transects. The most abundant chemically defended genera were Aplysina and Aiolochroia. The most abundant sponge in on the reefs in this study was Aplysina cauliformis. This sponge produces fistularin-3 (30), a defensive alkaloid that is also found in other sponges in the genera Aplysina, Aiolochroia and Verongula. In areas with high populations of spongivores, greater numbers of sponges were reported to be chemically defended. In Curaçao and Bonaire where there are small human population as well as strict marine preservation laws, reefs were rich in chemically defended species (>90% sponge population). In areas where overfishing occurs and spongivores are removed from the habitat, more than 50% of the sponge assemblages were considered palatable sponges. The presence of spongivores account for 32.8% of the differences observed between the protected and overfished areas. These data support the hypothesis that in areas of overfishing, palatable sponges which often grow faster may out compete the slower growing sponges and corals.
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Crude extracts and calcibacteria spherules from Atlanto-Mediterranean sponge Hemimycale columella were recently reported to deter feeding by the sea urchin Parablennius incognitus and the fish Paracentrotus lividus.46 Extracts, spherules, or both were incorporated into a 4% carragenate-Cystoseira mediterranea mix diet for P. incognitus and bread pellets for P. lividus. The crude extract, the calcibacteria spherules, and the combination of both did not significantly differ from one another in their deterrence effect of the sea urchin from feeding on H. columella. P. incognitus ate significantly fewer pellets with spherules compared to the crude extract and the controls. In the field experiment, Oblada melanura avoided crude extracts and spherules while Diplodus vulgaris, Chromis chromis, and Coris julis only avoided the spherule treatment. The authors suggest that sponge H. columella has more than one defense mechanism against potential predators.

5.1 Inducible defenses of sponges


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Dysidea avara from the northwest region of the Mediterranean Sea was the subject of a 23 month study from March 2009 to January 2011 that addressed concentration changes in the sesquiterpene hydroquinone avarol (31) and 5′-monoacetylavarol (32), the major metabolites reported from the sponge.47 Samples were collected monthly at depths of 10–14 m to explore the relationship of sesquiterpene production in D. avara with the type of neighbor adjacent to an individual sponge. Sponges were randomly sampled and sorted into three groups: (1) contact with invertebrates; (2) contact with algae; and (3) samples from the center of a sponge not in contact with other organisms. Extracts were prepared from the peripheral and central parts of the sponge and the concentrations of the metabolites in the extracts were determined by high performance liquid chromatography, coupled with diode array detection and electrospray ion trap tandem mass spectroscopy (LC/DAD/ESIMS) using avarol and 5′-monoacetylavarol as a standard. Compound 31 was the major compound in the extracts ranging from 2.09% to 4.83% of the sponge dry weight. Concentrations of the metabolites varied significantly each month with the highest concentrations in the spring and early summer. The highest concentrations correlated with the brooding season of D. avara. Additionally, 31 exhibited significant intra-individual variation with higher concentrations in the peripheral tissues of the sponges that were in close proximity of other sponges. Similar differences were not observed for sponges in close proximity of algae.

Induction and activation of herbivore and antimicrobial chemical defenses in response to artificial predation were studied for eight species of sponges in Guam.48 Of the eight species used in the study, the extracts from seven species were unpalatable to Canthigaster solandri before incurring physical damage. Induced defenses were observed for Stylissa massa and Melophlus sarasinorum, while M. sarasinorum also showed activated defense in response to wounding. Induced antimicrobial defenses were observed for Aplysinella sp., Cacospongia sp., M. sarasinorum, and S. massa. The authors proposed that wounding selects for induced antimicrobial defenses to protect sponges from pathogens that could invade the sponge tissue via feeding scars.

5.2 Antimicrobial and antifouling defenses of sponges


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A study of the cold-water marine sponge Geodia barretti reported antifouling activity of two new barettin derivatives.49 Settlement assays with the Balanus improvisus larvae were conducted with barrettides A (33) and B (34) at three concentrations, 0.06, 0.6 and 6 μM. Structurally, 33 and 34 contain two disulfide bonds and only differ by the substitution of leucine for valine at position 19 in 34. This substitution had a significant impact on the activity of the compound in the settlement assays. 34 was 10× more potent in inhibiting larval settlement (EC50 0.6 μM) than 34 (EC50 6 μM).

6 Cnidarians


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Historically soft corals, which lack physical or skeletal defenses, have been prolific producers of secondary metabolites. To date, there are over 1700 secondary metabolites reported in the literature originating from Cnidaria.2 Soft coral often utilize a chemical defense strategy in order to keep their surface tissue free of fouling by other sessile organisms. In a recent study of the soft coral Sinularia rigida, Lai et al.50 isolated 12 new cembranoids, sinulariols T-Z5 as well as the known compound (2E,7E)-4,11-dihydroxy-1-12-oxidocembra-4,11-diene (35). These thirteen compounds were tested for their antifouling activity against the larval settlement of the barnacle Balanus amphitrite and bryozoan Bugula neritina. All compounds were tested at 25 and 5 μg mL−1. Individual larvae were transferred to one well in a 24-well plate and exposed to the compounds for 48 hours. The larvae were examined under a dissecting microscope to be either attached, unattached or dead. 35 and sinulariol Z (36) showed the greatest dose-dependent activity with EC50 values against B. amphitrite of 4.57 and 4.86 μg mL−1 and EC50 values of 13.48 and 12.34 μg mL−1 against B. neritina, respectively.

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An additional role these secondary metabolites from soft corals play is to deter predation a recent example of this is highlighted in a study by Núñez-Pons et al.51 The lipophilic extracts of five species of Antarctic Alcyonium were evaluated for their antifeedant potential against the putative sympatric predators, the sea star Odontaster validus and the amphipod Cheirimedon femoratus. All shrimp feeding cubes treated with diethyl ether extracts of A. antarcticum, A. gradis, A. haddoni, A. paucilobularum and A. roseum were significantly rejected when fed to O. validus, suggesting these soft corals were chemically defended against predation. Further chemical analysis of A. grandis and A. roseum yielded alcyopterosins (37–49) in the illudalane class of sesquiterpenoids. 37–45 were isolated from A. grandis and 46 and 47 were isolated from A. roseum. The waxy esters 48 and 49 were common to all Alcyonium soft corals. Due to lack of material only the extracts of A. antarcticum, A. haddoni and A. roseum were tested against C. femoratus at natural concentration resulting in all three being unpalatable. A mixture of compounds 37 - 47 were tested against the predatory sea star and deemed to be highly deterrent at the natural concentration while the waxy esters 48 and 49 were only deterrent at high concentrations. The food pellets containing 48 and 49 were only significantly rejected by C. femoratus with respect to the untreated control at a concentration of 10 mg per g−1 dry weight.

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7 Ascidians (tunicates)


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There have been a number of studies since our last review towards understanding the complex and diverse secondary metabolome of ascidians. Chemotaxis is important for species recognition in fertilization of spawning invertebrates. The identification of these molecules has been very challenging often due the fact that these metabolites are active at very low concentrations in seawater. Ascidea-SAAF2 (50), a sperm activating and attracting factor, was isolated from the egg seawater of the Acidia sydneinsis using bioassay-guided fractionation via sperm mobility assays.5250 was obtained in low concentrations, therefore charge remote fragmentation patterns obtained by tandem FABMS spectrometry was used to determine the structure. Metabolite 50 is structurally related to the known sperm activating and attracting factor, Ciona-SAAF1 isolated from Ciona intestinalis.

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For sessile marine organisms, prevention of epibiont settling and overgrowth through mechanical methods or by the production of antifouling natural products is essential for survival. Recently, isolated from Synoicum pulmonaria, the bromotyrosines, synoxazolidinones A (51) and C (52), and pulmonarins A (53) and B (54) were tested for antifouling activity against a panel of 16 marine fouling species including ten bacteria, six microalgae, and one crustacean.53 Standard growth inhibition and adhesion inhibition assays were conducted with bacterial and microalgal strains in 96-well plates at four concentrations (0.01–10 μg mL−1). Settlement assays were conducted in the laboratory with the cyprid larvae of Balanus improvisus. 51 inhibited the growth of 50% of the bacterial strains and all microalgal species. Further, 51 prevented the settlement of four microalgal species. 52 exhibited growth inhibition activity against all tested organisms, as well as anti-settlement activity against B. improvises at concentrations similar to the positive control, Sea-Nine 211 (Dow). Compounds 53 and 54 inhibited the settlement and growth of the marine bacteria at low concentrations. Results from this study demonstrate that S. pulmonaria produces an array of compounds with the potential to prevent surface fouling.

Increasingly, chemistry isolated from marine invertebrates has been ascribed to symbiotic bacteria and fungi. In an effort to understand host/symbiont relationships in terms of secondary metabolite production, Kwan et al.54 investigated the tunicate Lissoclinum patella and its associated cyanobacterium, Prochloron didemni. Samples of L. patella collected over a large geographic and time range were described using 18S and mitochondrial c oxidase 1 (COX1) gene sequences, revealing individuals could be grouped into no less than three separate clades, with a number of cryptic species in each. LCMS profiling of the known cyanobactins and patellazoles in individuals confirmed the groupings in all but one case. The patellamides were limited to a divergent clade with a vertically transmitted symbiont, Candidatus Endolissoclinum faulkneri. Symbiotic P. didemni, however, exhibited very low genetic variability across hosts but nevertheless, even genetically similar P. didemni produced different secondary metabolites. Based on these findings, authors highlighted the implication that hosts may actively recruit symbionts from the environment. Further, symbiont communities in cryptic species are an untapped source of natural products.

Tianero et al.55 investigated the microbiomes and metabolomes of ascidians collected from different geographical regions. 16s rRNA analysis revealed that ascidians hosted species specific microbiomes that consisted of a few dominant bacteria that remained present across space and time. They also reported that ascidian microbiomes contained a group of less abundant bacteria that varied geographically rather than by species and likely came from the surrounding seawater. In toxicity assays against a suite of bacterial and fungal human pathogens, toxicity of ascidian extracts did not correlate with microbial diversity. However, there was species specificity in both the composition of the microbiome and the metabolome largely driven by secondary metabolites. This indicates that chemical diversity is not simply a result of a diverse microbiome within ascidians, but rather the result of a few, highly abundant, specific endosymbionts that produce potent secondary metabolites. Studies like this one that couple advanced molecular tools with metabolomics analyses are a good first step toward a better understanding of the role of host-associated microbes, which are often difficult to culture, in the production of secondary metabolites.

8 Crustaceans


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The identification of crustacean pheromones continues to be an important topic in chemical ecology. One recent study isolated and characterized the compound N-acetylglucosamino-1,5-lactone (55), a component of a pheromone mix released in urine of the blue crab, Callinectes sapidus.56 When male crabs were exposed to female urine they displayed courtship behavior including courtship paddling, grasping, standing high and spreading their chelae. Female crab urine was filtered into different sized fractions and courtship behavior was observed in both fractions smaller than <1000 Da. Through extensive HPLC and NMR analysis, 55 was isolated and structurally characterized. 55 was found in high concentrations in the premolt stage of both male and female crabs. Pure 55 was tested on 14 adult male crabs' receptor neurons and in 6 neurons 55 induced olfaction at similar intensities as food stimuli, 7 neurons only responded to food, and one did not respond to either stimuli. This compound is probably part of the molting process for blue crabs and the experiments suggest that it is also a component of a complex mixture of pheromones used for mating.

Waterborne chemical cues from predators can disrupt prey behavior. Different biomasses of blue crabs were used to test the interference of predation by a mud crab, Panopeus herbstii on its oyster prey.57 In both field and laboratory experiments oysters previously exposed to waterborne cues from a large blue crab or multiple small blue crabs were eaten by the mud crab 20–25% less than oysters exposed to a single crab or the no predator control. Indirect effects were also tested by manipulating the recent feeding history of blue crabs and measuring the effect on mud crab behavior.58 In field experiments the top predator Callinectes sapidus was fed different amounts of mud crabs and held in an enclosure that separated it from the mud crabs. Mud crabs were placed in enclosures with oyster clusters that form a refuge, with oyster spat attached to the clusters. Mud crabs were allowed to feed on the oyster spat while being exposed to blue crab chemical cues. After 24 hours mud crabs ate less oysters when blue crabs were 0.5 meters from the oyster refuges. Blue crabs that had consumed more food reduced the consumption of oysters by mud crabs up to 1 meter away from the refuges. There was no difference in the number of oysters eaten in either high or low fed crab treatments after 48 hours in the field. This study illustrates that predator cues can influence the foraging behavior of mud crabs, but these responses are dependent on predator biomass, feeding history and distance from prey.

Waterborne cues from predators are known to mediate prey behavior but different populations of prey may vary in their behavior. The dogwhelk Nucella lapillus was collected from different habitats across its range in the Gulf of Maine. Behavioral and morphological defenses were measured in response to the native crab Cancer irroratus and the invasive crab Carcinus maenas.59 Different dogwhelk populations were collected from eight sites along the coast of Maine that were either north or south and had high or low wave exposure. The amount of dogwhelk movement decreased in the presence of water-soluble cues from a green crab and a rock crab, regardless of collection location. The feeding rate of dogwhelks on mussels was affected by collection location and predator cues, with the greatest reduction in consumption rates observed in snails from the southern wave protected habitats. Snails from wave protected habitats consistently had reduced feeding in the presence of predator cues from each crab, but dogwhelks from wave exposed habitats did not change their feeding rates in the predator treatments. There was an effect of north versus south on total snail growth and new shell growth, but no effect of wave exposure. However, there was only an effect of predator cue on shell growth with snails collected from the southern site with low wave exposure showing a significant increase in shell thickness in the presence of both crab predators. These results suggest that habitat characteristics can influence chemically mediated predator prey interactions.

The foraging behavior of whelks, Nucella lapillus collected from wave exposure or wave protected sites was compared in the presence or absence of chemical cues from the potential predator Carcinus maenas.60 The collection sites were characterized and shown to have different flow and different abundance of the barnacle Semibalanus balanoides and the mussel Mytilus edulis, both of which are prey for N. lapillus. Whelks from both type of sites preferred to eat the barnacles and this preference did not change in the presence of chemical cues from the crab. The amount of energy in mussels was the same with and without the crab cues in both habitat types. The amount of energy consumed from barnacles by snails was consistently less in the presence of crab cues and also less in snails collected from wave exposed sites. These studies highlight that the environmental context is an important factor controlling predation.

Since oyster reefs can greatly influence local water flow patterns the effects of flow modification on predation, pressure was measured in patches of the clam Mercenaria mercenaria.61 The authors measured water turbulence downstream from an oyster bed in Romerly Marsh Creek, GA, USA and found that the flow was significantly slower directly adjacent to the oyster beds, but increased to the same velocity 3.3, 6.6 and 10 meters away from the oysters. The predation on the clam Mercenaria mercenaria by blue crabs was greatest adjacent to the oyster reef and decreased 10 meters downstream. But predation by the whelk Busycon carica was greatest 5 meters from the oyster bed. Experiments were conducted in a flume to confirm these field results, and blue crabs and whelks had the greatest foraging success in the presence of water passed over live oyster clusters with or without turbulence (in the flume experiment turbulence was created by the addition of dead oyster shells). Crabs were more efficient in the cue only treatment when compared to cues with added turbulence. The crabs walked more slowly in the turbulence treatments, but there was no effect of treatment on their path linearity. Whelk movement speed and path linearity did not differ among the treatments. These experiments show that turbulence can significantly alter the success of different predators and suggest that to best understand the detection of prey we must understand chemical cue dispersal in natural settings.

Water borne cues from crustacean predators can also cause inducible defenses in prey. The oyster Crassostrea virginica can alter its shell thickness in response to chemical cues from the crabs Callinectes sapidus and Panopeus herbstii.62 Newly settled oysters were exposed to each species of crabs or no crab for 8 weeks. Oyster shell diameter and mass increased in the blue crab treatment but not the mud crab treatment. The force required to break the oyster shells was greater in the mud crab treatment compared to the no predator control, and was the greatest in the blue crab treatment. The young oysters were exposed to mud crab predators and the no predator control were eaten more than the oysters raised in the presence of mud crabs and blue crabs. Water soluble cues from predators induced mechanical defenses in oysters that improved their ability to survive predation.

Even though it is rarely tested chemical cues from predators might have an impact on the next generation of a species. Reproduction in the mud snails Ilyanassa obsoleta was assessed in the presence of chemical cues from the predatory green crab Carcinus maenas and in the presence of the non-predatory urchin Strongylocentrotus droebachiensis.63 Mud snails were collected from Orr's Island, ME, USA and held in flow-through seawater in groups of 20 snails per container. Snails were exposed to chemical cues from crabs and urchins held in the same container separated by a mesh divider. The numbers of egg capsules laid and the number of eggs per capsule were not different if the female snails were exposed to crab or urchin cues. The larvae that hatched from the eggs were longer if the parents had been exposed to cues from both the green crab and the sea urchin. There was no effect of cue treatment on the time until hatching. The morphology of the egg capsules was measured and green crab cues increased the spine length to capsule height ratio. However, when fed to the green crabs, spine length had no effect on the number of egg capsules consumed. When egg capsules were offered to hermit crabs they did not eat capsules with longer spines in 7 of 10 trials. This study shows that predator cues in the natural environment affect egg capsule and larval morphology, but different morphologies were not always effective deterrents of predation.

A recent paper examined water soluble chemical cues from live reefs compared to dead reefs to see how these cues influenced the larval settlement of crustaceans, cephalopods and fishes.64 Ten-liter water samples were collected 0, 1, and 2 km away from two different reefs on Ishigaki Island, in the Ryukyu Archipelago in Japan. Water from a dead coral reef (Kabira) was shown to have fewer components than water collected from live coral reef (Oganzaki) using HPLC. As distance increased away from Oganzaki the HPLC peaks were reduced by 14-fold (1 km) and 17-fold (2 km) compared to the 0 km sample. In a modified flume experiment, larvae of Chromis viridis (fish), Paleomonidae sp. (crustacean) and Sepia latimanus (mollusk) were tested to determine their behavior in response to these water samples. Larvae from all species spent more time in the water collected 0 km from the live coral reef, but only C. viridis spent time in water 1 km from the reef, and none of the larvae stayed in water collected 2 km from the reef. The water from a dead coral reef never induced significantly different behavior by any of the larvae. Water soluble cues can act as indicators for appropriate larval settlement behavior but these behavioral modifications are less likely as the distance from the reef increases.

Chemical cues were tested for their role in larval settlement of the burrowing ghost shrimp Lepidophthalmus siriboia.65 Five treatments were tested for their effect on megalopae: seawater conditioned with 20 adult shrimp for 24 hours, filtered seawater and sand, filtered seawater and muddy sand, adult conditioned seawater with muddy sand and a filtered seawater control. In all treatments except the seawater control and the adult conditioned seawater, there was 100% larval settlement. There was the same percentage metamorphosis in all treatments and a faster time until metamorphosis in all treatments except for the filtered seawater control. This research shows that for these ghost crabs chemical cues do not drive their settlement and metamorphosis, they can settle in any treatment with appropriate benthic substrata.

Trade-offs in behavior between feeding and avoiding predators was recently tested with crab larvae.66 Using in situ larval collectors in Corral Bay, Chile the number of settled larvae per day were measured for the crabs Metacarcinus edwardsii and Cancer plebejus. For both crab species the presence of 5 g doses of salmon food pellets did not influence the number of larvae that settled. One-year-old M. edwardsii were put in the larval collectors and were placed in opaque pvc tube with holes in it to generate a chemical cue from a potential predator. Larvae of M. edwardsii responded to chemical and visual cues combined but neither cue alone. Larvae of C. plebejus did not have significantly different settlement in the presence of predator cues but had similar trends as those found in M. edwardsii. These results suggest that predator cues can influence settlement success into suitable settlement habitat.

9 Molluscs

Marine slugs often rely on chemical defenses and a recent paper argues that these chemical defenses have allowed radiation in the Antarctic slug species Doris kerguelenensis.67 Mitochondrial and nuclear gene regions showed multiple divergent lineages within D. kerguelenensis. Secondary metabolites were characterized using LCMS in the same individual slugs used in the phylogeny. These secondary metabolites were not characterized but are produced de novo and differed among different lineages. The authors hypothesize that D. kerguelenensis is a sibling species complex that has evolved multiple secondary metabolites for protection.
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The gastropod Dicathais orbita has been well studied because of the presence of Tyrian purple, an important dye used in ancient textiles. Mass spectrometry was used to quantify the distribution of choline esters and Tyrian purple precursors in the snail Dicathais orbita.68 These researchers use desorption–ionisation on porous silicon (DIOS) with mass spectrometry imaging (MSI) to characterize the location of choline esters and the Tyrian purple precursor. They found that the choline ester murexine (56) was biosynthesized in the hypobranchial glands. During egg laying 56 was transferred to the eggs themselves where the compound was modified to make Tyrian purple. 56 had a tranquilizing effect on the larvae and was hypothesized to be important for the egg laying process. There is great potential for modern technology to track compounds distribution within individuals to understand their ecological function.

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The extract from Tylodina corticalis, collected from the temperate Australian sponge Pseudoceratina purpurea, was investigated by high resolution LC-MS/UV-Vis spectrometry to determine if the mollusc sequesters metabolites from the sponge.69 Pseudoceratin A (57), aerophobin 2 (58), hexadellin A (59), purealidin L (60), P (61) and Q (62), and aplysamine 2 (63) were all present in the extract. The major compounds in the mollusc, 61 and 62, were only found in trace quantities in the sponge. The authors suggest mollusc selectively accumulates from the sponge and chemically modifies some of the metabolites. Further studies will be required to confirm this hypothesis as this study was limited to one specimen.

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A recent review highlights the source and ecological function of ink found in cephalopods.14 This review builds on previous work and also incorporates the recent results from the study of predatory responses to ink from the longfin squid, Doryteuthis pealeii.70 The ink and a synthetic ink mimic inhibited the rate of finding food by the flounder Paralichthys dentatus through visual effects. When the ink was incorporated into fish food, both P. dentatus and the sea catfish Ariopsis felis ate less food. Through bioassay guided fractionation the chemical deterrents in ink were localized to the fractions that contained melanin, although this fraction was not pure. Cephalopod ink continues to be an important system for studying the modality of chemical defenses in marine environments.

The ink from Aplysia californica has been reported to inhibit the feeding behavior of the spiny lobster Panulirus argus through sensory inactivation.71 The neuronal reception of food odors was measured for lobsters with the opaline component of sea hare ink coated on the antennules and controls without the coating. Lobsters were exposed to food odors prepared by soaking 1 g of shredded shrimp in 1 L of artificial seawater for one hour. Opaline reduced the intensity of the neuronal response to two different concentrations of food odors and the motor neuronal response in antennular movement. Carboxymethylcellulose (CMC), a mimic of the physical nature of opaline, also reduced the neuronal and motor neuronal response to food odors. Since CMC is similar to opaline in its “stickiness” and caused the same response, the authors argue that the response may be due to a physical change caused by the opaline.

Water soluble cues from predators might influence the recruitment of sessile benthic organisms. Water soluble chemicals from dogwhelks were used to assess barnacle recruitment with and without cues from potential predators.72 Recruitment tiles for barnacles were placed in the rocky intertidal habitats in Nova Scotia, Canada. Different densities of dogwhelks were held in cages 1.5 cm away from the tiles to expose barnacles to water soluble cues from dogwhelks. Tiles incubated with one and five dogwhelks showed no difference in recruitment compared to the no snail control. But tiles incubated with ten dogwhelks had approximately half as many barnacle recruits. Chemical cues from potential predators can deter barnacle settlement.

The chemical cues necessary for oyster larval settlement was recently studied for Crassostrea gigas.73 Shell fragments from 11 different molluscs all induced C. gigas larval settlement except for shells of Patinopecten yessoensis and Atrina pinnata. Shell fragments of C. gigas and C. nippona both induced the most settlement. Treating C. gigas shell fragments with antibiotics did not change its ability to induce larval settlement. However, the 2 N HCl extract from C. gigas shells induced the same amount of larval settlement as the shell fragments when the aqueous and organic extracts displayed not activity. Larval settlement activity in the HCl extract was maintained at 100 °C but was reduced at 200 °C and 300 °C. The extracted cue induced less settlement after the addition of pepsin, trypsin, PNGase F and trifluoro-methanesulfonic acid. Using gel-filtration, the active compound was determined to be between 45 and 150 kDa in size, which corresponded to a major band at 55 kDa on SDS-PAGE. The unidentified settlement cues were further characterized for C. gigas.74 Artificial substrates including nitrocellulose membranes, plaster plates, glass, filter paper all induced settlement when shell extract was added. When lectins (lentil lectin, concanavalin A, soybean lectin, and wheat germ agglutinin) were added to shell chips and filter paper with shell extract they all reduced settlement of the larvae except soybean lectin. The lectins inhibited settlement of oysters but this inhibition was eliminated with the addition of N-acetyl-D-glucosamine. The authors argue that settlement of oyster larvae is mediated by lectin like receptors so the added lectins showed that settlement relies on lectin sugar interactions. The authors suggest that the settlement inducing cues are glycoproteins found in the acid soluble protein matrix in the shell of C. gigas.

The ecology of chemical cues that drive larval settlement was studied with the oyster Crassostrea virginica in Wilmington, NC, USA.75 Recruitment of C. virginica was monitored on cement blocks with and without added tri-peptide glycyl-glycyl-arginine. This chemical cue did not increase larval settlement and neither did the presence of live adult oysters. However, excluding predators from the blocks did significantly increase oyster recruitment. Even though chemical cues are known to be important for oyster recruitment the ecological context is an important consideration to understand patterns of larval settlement.

10 Echinoderms

Two recent studies use Matrix-assisted laser desorption/ionization (MALDI) imaging for quantitative analysis of saponins in echinoderm tissues. Demeyer et al.76 employed MALDI imaging to characterize the inter-organ and intra-specific variability of the asterosaponins previously isolated from Asterias rubens. Their results demonstrate that there is a large diversity of molecules in this class and that many of them are concentrated in specific organs (tube feet, aboral body wall, gonads, stomach and pyloric caeca). Bondoc et al.77 conducted a similar study with semi-pure and membranolytic extracts from Holothuria impatiens, H. fuscocinerea, and H. scabra. MALDI-FTICR MS and nano-HPLC-chip Q-TOF MS analyses showed significant intraspecific variability in saponin concentration between species. Of the three, H. scabra extracts had the highest diversity of saponins and three compounds unique to this species, 24-dehydroechinoside A (64), holothurins A2 (65) and A3 (66). Going one step further, the authors used phylogenetic mapping to examine 32 holothurid species to show that the non-sulfated hexosidases glycone of the saponins is evolved to the ecologically active sulfated tetraosides.
image file: c8np00061a-u25.tif

Saponins, isolated from conditioned seawater from four sea cucumbers, Bohadschia vitiensis, B. subrubra, Holothuria scabra and H. lessoni, were shown to attract the symbiont harlequin crab, Lissocarcinus orbicularis.78 Using a Davenport olfactometer, Caulier et al.76 demonstrated that Harlequin crabs preferentially migrated toward the conditioned water from the sea cucumbers or the purified saponins in the two paired branches of the tube. The authors concluded that saponins, known to be chemical defenses against predators served, instead, as kairomones for the Harlequin crabs.

11 Other invertebrates

Broad surveys of concerning the ecological activities of Antarctic invertebrate and macroalgal extracts have been reviewed by Núñez-Pons and Avila.7 A related study was conducted by Angulo-Preckler et al.79 in which the extracts from Antarctic benthic marine invertebrates were investigated for antimicrobial activity against six allopatric bacterial strains. The hydrophilic fractions of 16 species (10 sponges, three soft corals, two bryozoans, and one holothurian) were incorporated into Phytagel® at natural volumetric concentrations. The hydrophilic extracts from 10 of the 16 invertebrate species tested (62.5%) inhibited growth of at least one bacterial strain. The authors suggest that antimicrobial defenses are a common trend among sessile or slow-moving marine benthic invertebrates from the Antarctic.

12 Vertebrates

Olfactory chemical cues have been the subject of recent studies of fish associated with coral reefs. In the first, authors examined whether fish larvae showed a preference for algal chemical cues versus coral chemical cues to determine if the observed shift of many coral reefs to algal rich habitats could affect the recruitment of fish larvae.80 Laboratory experiments were conducted with 10 species of larval fish from the reef crest of Ragniroa. Fish were exposed to water collected from the coral and algal reefs in a 2-channel choice flume. Experiments were conducted with one fish larva at a time. Of the ten species, Acanthurus triostegus, Chromis viridis, Aulostomus chinensis, Ptereleotris microlepis, Sargocentron spiniferum, Chrysiptera glauca and C. leucopoma demonstrated a strong preference for the coral reef water. The herbivore, Zebrasoma veliferum, was the only larva to prefer the algal reef water. A similar study conducted in Ishigaki Japan compared the chemical cues released from live versus dead corals.81 Seawater samples collected at the coral reef and at the distances of 1 km and 2 km seawards were subject to HPLC analysis. The extracts prepared from the water surrounded by the living corals were chemically diverse compared to the dead corals. One of the peaks in the chromatogram from the living coral increased in concentration at the 1 and 2 km distances. The compounds were not identified. In 4 channel flume experiments the larvae from Chromis viridis, Palaemonidae sp., and Sepia latimanus migrated toward the seawater collected directly at a live coral reef. These studies provide evidence that the increasing number of dying coral reefs and algal phase shifts could affect the ability of larval fish to navigate back to the benthic habitat.

Brooker et al. reported the first example of ‘chemical crypsis’ in a vertebrate.82 This study investigated the transfer of olfactory chemical cues from Acropora spathulata or Pocillopora damicornis to the corallivore Oxymonacanthus longirostris. Pairwise choice experiments with sympatric coral inhabiting crabs, the Pocillopora-obligate Trapezia cymodoce and the Acropora-obligate Tetralia glaberrima, showed the crabs favored filefish fed on their preferred coral over their non-preferred coral. In addition, predatory cod (Cephalopholis spp.) were less attracted to filefish odor when it was presented alongside the coral it had been fed on. These results suggest that the filefish has a diet-induced chemical defense.

13 Conclusions

Marine chemical ecology is an interdisciplinary field that continues to grow as a result of the successful collaborations between benthic ecologists, natural product chemists, pharmacologists, microbiologists, molecular biologists and other related disciplines. Advancement of field and laboratory techniques have led to a better understanding of the molecules that shape the interactions between algae, invertebrates, fish and microorganisms in the benthic environment. Sensitive analytical instrumentation can be used to identify compounds released in seawater at low concentrations while matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) can locate active metabolites within the tissues of the organisms or observe interactions between organisms.22,68 Laboratory studies have greatly advanced our understanding of the chemical nature of coral reef settlement cues however, the function of these cues in the natural environment is still not well understood. Continued investigation of the production and reception of marine natural products in situ is needed in order to fully comprehend complex ecological interactions.

The changing environment of benthic communities continues to be a concern. Ocean acidification and global climate change can result in widespread disease outbreaks sometimes causing phase shifts from coral reefs to algae dominated communities.83 The phase shifts have implications in chemically mediated interactions, notably the loss of chemical cues that disrupt the recruitment of juvenile fishes to habitats.80 Recent work also provides some evidence that there can be an effect of ocean acidification on the production of chemical defenses by marine algae.35,44 There are many opportunities for further studies in this area to address questions regarding the effects of habitat changes on the production of metabolites important for the survival and proliferation of organisms associated with benthic marine habitats.

Our knowledge of the relationship between marine natural products and microbiomes of many organisms continues to grow. This field will continue to advance as sequencing technologies improve and costs decrease. Much of the current work is based on profiling the taxonomic composition of microbiomes using targeted gene sequencing.55 While this has been a great advancement, these techniques are limited in their ability to distinguish between microbial taxa at fine scales because they often rely on small sections (<300 bp) of the 16S gene and therefore provide only limited information as to the likely function of these microbes. Studies are increasingly incorporating shot-gun metagenomics and transcriptomics in order to obtain a more in depth understanding of the role microbes play in ecological interactions. In addition, studies concerning marine microbes are beginning to bring to light the complexity of interactions between benthic marine organisms.

Brominated metabolites from different chemical classes are common among the active natural products covered in this review.21,39,40,53 Ecological activities reported for these molecules vary significantly including coral settlement cues, antifouling agents, antibacterial compounds and feeding deterrence. As the field continues to progress studies need to focus more on the isolation and characterization of active metabolites. This opens up opportunities for pharmacological studies that will allow us to expand the discussion to the significance of structure–activity relationships in ecological interactions.

In conclusion, our knowledge of the chemical ecology of benthic marine organisms in habitats from the tropics to Antarctica has expanded over the period of this review. The field will continue to grow with future studies addressing the ecological roles of marine natural products, the effects of climate change and the relationships between microorganisms and their hosts.

14 Conflicts of interest

There are no conflicts of interest to declare.

15 Acknowledgements

The authors thank Sara Abdourasul, Ernesto Marticoreno and Amanda Walker and the students from the Spring 2018 natural products class for their kind assistance with formatting and proofreading the manuscript.

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