Marine chemical ecology in benthic environments

NPRNatural Product Reportswww.rsc.org/npr

ISSN 0265-0568

REVIEW ARTICLEValerie J. Paul et al.Marine chemical ecology in benthic environments

Volume 31 Number 11 November 2014 Pages 1491–1664

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REVIEW

Marine chemical

aChicago State University, Department of PhbSmithsonian Marine Station at Fort Pierce,cEckerd College, Department of Biology anddUniversity of Hawaii at Manoa, Biology De

Cite this: Nat. Prod. Rep., 2014, 31,1510

Received 13th February 2014

DOI: 10.1039/c4np00017j

www.rsc.org/npr

1510 | Nat. Prod. Rep., 2014, 31, 1510–1

ecology in benthic environments

Melany P. Puglisi,a Jennifer M. Sneed,b Koty H. Sharp,c Raphael Ritson-Williamsd

and Valerie J. Paul*b

Covering: 2010 up to the beginning of 2013

This review covers the recent marine chemical ecology literature for benthic bacteria and cyanobacteria,

macroalgae, sponges, cnidarians, molluscs, other benthic invertebrates, and fish.

1 Introduction2 Microorganisms2.1 Bacteria2.2 Cyanobacteria2.3 Fungi2.4 Microalgae3 Macroalgae3.1 Chemical defense against herbivores3.2 Inducible defenses3.3 Antimicrobial and antifouling defenses of macroalgae3.4 Algal–coral interactions4 Seagrass5 Sponges6 Cnidarians7 Ascidians (tunicates)8 Crustaceans9 Molluscs10 Echinoderms11 Other invertebrates12 Vertebrates13 Conclusions14 Acknowledgements15 References

1 Introduction

In this report, we review the literature over the past 2 1/2 years inthe eld of marine chemical ecology, focusing on benthicspecies and communities. Research has continued at a rapidpace since we last reviewed this topic,1 with signicant advancesmade in almost every area of marine chemical ecology. Becauseof the large number of publications in this subject area over the

armaceutical Sciences, Chicago, IL, USA

Fort Pierce, FL, USA. E-mail: [email protected]

Marine Sciences, St. Petersburg, FL, USA

partment, Honolulu, HI, USA

553

past two years, this review will emphasize studies of benthicmarine algae and invertebrates, with additional coverage ofchemical ecology of marine vertebrates (mainly sh). Forcomprehensive reviews of advances in the chemical ecology ofplanktonic organisms see Sieg et al.2 for the 2009–2010 timeperiod and Roy et al. for the 2010–2012 time period. Thesereviews are organized by the type of ecological interaction, fromintraspecic interactions to allelopathic and predator–preyinteractions and ecosystem-level effects of natural products andmarine toxins from phytoplankton and other pelagic organ-isms. Research in marine chemical ecology continues inimportant areas such as predator–prey and seaweed–herbivoreinteractions, defenses against fouling organisms and infectionby microorganisms, competitive interactions, invasive speciesand settlement cues. We also discuss new insights into thebiosynthesis of bioactive marine natural products by symbioticmicroorganisms.

Several comprehensive reviews have been published over thepast two years that relate to marine chemical ecology. TheHandbook of Marine Natural Products published in 2012 (ref. 3)contains a section on Marine Natural Products and ChemicalEcology. This section includes reviews on antipredatorydefenses of marine invertebrates and planktonic organisms,antifouling activity of marine natural products, chemicallymediated competition, and marine metabolites and metal ionchelation.4–8

The November 2011 issue of the journal Integrative andComparative Biology contains a set of papers that resultedfrom a symposium on “Neuroecology: Neural determinants ofecological processes from individuals to ecosystems” at theannual meeting of the Society for Integrative and ComparativeBiology in January 2011.9–12 Topics range from the neuro-ecology of chemical defenses to the chemical ecology ofbenthic marine invertebrates along the Western AntarcticPeninsula to interfaces between bacterial and eukaryotic“neuroecology”.

This journal is © The Royal Society of Chemistry 2014

http://crossmark.crossref.org/dialog/?doi=10.1039/c4np00017j&domain=pdf&date_stamp=2014-10-08

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2 Microorganisms2.1 Bacteria

In the benthic environment, all surfaces – outer body surfaces ofanimals, surfaces of macroalgae and seagrass, and non-livingsubstrates including rock and rubble – are susceptible to colo-nization by bacteria and subsequent surface biolm formation.Although many studies have catalogued compositions of thehost-associated microbiota, or the microbiomes, and the collec-tive genomes of the host and microbiome (metagenome), little isknown about the mechanisms by which host-associated micro-biomes are structured and maintained. Recent reviews summa-rize examples in which bacteria produce small molecules to allowor prevent selective, specic bacterial infection of eukaryotictissues and/or surfaces.11,13–15 The molecules involved in this“cross-talk” between microorganism and eukaryotic host appearto be relatively conserved across a broad range of marine andterrestrial animals, regardless of whether the bacteria are

Melany P. Puglisi is an associateprofessor at Chicago StateUniversity. She received her BSfrom Southampton College, LIUin 1991 in Chemistry, her MSfrom the University of GuamMarine Laboratory in Biology in1995, and her PhD. In Pharma-cognosy in Marine ChemicalEcology in 2001 from theUniversity of Mississippi. Mela-ny's postdoctoral research atUCSD Scripps Institution of

Oceanography and Smithsonian Marine Station at Fort Piercefocused on microbial chemical defenses of macroalgae. She is co-author of 17 research papers and review articles. Here currentinterests include chemical defenses of macroalgae, fungi andsponges against co-occurring microorganisms.

Jennifer Sneed is a researchbiologist at the SmithsonianMarine Station at Fort Piercestudying chemically mediatedinteractions between organismsin the marine environment. Shespecializes in understanding theroles that marine microbes playin the ecological interactions ofother organisms. She has a MSin biology from the University ofSouth Florida and a PhD inanalytical chemistry from the

Friedrich Schiller University in Jena, Germany. Jennifer beganworking at the Smithsonian Marine Station as a PostdoctoralFellow in 2011 and was hired on as a full time research biologist inthe fall of 2012.


benecial, commensal, or pathogenic (reviewed in Bosch andMcFall-Ngai et al.16). Changing environmental conditions, espe-cially increasing sea surface temperature and ocean acidication,have been shown to alter the composition and metabolism ofbenecial bacterial communities associated with benthic hosts(reviewed in Mouchka et al.,17 Sharp and Ritchie18).

Recent research has focused on bacterial assemblages onmarine algal surfaces. In a meta-analysis of cloned 16S rRNAsequences representing bacteria from marine algae, Goeckeet al.19 propose that bacterial communities in marine macro-algae harbor diverse bacterial assemblages, which functionprimarily to degrade polysaccharides from the algal hosts. Incomparison, they propose that surfaces of microalgae aredominated by a-proteobacteria, which likely consume dime-thylsulfonioproprionate (DMSP) released by the microalgae.Seyedsayamdost et al.20 demonstrated that the physiologicalstate of a microalgal host Emiliania huxleyi, regulates bioactivemetabolite biosynthesis that mediate a “Jekyll-and-Hyde”

Koty Sharp received her PhD inMarine Biology at Scripps Insti-tution of Oceanography with DrMargo Haygood and Dr JohnFaulkner. She was a Smithso-nian Marine Science NetworkPostdoctoral Fellow with DrValerie Paul at the SmithsonianMarine Station at Fort Piercefrom 2006–2008 and a post-doctoral research fellow atOcean Genome Legacy with DrDaniel Distel from 2008–2011.

Koty is currently an assistant professor at Eckerd College in St.Petersburg, FL. Her interests include the microbial ecology andchemical ecology of marine invertebrate–bacterial symbioses andrecruitment and transmission of symbionts in marine inverte-brates.

Raphael Ritson-Williams iscurrently a PhD student in theBiology Department of theUniversity of Hawaii at Manoa.He received his BS in biology fromthe Evergreen State College andhis MS in marine biology from theUniversity of Guam. His researchinterests include coral reef organ-ismal and chemical diversity,especially those compounds thatinuence organisms' evolutionand ecology.

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switch in the bacterial–microalgal relationship frommutualismto parasitism. Seyedsayamdost et al.21 explored the chemicalecology of Phaeobacter gallaeciensis BS107, a member of theRoseobacter clade (a-proteobacteria) cultured from the micro-alga Emiliania huxleyi throughout its life cycle. Members of theRoseobacter clade have been shown to produce antibiotics foralgal host protection, but in response to algal senescence, thebiosynthesis of P. gallaeciensis BS107 shis from antibiotics andgrowth stimulants to the highly cytotoxic roseobacticides.During senescence, E. huxleyi releases products of lignin (cellwall) breakdown, including p-coumaric acid 1 (pCA). The pres-ence of 1 elicits synthesis of roseobacticides 2–10, which arehighly potent against E. huxleyi cells, lysing host cells, andultimately causing a shi from benecial associate to oppor-tunistic pathogen. Seyedsayamdost et al.21 propose that 1 maybe part of a signal that regulates roseobacticide biosyntheticgenes. Although these studies focus on planktonic microalgae,we include the results as an excellent example of the use ofnatural products chemistry to reveal signals that controlecologically signicant eukaryotic–prokaryotic interactions.

The bacterial community associated with the surface mucuslayer (SML) in tropical corals has recently been a major focus ofresearch, primarily because it has been shown to exert a stronginuence on disease resilience in tropical corals.22 Host-derived

Valerie Paul received her BAand PhD degrees from theUniversity of California SanDiego, where she studiedmarine natural products andchemical ecology under theguidance of Professor WilliamFenical at Scripps Institution ofOceanography. She served onthe faculty of the University ofGuam Marine Laboratory from1985–2002, and joined theSmithsonian Institution in 2002

where she is currently Director of the Smithsonian MarineStation at Fort Pierce.

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and microbially-produced compounds inuence the bacterialcommunity composition and metabolic activity in the coral SML(reviewed in Krediet et al.;15 Sharp and Ritchie18). Bacteriacommon to diverse coral hosts include the g-proteobacteriagenera Pseudomonas and Vibrio.23 Using cross-inhibition tests,isolates cultured frommucus of the coral Acropora millepora wereassayed for their ability to inhibit growth of other mucus isolates.The study suggests specic genera from the a-proteobacteriamayprovide additional, specic regulation of bacterial growth in theSML.23 Although the bacterial compounds have not yet beenidentied, this culture-based approach provides strong evidencethat some coral-associated bacteria, especially the proteobacteria,have the ability to inhibit growth of other bacteria.23

Benecial coral-associated bacteria inhibit growth andprevent pathogenic infection of eukaryotic tissues by targetingquorum sensing (QS) pathways in potential pathogens. In QS,small diffusible compounds called autoinducers, molecules thataccumulate to a threshold concentration, regulate synchronizedgroup behaviors, magnifying their ecological impact. Quorumsensing has been demonstrated to regulate many bacterialbehaviors, including biolm formation, antibiotic production,and bioluminescence, and bacterial–eukaryotic interactions,including symbiotic infection and pathogenesis (reviewed inKrediet et al.15). Both eukaryotes and prokaryotes have evolved torecognize and counter quorum sensing in pathogens. Alagelyet al.24 demonstrated that corals produce signal-mimics that canstimulate QS responses in bacteria, and other bacteria cancounter-attack by producing quorum quenching acylases or lac-tonases that break down signaling molecules. Quorum sensingmay inhibit or activate pathogenesis, antibiotic production,exoenzyme production, and attachment by benecial bacteriawithin coral tissues and on surfaces. Coral extracts containcompounds capable of interfering with quorum sensing activi-ties24 that may be involved in regulating the colonization of coralmucus by pathogens, commensal bacteria, and/or benecialbacteria. The source of this activity is difficult to pinpoint andcould originate from the coral, the dominant endosymbiont, orany associated bacteria. Alagely et al.24 recently showed that bothcoral and Symbiodinium-associated bacteria alter swarming andbiolm formation in the coral pathogen Serratia marcescens.


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These phenotypes are typically controlled by QS, although inhi-bition of QS by these isolates remains to be demonstrated. Whileit is clear that at least some coral-associated commensals andpathogens produce QS signals under laboratory conditions,24,25 itis not clear whether these signals accumulate to thresholdconcentrations in natural environments.

The bacterial communities associated with benthic organismsnot only have direct ecological impacts on the encrustingorganisms, but they also inuence the ecology of other eukary-otes in the same ecosystem.26 For example, particular species ofcrustose coralline algae (CCAs) have been shown to facilitatelarval settlement of the threatened coral species Acropora cervi-cornis and A. palmata in the Florida Keys and the Caribbean.27

Webster et al.28 studied the potential impact of ocean acidica-tion on bacterial community composition on the CCA Hydro-lithon onkodes, previously shown to facilitate coral larvalsettlement. Decreased pH levels correlated with a shi in bacte-rial communities on the CCA surface, and with a decline in corallarval settlement rates. This study further demonstrates that coralsurvival, specically coral recruitment, may be signicantlychallenged by declining seawater pH, not only because of alter-ations in cover of carbonate structures on which to settle, but alsobecause the facilitating bacterial communities are lost.

The integration of microbiological and chemical ecologyapproaches indicates that bacteria, primarily from the g-pro-teobacteria, present on the CCA surface exert inuence on thefacilitation of larval attachment and/or metamorphosis in somespecies of corals.29,30 In laboratory assays, Tebben et al.29

demonstrated that exposure to Pseudoalteromonas isolatescultured from the crustose coralline algae Negoniolithon foslieiand Hydrolithon onkodes signicantly increases rates of meta-morphosis of the Pacic coral Acropora millepora. The meta-morphosis-inducing compound was identied astetrabromopyrrole (TBP) 11.29 In their ndings, TBP inducedmetamorphosis, but not settlement (attachment and meta-morphosis). Through comparative transcriptomics in TBP-treated larvae (metamorphosed larvae) and CCA-exposed larvae(settled larvae), Siboni et al.30 determined that the membraneprotein Apextrin is involved in larval adhesion and attachment.They propose that in response the levels of TBP tested in theirassays, attachment is potentially inhibited by downregulation ofproduction of Apextrin and other surface adhesion proteins. Inaddition, the transcriptomic data in this study suggest thatmorphogenesis is controlled via MAPK signaling pathways.

Other strains of Pseudoalteromonas and Thalassomonas havealso been shown to induce larval settlement and meta-morphosis in the Pacic coral Pocillopora damicornis.31 Thisstudy also highlighted that bacterial function cannot be infer-red by phylogenetic affiliation; not all tested isolates of Pseu-doalteromonas and Thalassomonas were inductive in this study.


The variation in ability to induce settlement among closelyrelated bacterial strains indicates that the ability to inducesettlement is strain-specic, likely involving other components(i.e. bioactive compound biosynthetic gene clusters) of thegenome outside of the ribosomal rRNA operon. In addition, theisolation source of the bacteria (algal surface versus coralsurface) was not linked to the inductive properties of differentstrains.31 Together, these studies indicate that specic bacteriaproduce compounds that direct successful larval attachmentand metamorphosis in some coral species. The overall diversityof bacteria capable of production of TBP, or other compoundsthat regulate coral larval settlement in the benthic environment,is not yet well characterized. In addition, the concentrations ofthese inductive compounds occurring on surfaces in reefecosystems remains to be characterized. Future studiesfocusing on the natural distribution and abundance and theprocesses that regulate distribution, abundance, and metabo-lite production of inductive bacteria in reef environments, willfurther our understanding of the potential for bacteria toenhance coral larval recruitment.

Identication of bioactive metabolite biosynthesis bysymbiotic microorganisms remains a daunting task becausemany bacterial symbionts are not yet cultured but are onlyidentied and visualized with culture-independent techniques.However, in recent publications, researchers have used tech-niques of genomics, DNA sequence analysis, and advancedmicroscopy to explore the bacterial biosynthesis of bioactivecompounds in the ascidian Lissoclinum patella.

The symbiotic cyanobacterium Prochloron didemni has beenshown to produce the patellamides, cyclic peptides found insome specimens of the Pacic ascidian Lissoclinum patella.32

Some individuals of L. patella contain the highly cytotoxicpatellazoles 12–14, cyclic polyketides that have been hypothe-sized to also be microbial in origin. A characterization of theL. patella microbiome revealed a complex assemblage foundacross different specimens.33 Although patellazole structurallyresembles other bacterial secondary metabolites, identicationof candidate patellazole biosynthetic gene clusters fromL. patella were unsuccessful until entire bacterial genomes wereassembled via next-generational sequencing on cell prepara-tions from patellazole-containing L. patella.34 Using shotgunsequencing from a zooid cellular fraction, the genome of anuncultured symbiotic g-proteobacterium, Endolissoclinum faul-kneri, was assembled. The genome includes a biosynthetic genecluster that matches the predicted sequence for patellazoles.34

The genome of E. faulkneri is highly reduced; many of the genesin the E. faulkneri genome are lost or non-functional. Theexception is the large (86 kbp) ptz gene cluster encoding forpatellazole biosynthesis, which remains intact within anotherwise extremely small (1.48Mbp) genome. This streamlinedgenome highlights that the bacterium has specialized overevolutionary time just to produce patellazoles, whose highpotency and concentration in L. patella implies a defensive rolefor the compounds. The patellazole-producing symbionts arelocalized within bacteriocytes, and are likely transmitted verti-cally, given their localization and their reduced genome.34 Thediscovery of other bacterial sources of bioactive compounds is

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an excellent example of a single eukaryotic species harboringdiverse bacteria that each provides unique metabolites to thehost's potential arsenal of active compounds.

Recent work35 attempted to determine bacterial origins ofbioactive compounds by exploring potential links betweenbacterial community composition and “natural productproles” in the sponge Aplysina aerophoba. In order to prole thebacterial communities in the sponge specimens, the authorsused 16S rRNA gene-based polymerase chain reaction (PCR) anddenaturing gradient gel electrophoresis (DGGE) analysis. Basedon the bacterial community and bioactive compound proling,they found a number of different correlations, including a linkbetween the presence of a Chloroexi sp. with aplysinamisin-115, and with yet unclassied bacterial strains and aerophobin-216 and isostularin-3 17. Although the data do not conclusivelydemonstrate specic bacterial origin, this approach may bevaluable for “siing” through the notoriously complex bacterialassemblages in sponges in order to identify candidate producersof compounds of ecological signicance.

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In a recent study on the encrusting bryozoan Membraniporamembranacea, diverse bacteria were isolated on various culturemedia.36 Of the 96 cultured isolates, which included represen-tatives of a- and g-proteobacteria, Bacillus, and Actinobacteria,nearly half of them (47 isolates) exhibited antibacterial activityagainst test strains such as the gram-positive strains Bacillussubtilis and Staphylococcus lentus, gram negative E. coli andPseudomonas uorescens, and the yeast Candida glabrata. Thisstudy highlights the importance of using diverse media andculture conditions – the authors note the variable occurrence ofantibacterial activity depending on test media used. Althoughthe structures of the compounds responsible for the observedantibacterial activity have not yet been elucidated, the assayspoint to several bacterial genera and groups that play aprotective role to the host bryozoan and/or regulate the associ-ated bacterial community structure.

The wood-boring bivalve mollusc Lyrodus pedicellatusharbors a community of closely related intracellular symbioticg-proteobacteria within symbiosomes within the gills. Thesymbionts have been shown to degrade lignocellulose and xnitrogen for the host.37,38 Recent genomic analysis of Ter-edinibacter turnerae, one of the cultivated representatives of thecellulose-degrading symbionts, shows that T. turnerae possessesgene clusters encoding for biosynthesis of polyketides and non-ribosomal peptides.39 Using reverse-transcription PCR, theauthors demonstrated that biosynthetic genes for tartrolon


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antibiotics 18–22 are expressed within the shipworm, and thecompounds were detected in the shipworm via liquid chroma-tography and mass spectrometry. Elshahawi et al.39 proposedthat these antibiotics play a role in structuring the bacterialcommunity within the shipworm hosts. The chemical regula-tion of bacterial growth in the host cecum, which has aremarkably low bacterial cell density, may enable the uniquelignocellulose degradation that occurs in shipworm symbioses.

Like the study of benecial bacterial assemblages in marineorganisms, the study of pathogenic bacteria in marine organ-isms has been challenging, due to the diversity and complexityof marine microbial communities. In addition, identication ofpathogens is difficult due to the lack of cultured representativesof most marine bacterial groups.40,41

Several recent reviews offer comprehensive overview ofprocesses governing pathogen dynamics, abundance, andenvironmental determinants of coral diseases,40–42 but still verylittle is known about the bioactive compounds involved inpathogenesis. In some cases, such as in the coral disease whitepox, a single potential pathogen (Serratia marcescens) has beenidentied.43 Serratia marcescens has been detected in seawater,several species of corals, corallivorous snails, human waste-water, and marine sponges.43,44 Isolates were used in infectionexperiments to satisfy Koch's postulates and demonstrate thatS. marcescens causes white pox. Though bioactive compoundshave yet to be linked to pathogenesis by S. marcescens, it hasbeen demonstrated that many strains of S. marcescens producethe prodigiosins 23, active against a broad range of bacterialtaxa.45

Another bacterial disease in corals, black band disease(BBD), has been shown to be a polymicrobial disease, contain-ing cyanobacteria and diverse heterotrophic bacteria. Themajority of tested cyanobacterial cultures, including strainsisolated from BBD disease bands and other free-living marinestrains, inhibited growth of heterotrophic strains isolated fromcoral SML.46 In addition, lipophilic extracts of the cyanobacteriainhibited growth of known coral pathogens Vibrio coralliilyticusand Aurantimonas corallicida.46

The coral pathogen Vibrio coralliilyticus is a putative path-ogen involved in coral bleaching and disease, and severalcultured strains of V. coralliilyticus exhibit multi-drug resistanceto a range of commercial antibiotics.47 In addition, very fewcultured isolates from coral mucus have been shown to inhibitV. coralliilyticus growth. V. coralliilyticus has been shown toproduce the antibacterial compound andrimid 24, and andri-mid synthesis is increased two-fold under exposure to chitin.48

The ecophysiological consequences of chitin regulation ofantibacterial production remain poorly understood.


2.2 Cyanobacteria

A general review of the chemical ecology of cyanobacteria coverscyanobacteria from marine and freshwater habitats.49 Thefunctions of cyanobacterial secondary metabolites in UVprotection, feeding deterrence against consumers, allelopathyand competition, and chemical communication (especially asinhibitors of bacterial quorum sensing) are reviewed. Thebiodiversity of cyanobacteria, their chemical diversity, andevolution of biosynthetic pathways for natural products are alsodiscussed. Biosynthetic pathways have been elucidated formany known cyanobacterial toxins, and this has led to thedevelopment of molecular techniques for detecting and quan-tifying the producing cyanobacteria in different aquatic envi-ronments under different environmental conditions. Dittmannet al.50 wrote a comprehensive review of toxin biosynthesis infreshwater and marine cyanobacteria with an emphasis on theevolution of different biosynthetic pathways in cyanobacteria.

The temporal dynamics of natural product production inthree cultured cyanobacteria were investigated with 15N stableisotope labeling and matrix-assisted laser desorption ionization(MALDI) imaging techniques.51 The sensitivity of MALDIimaging allowed the measurement of turnover rates of differentprimary and secondary metabolites from small amounts ofbiomass. These techniques could be applied to studyingbiosynthesis of natural products in cyanobacteria growingunder different environmental conditions in nature.

Several studies examined the functions of marine cyano-bacterial metabolites as inhibitors of bacterial quorum sensing.Malyngolide 25 appears to function in this manner at ecologi-cally relevant concentrations.52Other cyanobacterial compoundsthat interfere with bacterial quorum sensing include micro-colins A 26 and B 27 and malyngamide A 28. Malyngamide B 29and lyngbyastatin 3 30 inhibited quorum sensing but alsoinhibited growth of Chromobacterium violaceum CV017 indi-cating that these compounds also had some antimicrobialactivity.52 Screening of a large chemical library from marineorganisms identied kojic acid 31 as a potent inhibitor ofquorum sensing. In addition, experiments conducted within amicrocosm showed that this compound inuenced the micro-bial communities that formed on a glass slide immersed in 1 l ofunltered seawater for 7 days. The authors suggest that this maybe useful in the control of biofouling communities.53

Two studies of Lyngbya majuscula from Florida collectionsyielded two new molecules that affect LasR, a receptor involvedin quorum sensing in Pseudomonas aeruginosa. A new stereo-isomer of malyngamide C, 8-epi-malyngamide C 32, and lyngbicacid 33 inhibit bacterial quorum sensing in a reporter geneassay using the plasmid pSB1075.54 The cyclopropane-contain-ing fatty acid, lyngbyoic acid 34, major metabolite of Lyngbya cf.majuscula from various sites in Florida, strongly affects LasR

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and reduces pyocyanin and elastase (LasB) both on the proteinand transcript level in wild-type Pseudoalteromonas aeruginosaand directly inhibits LasB enzymatic activity.55

The antibacterial activities of 10 strains of black banddisease (BBD) cyanobacteria and 10 other strains of marinecyanobacteria were tested against heterotrophic bacteria iso-lated from the black band disease consortium and the surfacemucus layer of corals. Lipophilic extracts from the cyano-bacterial strains inhibited bacterial growth, and inhibition washigher for BBD cyanobacteria compared to the other marinecyanobacterial strains.46 The authors suggested that the BBDcyanobacteria produce antimicrobial compounds to help

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structure the polymicrobial BBD consortium,46 which has beenfurther characterized by Casamatta et al.56

Progress has been made in understanding the diversity andtaxonomy of marine benthic cyanobacteria, which has impor-tant implications for marine natural products, chemicalecology, and harmful algal bloom monitoring.57,58 Phylogeneticanalysis of cyanobacterial strains that produce over 100 bioac-tive compounds indicate that there is considerable biodiversitythat was not previously recognized by traditional morpholog-ical-based approaches to cyanobacterial taxonomy. Theproduction of natural products appears to be specic toparticular clades and may be species specic.58 These studiesprovide a framework for further classication and claricationof the taxonomy of the chemically rich tropical and subtropicalbenthic marine cyanobacteria.

Cyanobacterial toxins such as microcystins are usuallyassociated with fresh water cyanobacteria, but these toxins canenter marine ecosystems by runoff of microcystin-contaminatedfresh water. Miller et al.59 showed that deaths of 21 southern seaotters were linked to microcystins entering the Monterey BayNational Marine Sanctuary from ocean discharge of threenutrient enriched rivers. High concentrations of microcystinsup to 2900 ppm were found in a freshwater lake and down-stream tributaries that feed into the rivers within 1 km of theocean. Clams, mussels and oysters, which are food for sea ottersand humans, exhibited bioaccumulation of microcystins, andthese contaminated bivalves were suggested to be the mostlikely source of the toxins. This study demonstrates the inter-connections of freshwater sources of cyanotoxins and marine


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organisms and highlights this as a serious environmental andpublic health consideration.

2.3 Fungi

Fungi are abundant in the marine environment and recentstudies demonstrate diverse communities associated withinvertebrates. Zhang et al.60 cultured 41 fungal species from 20genera from six gorgonian species. Aspergillus and Penicilliumwere the most common genera. Antibacterial and antifungalscreening against marine microorganisms and known patho-gens showed that 38% of fungal extracts were effective in growthinhibition. The authors suggest that associated fungi mayprovide protection against pathogens.60 A similar study of thefungi from the marine sponge Psammocinia sp. from theMediterranean Sea yielded 85 fungi.61 Strains in the Acre-monium, Penicillium and Trichoderma were common as well asseveral undescribed strains in the genera Phoma and Tricho-derma. In vitro screening demonstrated that many of the iso-lated strains inhibited the growth of other fungi.

A study of the ascomycete Aspergillus sydowii, a knownpathogen of sea fan corals in the Caribbean, demonstrated thatthis fungus produces dimethyl sulde (DMS) from dime-thylsulfoniopropionate (DMSP). A. sydowii strains containedhomologs of the dddP gene, which encodes an enzyme thatreleases DMS from DMSP.62

Recent advances suggest that fungi associated with marineinvertebrates may play an active role in the formation of bio-lms and defense against potentially harmful pathogens.

2.4 Microalgae

The chemical ecology of microalgae was recently reviewed bySieg et al.2 and Roy et al.63

3 Macroalgae3.1 Chemical defense against herbivores

Brown algae of the family Dictyotaceae (e.g. Dictyota and others)produce diverse classes of diterpenes that function as defensesagainst herbivores and competitors. Bianco et al.64 report thatnatural concentrations of the dichloromethane extract and themajor secondary diterpene of the dolastane class 35 from thebrown alga Canistrocarpus (formerly Dictyota) cervicornis reducefeeding by the sea urchin Lytechinus variegatus.

Sea urchins are generalist herbivores in tropical andtemperate regions. Cra et al.65 investigated the palatability oflipophilic extracts from nine subtropical algae to foursubtropical and three temperate sea urchins at two differentconcentrations. The extracts from the three seaweeds Caulerpasertularioides, Dictyota ciliolata, and D. pulchella deterred all of


the urchins in the study. For the other seaweeds, there werepatterns of feeding resistance against the lipophilic extractswithin genera as well as the biogeographic origin of urchins.Subtropical species of urchins exhibited greater feeding resis-tance compared to the temperate urchins.

The importance of consumer diversity in consumption ofchemically defended tropical seaweeds was explored on reefs inFiji, taking advantage of adjacent no-take marine reserves andshed reefs. No-take reserves had greater biomass and speciesrichness of herbivorous shes, higher coral cover and lowerabundance and species richness of macroalgae than adjacentshed areas of the reef.66 Video taped feeding activity on sevenmacroalgae transplanted into no-take reserves showed thatrelatively few species of herbivorous sh were responsible forgrazing macroalgae, and these had specic feeding preferences.Chemical defenses of macroalgae largely explained the patternsof feeding observed for different herbivores, with specicherbivores consuming different types of algae.66 The feedingcomplementarity among different herbivores with different dietbreadths observed in this and other studies highlights theimportance of herbivore diversity on reefs, which has implica-tions for coral reef management.

3.2 Inducible defenses

Plant defense theory predicts that both constitutive andinducible defenses can be inuenced by energetic limitation orother conditions that limit resources available to plants. Kubi-cek et al.67 examined whether herbivory by two mesograzers, theisopod Idotea ochotensis and the snail Lacuna smithii, on theJapanese red alga Chondrus yendoi was affected by light avail-ability to the alga. The alga was grown in outdoor aquaria for 10days and exposed to 6 different levels of sunlight (0–99%reduction) by covering the sides of aquaria with black foil andthe tops with black mesh. The light compensation point wasdetermined by measuring dissolved oxygen in aquaria, andsome of the experimental irradiance levels were designed to fallbelow this level. During the light manipulations, moderatenumbers of grazers were added to the aquaria to attempt toinduce defenses in the alga. The rst experiment used foursnails (L. smithii), and a second experiment used one isopod(I. ochotensis) to induce algal defenses. Additional aquaria wereused without added herbivores to determine algal growth ratesunder the different light regimes. No choice feeding assays wereused to assess inducible defenses; grazers were offered previ-ously grazed and ungrazed algae. Low light levels negativelyaffected growth of Chondrus yendoi, although there wasconsiderable variation, with some thalli still growing at 1% ofnatural light intensity. Aer the 10 day experiment, the palat-ability of the alga to the snail was signicantly inuenced bylight conditions, but prior grazing had no effect. Consumptionrates were lower on the seaweed grown under natural light andmoderate shading relative to heavily shaded thalli. In contrast,the isopod grazed signicantly less on previously grazedseaweed, but did not differentiate among seaweed grown underdifferent light levels. The authors suggested that the algaexperiences species-specic reactions to herbivores and is more

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responsive to grazers that have higher rates of consumption.This specicity in grazer–algal interactions can be differentiallyinuenced by environmental conditions.

A similar study was conducted with the brown alga Fucusvesiculosus to examine the effects of light limitation andtemperature on palatability and inducible defenses of thisalga.68 Several different two-week long defense inductionexperiments were conducted under different temperatures (8–22 �C) and light intensities. Five to eight individuals of theisopod grazer Idotea baltica were used to induce defenses in thealga. Paired aquaria were used for each temperature or lighttreatment to assess inducible defenses, with one aquariumcontaining isopods and one without isopods. At the end of thetwo-week experiments, growth was determined as wet weightchange, C : N ratios and mannitol content was determined.Grazing assays with both live and pelleted, powdered F. ves-iculosus were conducted to test for treatment effects andinducible defenses. Mannitol, a primary product of photosyn-thesis in F. vesiculosus, was signicantly reduced in the presenceof grazers and very low light levels. Net growth of previouslygrazed algae was lower than in previously ungrazed algae atmost light treatments, and almost no consumption wasobserved on previously grazed F. vesiculosus at all light levels.The consumption of previously ungrazed algae increased withirradiance. No secondary metabolites, such as phlorotannins,were measured during this study, and thus the reduced palat-ability could not be directly attributed to induced chemicaldefenses. The various experiments demonstrated how palat-ability of F. vesiculosus can be affected by interactions betweenbiotic (grazing) and abiotic factors.

The effects of six levels of irradiance on palatability andantifouling defenses of ve species of Brazilian seaweedsshowed no effects of irradiance on the attractiveness of theseaweeds to amphipod mesograzers and a fouling organism,the mussel Perna perna.69 The authors had predicted that low-light stress would reduce the ability of the seaweeds to defendagainst grazers and macrofoulers.69 Generally, results of the 10–14 day irradiance exposures showed that growth and photo-synthetic rates of the seaweeds were reduced under low lightintensities. However, there was generally no effect on palat-ability of articial food pellets to an amphipod assemblagedominated by Elasmopus brasiliensis. Similarly, assays con-ducted with extracts of the algae coated onto lter paper toassess number of byssus threads for attachment by the musselP. perna did not discern any differences in activity across irra-diance levels. The authors discussed various reasons for thelack of response of algal defenses to light limitation, includingchanges in nutritional value of the algae and the allocation ofresources toward defense instead of growth.

The red alga Gracilaria chilensis is commercially farmed inChile for its agar production; infection by algal epiphytes is amajor nuisance to farmers. Weinberger and colleagues70

demonstrated that a dichloromethane extract and fractions ofG. chilensis exposed to epiphytes could trigger a defenseresponse against further epiphyte attack in the alga.HPLC analysis showed that treatment by the extract atconcentrations as low as 1 mg ml�1 induced an increase of 8-

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hydroxyeicosatetraenoic acid (8-HETE) 36 and 7,8-dihydroxy-eicosatetraenoic acid (7,8-di-HETE) 37, two metabolites previ-ously identied as playing a role in the activated defenseresponse of this alga. Upon fractionation of the dichloro-methane extract, one fraction was identied that contained theinductive activity. The chemical nature of the active moleculewas not determined, but several plant hormones such as methyljasmonate, linolenic acid, prostaglandin E2 and 7,8-di-HETEwere tested and were not active. Settlement of spores from theepiphytic red alga Acrochaetium sp. was developed into an assayto test whether an induced chemical defense against epiphyteswas present. Resistance to settlement of algal spores wasobserved when an initial 18 hour treatment of G. chilensis thalliwith the active extract or fractions was followed by a 54 hourincubation period. Resistance was not induced under shorterincubation time periods. The authors further explored theactivity of a putative lipoxygenase gene and key enzymesinvolved in formation of 7,8-di-HETE and found that inductionin the alga occurs aer an increase in lipoxygenase and phos-pholipase A2 activity.

3.3 Antimicrobial and antifouling defenses of macroalgae

A comprehensive review of chemical interactions betweenmacroalgae and bacteria has been published.14 This papercovers 40 years of research on macroalgal–bacterial interac-tions. Chemical interactions mediate many of these interac-tions between macroalgae and bacteria. Positive interactions,such as phytohormone activity, control of morphogenesis andcues for algal settlement and germination, as well as antibioticand antifouling activities are discussed. Perhaps nomarine algahas been better studied for its antimicrobial and quorumsensing inhibitory activity on bacteria than the red alga Deliseapulchra. Harder et al.71 provided an excellent review of twodecades of research on macroalgal–bacterial interactions in thisalga, based on the production of halogenated furanones by thealga. The halogenated furanones deter feeding by herbivoresand fouling by eukaryotic organisms, epiphytic bacteria, and


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potential bacterial pathogens. The antimicrobial activity ismediated by the interference of the furanones with bacterialquorum sensing. The halogenated furanones mimic the acylhomoserine lactones, one class of compounds that mediatequorum sensing in bacteria, and they occupy the same receptorbinding sites. The ability of the halogenated furanones fromD. pulchra to specically interfere with QS signaling in bacteriacan prevent or modulate bacterial colonization, biolm forma-tion, and bacteria-caused bleaching and disease.71

Populations of Delisea pulchra on the east coast of Australiashow signs of a seasonal bleaching disease in the summermonths, which is positively correlated with elevated seawatertemperatures but not with light levels. Campbell et al.72 inves-tigated the role of water temperature, algal chemical defensesand bacterial pathogens on bleaching in D. pulchra. Bleachedalgae had whitened areas that initially appeared anywhere onthe thallus (not just in sunlight exposed areas) and then spreadover the thallus. Case et al.73 showed that bleaching can beinduced in cultured D. pulchra in the laboratory. The productionof halogenated furanones can be manipulated in the laboratoryby growing sporelings in bromine-free articial seawater, whichlimits their ability to produce the halogenated compounds.Bleaching was induced when the production of halogenatedfuranones was inhibited in this way, temperatures wereincreased and algae were inoculated with certain strains ofbacteria that colonize algal surfaces.73 Campbell et al.72

measured concentrations of halogenated furanones by GC-MSin bleached and healthy algae collected in the eld duringsummer months and found that bleached individuals hadsignicantly lower levels of total furanones than healthy algae,and this pattern was more pronounced in deep than shallowwaters. Surface associated microbial communities of bacteriawere also signicantly different between healthy and co-occur-ring bleached algae based on analysis by terminal restrictionfragment length polymorphisms (TRFLP). Sporelings that weregrown in bromine-free and regular articial seawater (ASW) for4–6 weeks were outplanted to the eld at 3 m depth inside ofow-through growth chambers for 24 hours. The ones grown inbromine-free media did not produce furanones, and these algaebleached signicantly more than the control algae grown inregular articial seawater with bromine that did produce halo-genated furanones. In a laboratory experiment manipulatingtemperatures, microbial communities and furanone concen-trations (by using sporelings grown in regular and bromine freeASW), bleaching was more extensive in higher temperaturetreatments, consistent with eld observations. Additionally,thalli grown in non-sterile media bleachedmore than those thatwere grown in sterile media, and algae that produced furanonesthat were treated with antibiotics bleached less than untreatedalgae. The authors concluded that bleaching in D. pulchra is aresult of bacterial infections that are exacerbated by higherwater temperatures.

The bacterial community on the green macroalga Dictyos-phaeria ocellata was proled using denaturing gradient gelelectrophoresis (DGGE) and the alga was found to have a uniqueassemblage of bacteria on its surface compared to other closelyrelated algae (Batophora oerstedii and Cladophoropsis


macromeres) and inanimate surfaces collected in the samelocation.74 Compounds extracted from D. ocellata using thehexane dipping method were coated onto standard Petri dishesand had no effect on the composition of the bacterial commu-nity that formed on the surface compared to dishes coated insolvent alone. Crude whole-cell methanol and ethyl acetateextracts of D. ocellata were incorporated into Phytagel™ andtested for their effects on the development of bacterial biolmcommunities. The bacterial community that formed on thePhytagel™ plate containing D. ocellata methanol extracts wassignicantly different from the solvent control indicating thatrelatively polar organic compounds produced within the algamay be responsible for the development of unique bacterialassemblages on the surface of the alga. It was also found thateld enclosures containing D. ocellata had different bacterialcommunities than those without the alga indicating thatD. ocellata is also capable of altering the bacterial community inits closely surrounding seawater. D. ocellata inhibited thegrowth of three out of four naturally co-occurring bacterialstrains tested in laboratory co-culture experiments, but affecteddifferent bacteria during different parts of their growth curve.75

The activity of waterborne compounds produced by the algaunder co-culture conditions was tested by treating liquidbacterial cultures with either sterile media ltrate or organicextracts of the media from each co-culture treatment. Algalwhole-cell extracts were also tested to determine if activity couldbe attributed to compounds not released into the water. Thegrowth of each bacterial strain was affected by at least onecomponent tested; however, no two bacterial strains respondedin exactly the same way to all treatments, demonstrating adifferential response to algal compounds by different bacterialstrains. The authors suggest that this differential response maybe a mechanism by which the alga can regulate the compositionof its associated bacterial community.

The brown alga Fucus vesiculosus was further explored forpatterns of antifouling activity and antimicrobial protectionunder various environmental stresses, such as low light, hightemperature and high grazing.76 The density and compositionof the biolm was analyzed, primarily by cloning andsequencing the bacterial community. These results, because oflow replication, were mainly descriptive but showed somevariability among treatments as might be expected. Surfaceextracts (1 : 1 hexane–methanol) as well as whole algal extracts(1 : 1 dichloromethane–methanol) were tested in antimicrobialassays against marine bacteria and against the diatom Amphorasp. Both settlement and growth of the bacteria and diatom weremeasured. F. vesiculosus showed antibacterial activity, andinhibition of both growth and settlement of bacteria wasobserved, although the effect on growth was usually strongerthan on settlement of bacteria. Extracts of algae exposed to fullsunlight were more resistant to bacterial settlement than thosefrom shaded algae; but temperature and prior grazing treat-ments had no discernible effects on the activity of the algalextracts in the bacterial settlement assays. There was also astrong seasonal pattern, with the strongest anti-settlementactivity found in spring and early summer, which correspondedwith maximum reproductive periods for F. vesiculosus. This

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seasonal pattern was much stronger than patterns seen for theresponses of the alga to the different environmental stresses.

Phlorotannins were fractionated based on polarity andmolecular size from the brown alga Ascophyllum nodosum.77

Phenolic content of each fraction was assayed by the Folin–Ciocalteu method with phloroglucinol as standard. Thesefractions were assessed for radical scavenging activity with aDPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical method.Concentration of phenolics in the different fractions correlatedwith their radical scavenging ability, although the authorsproposed that the molecular size and structure could also play akey role in the radical scavenging activity of the differentfractions.

3.4 Algal–coral interactions

Coral reefs worldwide are undergoing phase shis from coraldominated ecosystems to algal dominated ecosystems. Withincreasing algal abundance on reefs, corals must compete withalgae for space and resources and in some cases defend them-selves from allelopathy. Combinatorial approaches frommicrobiology, molecular biology, microscopy, and chemicalecology have been used to identify the role of bacterialcommunities in ecosystem-wide responses to increased seasurface temperature, localized nutrient loading, and overshingof herbivores, all of which can lead to increased macroalgal andcyanobacterial cover on reefs. There has been a growing concernfor how overgrowth by algal turf, macroalgae and benthic cya-nobacteria will affect the health and resilience of benthicecosystems.78,80,81

Over the past three years a comprehensive body of literaturehas been published that demonstrates the negative impactsthat some macroalgae can have when they overgrow or competewith corals. These negative impacts can occur at all life historystages and can affect coral, their zooxanthallae, and the bacte-rial communities found in coral mucus. Algal chemicaldefenses can mediate these interactions. Rasher and Hay82

demonstrated that some macroalgae and their algal extracts(including algal surface extracts) embedded in Phytagel causedbleaching and death of coral tissue when in direct contact onreefs both in Panama and Fiji. They suggested that the presenceof lipid soluble compounds and direct contact were importantfor these interactions to occur. Many of these same algae werecontrolled by herbivores when placed on a reef protected fromshing, illustrating the importance of herbivory in controllingmacroalgal abundance on reefs and controlling the harmfuleffects of macroalgae on corals. Further studies of the effects of8 macroalgae on 3 different corals in Fiji highlighted the speciesspecicity of these interactions.83 All macroalgae had negativeeffects (bleaching, decreased photosynthesis) on the coralAcropora millepora, while another coral, Montipora digitata, wasonly negatively affected by three macroalgae. Bioassay guidedfractionation of crude extracts of two macroalgae that demon-strated the strongest allelopathic activities (bleaching anddecreased photosynthesis was observed aer extracts wereapplied to corals in Phytagel for 24 h) led to the isolation of twololiolide derivatives from the red alga Galaxaura lamentosa 38–

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39 and two diterpenes from the green alga Chlorodesmis fasti-giata 40–41. The isolated compounds were detected in surfaceextracts of the algae. The authors suggested that allelopathicinteractions between some macroalgae and corals may helpexplain the lack of coral recovery on many present-day reefs thatare dominated by macroalgae.

Dixson and Hay84 discovered that the common branchingcoral Acropora nasuta chemically signaled its symbiotic gobyshes to defend it from a competitive macroalga, Chlorodesmisfastigiata. When C. fastigiata was attached to A. nasuta coloniesoccupied by one of four different commensal shes, algalabundance declined by 30% on those corals hosting symbioticgobies (Gobiodon histrio or Paragobiodon echinosephalus).C. fastigiata was found in the gut of 85% of G. histrio occupyingcorals with C. fastigiata attached and in none of the G. histriooccupying corals without the alga. None of the P. echinosephalushad C. fastigiata in their guts, indicating that G. histrio eats thealgae, while P. echinosephalus removes the algae but does notingest it. G. histrio produce a toxic skin secretion, and mucusfrom G. histrio occupying corals with C. fastigiata attached wastwice as potent against predatory shes compared to mucusfrom G. histrio occupying corals without C. fastigiata. Damagefrom the toxic alga (assessed using coral photophysiology as aproxy) was reduced by 70–80% on corals occupied by gobiescompared to corals that had no sh symbionts. The symbioticgobies were triggered to defend their coral hosts in response tochemical cues released from the coral aer contact with thetoxic alga or contact with the lipid soluble extracts from thealga. Gobies presented with water collected at the site of theA. nasuta/C. fastigiata interface (before and aer the alga wasremoved) swam toward the cue while those presented withwater collected fromwithin the algal laments had no response.Gobies had no response to the same set of water samplescollected from interactions between a related coral, A. millepora,and C. fastigiata indicating that the gobies are responding tocues specic to their host species. The goby response was alsoelicited when algal mimics treated with lipid-soluble extractsfrom C. fastigiata were attached to the host coral.

Gene expression responses of the coral Acropora milleporaaer long term (20 days) contact with four of the macroalgae


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previously tested,85 and short term contact (1–24 h) withChlorodesmis fastigiata and its hydrophobic extract in Phytagelwere studied to better understand the effects of macroalgalcontact on gene expression and physiological responses ofthe coral. Custom microarrays with 148 holobiont genes (coraland zooxanthallae) were used to detect changes in geneexpression; an expanded array with 820 genes was used for thestudies with C. fastigiata. Interestingly, the algae that causedthe most noticeable effects on bleaching and photophysiologyof corals, including Chlorodesmis fastigiata, Dictyota bartayr-esiana and Galaxaura lamentosa, elicited fewer changes ingene expression patterns relative to Turbinaria conoides, whichdid not cause changes in bleaching or photophysiology of thecoral. Most genes that were affected were related to biosyn-thesis, cellular processes and metabolism. Upregulated genesincluded those related to heat shock proteins and transportgenes. Short term exposure to C. fastigiata and its hydrophobicextracts increased gene expression related to proteindegradation, catalytic activity and cellular metabolicprocesses, and decreased gene expression in signal trans-duction genes. Zooxanthellae genes that changed in theextract treatments were involved with protein degradation,mRNA processing and synthesis of the ribosomes. The authorssuggested that these molecular responses by the coral hol-obiont indicate changes in signal transduction and animbalance between reactive oxygen species and antioxidantcapabilities of the coral and its zooxanthellae when exposed tomacroalgal allelochemicals.

Field experiments with an additional four common speciesof macroalgae were conducted in Fiji to further explore thechemically mediated interactions between coral (Porites rus)and macroalgae.86 Contact with all four seaweeds for 7 dayscaused bleaching in P. rus, whereas contact with plasticmimics of seaweeds did not cause bleaching. The allelopathiceffects of the red alga Phacelocarpus neurymenioides, whichcaused moderate bleaching, were further explored. Bioassay-guided fractionation was used to determine whichcompounds caused bleaching in Porites rus, and the activitywas traced mainly to neurymenolide A 42, a known antibioticmetabolite in this alga.87 To better understand the distribu-tion of the compound on the surface of the alga where it couldcome in contact with corals and other organisms, desorptionelectrospray ionization mass spectrometry (DESI-MS) wasused to visualize and quantify neurymenolide A 42 on thesurface of live P. neurymenioides. This alga had the robust andat surface necessary for analysis by DESI-MS. Surfaceconcentrations were estimated by comparing integrals ofnatural surface concentrations to a standard curve. Theseanalyses revealed that concentrations of 42 on the older, lowerportions of the blade were more than double the concentra-tions on top or middle portions, and concentrations did notdiffer between edges and centers of blades. This studyprovided conrmation that the allelopathic compound occurson the algal surface, which allows it to transfer via directcontact and damage corals and other competitors when incontact on the reef.


Morrow et al.88 demonstrated that extracts of six macroalgaeand two cyanobacteria had species-specic effects on 54bacterial isolates from reef corals and macroalgae. Growth ofthe bacteria was monitored in the presence of nonpolar (1 : 1EtOAC–MeOH) and polar (1 : 1 EtOH–H2O) extracts from thealgae and cyanobacteria in 96-well plate bioassays. Thenonpolar extract from the brown alga, Dictyota sp., and polarextracts of Dictyota menstrualis and Lobophora variegata werehighly inhibitory to bacteria. Most of the other extracts stimu-lated bacterial growth. Bacteria cultured from different loca-tions on the reef (coral surfaces, macroalgal surfaces)responded differently to algal extracts, and bacteria in the genusPseudoalteromonas and other members of the order Alter-omonadales demonstrated the most growth in response to algalextracts. Some of these common algae produce broad spectrumeffects on bacteria, while the others demonstrated more speciesspecic interactions. Shis in the microbial assemblages oncorals may play a role in coral health and coral diseases.

Morrow et al.89 further explored the effects of macroalgae onmicrobial assemblages of corals by applying both live algae andnonpolar and polar algal extracts embedded in Phytagel for 72 hto two species of corals, Porites astreoides and Montastraeafaveolata, in Florida and Belize. The extracts used (nonpolarextracts of Dictyota sp., Halimeda tuna, Lobophora variegata, andpolar extract of L. variegata) were some of those that showedeffects on the 54 bacterial isolates tested in well plate assays.88

Surface mucus was collected before and aer application oftreatment and coral gels in sterile syringes for bacterialcommunity analysis by denaturing gradient gel electrophoresis(DGGE) of 16S rRNA gene amplicons. Coral tissue was alsosampled aer the application of the extracts and live algae forcellular stress enzyme assays. Some of the extracts and live algaecaused changes in bacterial assemblages in coral mucus layers.The polar extract of Lobophora variegata had the mostpronounced effect, causing a shi to an entirely new bacterialassemblage over the entire coral colony, whereas the nonpolarextract of Dictyota sp. had little impact on the coral colony.Macroalgal extracts more frequently caused sublethal stressresponses in M. faveolata than in Porites astreoides. GlutathioneS-transferase (GST) activity was elevated inM. faveolata exposedto all extracts except nonpolar L. variegata, whereas GST levels inP. astreoides were only affected by the polar L. variegata extract.None of the extracts caused changes in superoxide dismutase(SOD) or catalase (CAT) activities; however, the application oflive L. variegata to P. astreoides caused a decrease in both SODand CAT activity compared to controls. Some of these macro-algae and their extracts impacted the composition of microbial

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assemblages associated with corals and affected coral detoxi-cation enzymes, further illustrating the diverse impacts thatmacroalgae can have on corals and the species specicity ofthese interactions.

Not only the adult life history stages of corals are affected bymacroalgae, but larval settlement and recruitment processescan be affected as well.90,91 Two common brown macroalgae,Dictyota pulchella and D. pinnatida, were tested for their alle-lopathic effects on different life history stages of the coral Por-ites astreoides. The organic extracts of both algae signicantlyreduced larval survival, and the extract of D. pulchella alsonegatively affected larval recruitment.91 There was no effect ofthe extracts on the photophysiology of adult corals. The authorssuggested that these macroalgae can maintain their dominanceon degraded reefs by chemically inhibiting coral recruitment.Ocean acidication can also inhibit settlement and recruitmentof the coral Acropora millepora by disrupting the settlementprocess of coral larvae on preferred species of crustose corallinealgae, reducing settlement rates and disrupting larval behaviorby mechanisms that are not well understood.92

4 Seagrass

Several studies have investigated the phenols found in sea-grasses. Grignon-Dubois et al.93 report notable variability ofrosmarinic 43, caffeic 44 and zosteric 45 acids from Zosteranoltii collected in four locations across the Atlantic and theMediterranean coast. The concentrations of rosmaric and zos-teric acids were high depending upon the site; however, theconcentrations in caffeic acid were low at all sites.93

In a feeding study of seagrass species Thalassia testudinumand Halodule wrightii, the phenolic concentrations weremeasured over time prior to and aer exposure to the herbivoreLytechinus variegatus.94 Overall, the phenolic concentrationswere higher in both species in summer compared to fall.Following grazing, condensed tannin concentrations increasedin T. testudinum while in H. wrightii. p-hydroxybenzoic 46 andgallic 47 acids concentrations increased. Sea urchins did not

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show a preference in choice feeding experiments in which theanimals were offered agar food made with high- and low-phenolic seagrass tissue. The authors suggest that the change inphenol concentrations may have an effect on mesograzersinstead.94 Similar results were observed in choice and no choicefeeding assays conducted with grazed and ungrazed T. testudi-num and L. variegatus and the parrotsh Sparisoma radians.95

While the parrotsh did show a preference for ungrazed tissues,the sea urchins consumed both grazed and ungrazed seagrass.Darnell et al.95 suggested that variability in total reactivephenolics, nutrients and tissue toughness did not correlate withherbivore feeding behavior. The sh Sarpa salpa is reported tobe better adapted to consume polyphenolics and have a higherimpact on populations of the temperate seagrass Posidoniaoceanica than the sea urchin Paracentrotus lividus.96 S. salpashowed preference for younger leaves while P. lividus preferredthe older leaves. Additional experiments using an articial dietshowed that the urchins were deterred by structural rather thanchemical defenses.96

In feeding studies of Zostera marina with mesograzers, sea-grass palatability to isopods was shown to vary signicantly withthe genotype of the plants and decreased when the plant wereenriched with nitrogen.97 Other environmental factors such asocean acidication have been shown to affect concentrations ofphenolic substances in seagrasses.98 Concentrations of simpleand polymeric phenolics were lower in the seagrass Cymodoceanodosa near CO2 vents in Italy. Similar responses were observedin two estuarine seagrass species, Ruppia maritima and Pota-mogeton perfoliatus, in in situ CO2 enrichment experiments inthe Chesapeake Bay.98

5 Sponges

Sponges are becoming the dominant species, especially onCaribbean reefs, where reef-building corals are succumbing todisease and environmental changes in the ocean (i.e. pH,salinity and temperature). Studies clearly demonstrate thatsponges are chemically defended from predation and marinepathogens either by the compounds they produce or thoseproduced by symbionts or associated microorganisms.99–101

Sponges and their associated microorganisms continue to be aprolic source of novel natural products.102

Recent studies of the chemical ecology of sponges have beenaimed at understanding the evolutionary advantages thatsecondary metabolites and other defenses may have on thesurvival of sponges. Sponges in the same genus and oen in thesame species have been reported to produce different secondarymetabolites with distinct chemical structures. There are two


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studies that have addressed the intra-specic chemical diversityof the Mediterranean bath sponge Spongia lamella. In the rststudy, the authors compared the composition and concentra-tion of the major metabolites produced by samples collectedfrom seven populations spanning 1200 km in the NorthwesternMediterranean Sea. Specimens were collected by SCUBA fromsites in Marseille, France, Catalan, Spain and Cueta, Straits ofGibraltar. The compounds present in the extracts were identi-ed by LC-MS spectra and comparison with literature data.Individual samples were quantied by HPLC-electron lightscattering detection (ELSD). A total of 25 compounds wereidentied in the extracts; nintenin 48 and ergosteryl myristate49 were the major metabolites. Qualitative variation of thenumber of compounds in the sponge extracts and quantitativevariation in the concentration of these metabolites wereobserved among the populations, increasing with distance,while there were consistent patterns within regions. Thesevariations are suggested to be a result of environmental andgenetic factors. The authors noted that the ecological functionof these compounds is still unknown.103

In the second study of Spongia lamella, the authors investi-gated the possible association of genetic, bacterial and chem-ical diversity of nine sponge populations collected from veregions: South France, Catalonia, Baleares, Gibraltar and


Portugal. Diversity was estimated at three levels, individual,population and interpopulation, for genetic, bacterial andchemical diversity. Overall the data showed that the spongepopulations differed signicantly in genetic, bacterial, andchemical diversity with considerable variation among the threelevels. Similar to their previous study of S. lamella, the authorsobserved a strong geographic pattern of increasing genetic,bacterial, and chemical variation with increasing geographicdistance between populations.104

An investigation of the intra-specic variability in secondarymetabolites among population of the Indo-Pacic sponge Styl-issa massa collected in American Samoa, Pohnpei, Saipan, andGuam reported both qualitative and quantitative variationamong the populations.105 Sponge extracts were analyzed byhigh-pressure liquid chromatography (HPLC). Six metaboliteswere detected and quantied in the sponge samples includinghymenidin 50, sceptrin 51, oroidin 52, debromohymenialdisine53, hymenialdisine 54 and palau'amine 55. In the Guam pop-ulations the concentrations of 50–52 varied among the reefswhile the concentrations of 53 and 54 were consistent amongpopulations. Sponges from two reefs on Guam had signicantlylower concentrations of all ve compounds compared to theother sponges collected for the study. The concentration of 50and 54 did not vary signicantly within the Indo-Pacic pop-ulations, whereas there were signicant qualitative and quan-titative differences in the concentrations of 51, 52, and 55.Palau'amine 55 was only detected in the American Samoacollections. A eld experiment conducted in the same study, inwhich specimens from 3–4 m were transplanted to 23–24 m andvice versa, showed that there was no change in concentrations of50 aer six weeks; however, the concentration was signicantlyhigher in all samples originally collected from the deep reef.The ecological role of these metabolites is unknown for thesponge. The authors conducted feeding experiments against thepuffersh Canthigaster solandri with the crude extract andindividual compounds and combinations of the compounds.While the crude extract signicantly deterred feeding, 50–55 didnot account for the feeding deterrent activity.106

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Chemical defenses of sponges have been demonstrated to beeffective against diverse consumers. In a study of Hymeniacidonheliophila collected in Rio de Janiero, crude extracts wereassayed in the laboratory against the hermit crab (Calcinustibicens), sea urchin (Lytechinus variegatus) and in the eldagainst generalist shes. Three extracts were prepared withn-hexane, ethyl acetate and acetone/methanol for use in theassays. Feeding by the hermit crab C. tibicens was signicantlyreduced by all three extracts while only the n-hexane extractdeterred feeding by the sea urchin L. variegatus. In the eldassays, only the ethyl acetate and acetone/methanol extractsdeterred sh feeding.107

The authors of the previous study also reported the anti-foulant properties of the three crude extracts prepared fromH. heliophila. The n-hexane, ethyl acetate and acetone/methanolextracts were tested at natural concentrations against the byssalattachment of the mussel Perna perna in 24 hour laboratoryexperiments. In these experiments, all three extracts signi-cantly inhibited the attachment of the byssal threads. Theantifouling chemical defense in this sponge may provide it witha competitive edge in an environment where there is ercecompetition for space.108

Early studies of sponge chemical ecology have shown that thedistribution of chemical defenses is not ubiquitous to thisphylum and may control sponge distributions on temperatereefs. One study investigated the relationship between spongepredators and the distribution of sponges on temperate reefs inthe South Atlantic Bight (SAB), Georgia. Transects at sites alongtwo different reefs showed that both habitats have high spongedensity and similar sponge diversity. Amorphous and encrustingsponges were found in the scarp (vertical, rocky outcroppings)sponge community, while the pedunculate, digitate, and arbo-rescent growth forms occurred in the plateau (sediment-ladenreef top) sponge community. Feeding assays were conducted inthe eld with crude extracts or structural components from 19sponge species against natural assemblages of generalist sh.The crude extracts from sponges collected from the scarpdeterred signicantly more sh than the structural components,while the structural components of more than half of thesponges collected from the plateau deterred signicantly moresh than the extracts. Transplant experiments with Chondrillaaff. nucula, Chondrosia collectrix, and Hyrtios violaceaus from thescarp and Axinella waltonsmithi, Axinella pomponiae, Desmap-samma anchorata, and Ptilocaulis walpersi were collected fromthe plateau showed signicant volume changes and scarring ofthe sponge tissue from predation were observed for 3 of the 4sponges transplanted from the plateau to the scarp.109

Structural defenses have been less commonly reported forsponges relative to chemical defenses. In a study of the defensemechanisms of the non-siliceous spongeMelophlus sarasinorumcollected fromWestern Shoals in Apra Harbor, Guam both eldand laboratory experiments were used to investigate thechemical and structural defenses of the sponge in two distincttissues, the ectosomal and choanosomal tissues of the sponge.The ectosomal tissue of M. sarasinorum is 3–4 mm thick, madeof a tough brous material, and can be easily distinguishedfrom the choanosomal tissue by color. Individual specimens

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were sectioned and the resulting tissues were powdered andextracted for chemical assays or extracted whole, dried andchopped into small pieces for structural assays or measured fortoughness and protein concentration. In laboratory feedingassays against Canthigaster solandri and eld feeding assayswith the crude extracts from both the ectosome and the chao-nosome, all of the extracts signicantly deterred sh feeding. Inthe laboratory feeding assays only the tissue from the ectosomewas unpalatable to C. solandri, suggesting that the structuraldefense is restricted to the top layer of the sponge. In additionto the feeding assays, the authors conducted a eld transplantexperiment in which individual sponges were collected fromApra Harbor and transplanted to another reef at the MarinePreserve at Tumon Bay. Videos of the caged and control speci-mens outside of cages suggested that sh predation is notcommon for M. sarasinorum; however, eld transects did showsmall hermit crabs and shrimp associated with the surface ofthe sponge. The authors propose that the distribution of thechemical defense and structural defenses of M. sarasinorummeet the predictions of the Optimal Defense Theory. In thismodel, the chemical defense is distributed throughout thesponge to limit sh predation, and structural defense at thesurface layer prevents predation by mesograzers in the mostvulnerable tissues.110

A similar study of the Brazilian sponges Tethya rubra andTethya maza also addressed chemical and structural defenses.T. rubra and T. maza were collected at Todos os Santos Bay,Salvador and Tarituba Beach, Brazil, respectively. The chemicalcomposition of the extracts was compared using thin layerchromatography (TLC), high-resolution gas chromatographyfollowed by mass spectrometry (HRGC-MS) and nuclearmagnetic resonance (NMR) spectroscopy. The chemical prolesof T. rubra and T. maza were similar and were reported tocontain approximately 45–47% sterols as themajor metabolites.The crude extracts and spicules isolated from the sponge tissuewere assayed alone or in combination against the hermit crabCalcinus tibicen using a squid paste food. The crude extract fromT. maza signicantly deterred feeding by the hermit crab whilethe extract from T. rubra did not. Sclerites from T. rubra andT. maza signicantly deterred feeding by the hermit crab atnatural concentrations (0.09 and 0.04 g ml�1, respectively). Therewas no evidence of additive or synergistic effects in the feedingassays using crude extracts in combination with the sclerites.111

The distribution of defenses within the tissues of thesponges is a topic that has received considerable attention sincethe last review. In a study of the aspiculate sponges Ircinia felix,I. campana, and Aplysina fulva, the authors addressed a similarhypothesis predicting that chemical defenses would beconcentrated in the outermost 2 mm layer of the sponge andhave a positive correlation with tissue nutritional quality andnegative correlation with structural components such asspongin bers. I. felix, I. campana, and A. fulva were collectedfrom J Reef off the coast of Georgia, U.S.A., and the outer 2 mmof sponge (outer region) was removed from the inner region.Gas-chromatography-mass spectrometry (GC-MS) was used toidentify the furanosesterterpene tetronic acids 56 from theIrcinia species, and liquid chromatography-mass spectrometry


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(LC-MS) was used to identify the brominated tyrosine 57derivatives from A. fulva. The concentrations of the fur-anosesterterpene tetronic acids from the Ircinia species andbrominated tyrosine derivatives from A. fulva were determinedin the outer region and inner region using HPLC. Similarly, theprotein and carbohydrate concentrations and total structuralcontent were determined for the distinct tissues. The bromi-nated tyrosine derivatives from A. fulva were concentrated in theouter 2mm of the branched sponge, while the concentrations ofthe furanosesterterpene tetronic acids from I. felix were highestin the inner region of the sponges and evenly distributed in thetissues in I. campana. Protein concentrations were higher in theinner region of Ircinia species but did not differ for A. fulva, andconsiderable variability was observed for carbohydrate contentamong the species and within the sponges themselves. Overall,there were no signicant positive or negative correlationsbetween chemical defenses and nutritional quality or levels ofstructural components in these sponges. The authors suggestedthat allocation patterns cannot be predicted and further studiesare warranted to determine the effects of the observed patternson feeding deterrence by sh and mesograzers.112

In a follow up study, Freeman and Gleason113 explored thehypothesis that the higher levels of chemical defenses observedin the outer tissue regions of Aplysina fulva and the inner regionsof Iricina felix would result in enhanced feeding deterrence, whilethe similar concentrations found in both regions of I. campanawould deter predators equally. The sponges A. fulva, I. felix, andI. campana were collected from a temperate hard-bottom reef inthe South Atlantic Bight and separated into the outer and innerregions similar to the previous study. Field assays with naturalsh assemblages were only conducted with the extracts fromA. fulva and I. campana due to the limited availability of I. felix.Extracts from all three species were assayed with the sea urchinArbacia punctulata in laboratory assays. All of the extracts fromthe outer and inner regions of the sponges signicantly deterredsh feeding in the eld and laboratory experiments. While it waspredicted that higher concentrations of defensive metabolites inthe outer and inner regions A. fulva and I. felix, respectively,would have a greater effect on sh feeding there was no differ-ence observed between the amount of food consumed containingthe outer and inner treatments in the feeding assays. Thechemical defenses were effective deterrents at the lowerconcentrations observed in the sponge tissues, and the authorssuggested that observed distribution patterns of compoundsamong these sponges may result from other ecological stressorsother than generalist sh predation.113


Chemical defenses of sponges against emerging diseases inthemarine environment has become a topic of particular interestto biologists and chemical ecologists in recent years, raisingquestions with regard to adaptability and survival of specicpopulations. In a study of Aplysina cauliformis collected from theBahamas, the chemical defenses of healthy sponges and speci-mens infected with Aplysina Red Band Syndrome (ARBS) werecompared. HPLC analysis of extracts from healthy and diseasedAplysina cauliformis infected with ARBS demonstrated qualitativeand quantitative differences in the chemical proles. Aplys-amine-1 58 and stularin-3 59 were present in signicantlyhigher concentrations in healthy sponges, whereas aerothionin60 and 11-oxoaerothionin 61 were found only in diseasedsponges. The organic and aqueous extracts from the healthy anddiseased sponges signicantly deterred feeding by the puffershCanthigaster rostera. In the case of 59, sh feeding was deterred atthe higher concentration present in healthy sponges. Theconcentration of 60 in diseased sponges was sufficient to deterfeeding at levels similar to 59, while 58 did not deter sh feeding.In addition to the feeding assays, the extracts from healthy anddiseased sponges were assayed against four known coral bacterialpathogens, one human enteric bacterium, and three otherbacteria isolated from the surface of corals in disc diffusionassays. The organic extracts from healthy and diseased spongesexhibited similar antibacterial proles and inhibited the growthof all eight of the panel organisms, while 58 inhibited the growthof 3 of the eight bacteria and 59 inhibited the growth of four ofthe eight bacteria. The healthy and diseased sponges appear toproduce different and effective chemical defenses againstpredation and bacterial infection.114

Two studies of the Aplysina aerophoba address the biosyn-thetic source and temporal variation in the production of

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brominated alkaloids reported from this genus. Studies ofA. aerophoba suggest that the brominated alkaloids (BAs) arebiosynthesized within the sponge cells; however, the bromo-peroxidase enzymes that bind halogen elements to thecompounds have been exclusively described in algae, fungi, andbacteria. In this study, the authors explore the hypothesis thatthe biosynthesis of the brominated alkaloids occurs in a coop-erative process between the sponge andmembers of its complexassociated bacterial community by investigating the correlationin changes of the concentration of BAs and the bacterialassemblages. Sponges were collected in Costa Brava in theNorthwestern Mediterranean. Samples were taken from onesponge from every site and samples were taken from the ecto-some and choanosome for chemical and bacterial assemblageanalysis. The brominated alkaloids were quantied by high-performance liquid chromatography (HPLC), and bacterialassemblages were analyzed using denaturing gradient gel elec-trophoresis on the 16S rRNA gene. Four of the six majormetabolites in the sponge proles were identied as aero-phobin-1 62 aerophobin-2 16, aplysinamisin-1 15, and iso-stularin-3 17. The concentrations of 15–17 were signicantlyhigher in choanosome tissues compared to the ectosometissues. There were no differences observed between individualsponges. The bacterial diversity of the choanosome and theectosome tissues mirrored the observed chemical diversity.Twenty-four bacteria were identied, and seven varied betweenthe two tissues. Factor analysis revealed positive correlationsbetween some Chloroexi strains and the concentration of 15,and a negative correlation with a cyanobacterial strain to thesame compound. These bacterial strains were found in thechoanosome and ectosome of the sponge, respectively. Ag-proteobacteria strain and one unidentied strain (OTU19)were positively correlated to the concentration of 16 and 17. Inaddition, the concentration of 62 was correlated with an Acti-nobacteria strain. The authors suggested that the bacteriaidentied in this study could either be involved in the produc-tion of brominated alkaloids or be directly affected by them.35

Temporal changes in the production of brominated alka-loids in the ectosome and the choanosome of Aplysina aero-phoba were quantied over a two-year period. Sponge samplingtook place monthly by SCUBA in Portbou in the NorthwesternMediterranean. Samples were sectioned into the ectosome andthe choanosome for chemical analysis. The concentrations ofaerophobin-2 16, aplysinamisin-1 15, and isostularin-3 17werequantied by HPLC using standard curves. Temporal variationwas observed in the ectosome, showing high concentrations of15 and 17 during the summer months, while the concentrationof aerophobin-2 increased in the month of August anddecreased in the month of February. In the choanosome, 15 and

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17 concentrations were high in August and decreased inNovember and the concentration of 16 was higher in Januaryand lower in March. Changes in water temperature correlatedwith changes in the concentration of 16 and 17 in the ectosome.The authors conclude that studies of this kind may lead to thebetter understanding of the production of target compoundswithin the sponge and could be used to maximize the supply ofthe compounds by harvesting at peak production times while atthe same time reducing the impact on the populations.115

The allocation of resources and thus difference in growthstrategy has been considered a possible explanation for thepresence or absence of chemical defenses among spongespecies. To further explore this hypothesis, an indirect study ofthe reproductive output of known undefended and defendedsponges from the Conch Wall and the adjacent shallow patchreefs in Key Largo, FL was conducted from November 2007 toOctober 2008. Monthly samples were taken from undefendedspecies Iotrochota birotulata, Niphates erecta, Callyspongiaarmigera and C. vaginalis, and the defended species Aplysinacauliformis, A. fulva and Amphimedon compressa. Samples wereprocessed for histology, sectioned, stained, and photographedfor the presence of reproductive propagules (oocytes, embryosor larvae). When the reproductive output index (ROI: % area ofpropagules/total area of tissue scanned) was compared amongthe sponge species there was considerable inter-species vari-ability with no signicant differences between undefended andchemically defended species. The authors suggested that amajor limitation to this kind of study is the challenges associ-ated with measuring and comparing trade-offs among spongespecies with different modes of reproduction.116

The use of environmental strains of bacteria as potentialscreens for novel compounds is becoming of greater interest tochemical ecologists. Bioassay-guided fractionation of then-butanol extract of the Mediterranean sponge Axinella verru-cosa, using the environmental marine bacterium Lysinibacillussp. ESY 9 (GenBank accession no. GU059941) yielded a novelantibacterial alkaloid debromo-carteramine A 63 as well as theknown compound hymenidin 64.117

Secondary metabolites may have multiple defensive roles forthe producer. A study of co-occurring Mediterranean sponges,


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Axinella polypoides and A. verrucosa, collected from the Gulf ofNaples, Italy investigated two solvent partitions and pyrrole–imidazole compounds, hymenidin 64 and debromo-carter-amine A 63, for microbial antifouling activity, antibacterialactivity and feeding deterrence activity against the generalistshrimp Palaemon elegans. Proton nuclear magnetic resonance(1H NMR) quantication was used to conrm the presence of 63and 64 in the n-butanol partition of the A. verrucosa extract.Bacterial settlement assays were carried out in the eld byincorporating the ethyl ether (Et2O) partition or the n-butanolpartition into a 5% agar solution at 10� the natural concen-tration and deploying the Petri dishes at the site where thesponges were collected. Plates were retrieved aer 24 hours andsettlement was measured using DAPI staining and counting thecolony-forming bacteria. Colony-forming bacteria were culturedon additional plates using Marine Broth 2166 agar. Similarresults from both assay methods showed that only the n-butanolpartition of A. verrucosa signicantly inhibited bacterial growthin the eld experiment. Disc diffusion assays with laboratoryand environmental strains of Gram-negative and Gram-positivebacteria displayed some similarity to the results observed in theantifouling assays. The partitions from A. polypoides did notinhibit the growth of any of the test strains while the n-butanolpartition of A. verrucosa inhibited the growth of Escherichia colistrain GM1655 and all strains of Gram-positive bacteria. TheEt2O partition of A. verrucosa also inhibited the growth of 3Gram-positive bacteria. Additional assays with the purecompound 64 exhibited an identical antimicrobial prole as then-butanol partition; however, the activity levels appeared to betoo low to account for all of the activity observed for the extract.Finally, feeding assays were conducted in tanks in the labora-tory in which the Et2O partition, n-butanol partition or 64 wasincorporated into a squid pellet at natural concentration.Similar to the other assays in the study, the n-butanol partitionof A. verrucosa deterred feeding by the shrimp while the Et2Opartition of A. verrucosa and both extracts from A. polypoides didnot have an effect. Hymenidin 64 effectively deterred feeding atthe natural concentration of 3 mg ml�1 or above. Multipledefensive roles were found for 64 produced by A. verrucosa;however, it is unclear whether A. polypoides produces chemicaldefenses and further studies are warranted.118

A survey of the antimicrobial activity of lipophilic andhydrophilic crude extracts was conducted with extracts from 25Antarctic sponges collected from Palmer Station, Antarctica. Apanel of 20 bacterial isolates cultured from the frozen tissuesamples of demosponges collected from the same habitat con-sisted of 16 strains of g-proteobacteria, one Flavobacterium,and three unidentied isolates. Antibacterial activity wasassessed at 1� and 3� natural concentration using a discdiffusion assay. Extracts were also assayed against the diatomSyndroposis sp. isolated near Palmer Station in a 96-well plateformat at 0.3�, 1�, and 3� natural concentration. While therewas sporadic or no antibacterial activity observed for almost allof the extracts, 96% of the lipophilic extracts caused signicantdiatom mortality at the estimated natural concentration and60% causing mortality at 30% of the natural concentration.Sixty percent of the hydrophilic extracts resulted in signicant


diatom mortality at the natural concentration, while 24% of thehydrophilic sponge extracts resulted in signicant diatommortality at 30% of the natural concentration.119

Larval settlement of sponges has been the topic of two recentstudies. The rst study investigated the role tannins play insponge recruitment in a mangrove-sponge. Tedania ignis wascollected in Spaanse Water, Curaçao and used in two eldexperiments. To assess larval recruitment by tannins, tannicacid embedded in a polymeric gel as an articial substrate wasplaced in the shallow mangrove fringes for seven weeks. Toassess the effect of sponge colonization on the production oftannins in mangrove roots, T. ignis collected from the adjacentbenthic community was transplanted to bare mangrove roots.Tannin concentrations and total phenolics were determined forthe prop roots. Signicantly more T. ignis larvae settled on proproots with high tannin concentrations (1.8 nM) than the controland prop roots with lower concentrations (0.3 nM). The resultsfrom the transplant experiment demonstrated a signicantincrease in tannin and polyphenolic concentrations in rootswith sponges attached either naturally or articially during theexperiment. The authors suggest that there is a positive feed-back in recruitment of T. ignis larvae.120

The second study assessed larval settlement in two commonGreat Barrier Reef sponges, Coscinoderma matthewsi and Rho-paloeides odorabile. Female C. matthewsi were collected fromPioneer Bay, Orpheus Island and female R. odorabile werecollected from Rib Reef. Larvae were collected usingmesh traps.Settlement experiments were conducted with methanol extractsprepared from the crustose coralline alga (CCA) Porolithononkodes collected from Bramble Reef, live CCA and cnidarianneuropeptides (GLW-amide neuropeptides) purchased fromSigma Genosys. Settlement times varied between the larvaefrom C. matthewsi and R. odorabile. Settlement was observed forboth sponge species at increased rates in response to live CCAand optimum concentrations of CCA extract of 1–30 mM and 10–30 mM for C. matthewsi and R. odorabile, respectively whencompared to 0.2 mm ltered seawater controls. The presence ofGLW-amide neuropeptides increased metamorphosis inR. odorabile, 42% compared to 16% in seawater controls, and inC. matthewsi, 68% compared to 36% in seawater controls. Theresults of these experiments demonstrate that sponge larvaehave the ability to identify suitable habitat for successfulrecruitment. The effect of CCA on larval recruitment alsosuggests a potential overlap with signal transduction pathwaysinvolved in metamorphosis.121 The recent advances in this areasuggest that sponges are rich in chemistry with the potential toprotect them from predators, infection and act as signalingmolecules for larval recruitment.

6 Cnidarians

Cnidarians continue to be rich sources of novel secondarymetabolites. In the rst decade of the 21st century over 2000 newsecondary metabolites were described from cnidarians.122 In thelast two years, several review articles have highlighted the vastdiversity of natural products produced by cnidarians, focusingon their pharmaceutical and industrial potential.122–125 Reviews

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on cnidarian venoms and the toxins comprising them havebeen published recently, including a paper on Mediterraneanjellysh venoms126 and an overview of what is known to dateabout the structure and function of sea anemone toxins,including descriptions of the genes involved in toxin produc-tion.127 Rachamim and Sher128 reviewed the main toxinsproduced by Hydra and made the case that natural productsproduced by cnidarians have a large range of activities with thepotential for ecosystem-wide impacts and that Hydra are goodmodel organisms to study the chemical ecology of theseanimals.

The production of potent toxins associated with nematocystsis an area of much interest in the chemical ecology of cnidar-ians. A new method for examining the identity of neurotoxinspresent in different types of nematocysts revealed that two typesof nematocysts found within the jellysh Aurelia aurita con-tained different toxins.129 The two types of nematocysts (eur-yteles and isorhizas) were isolated by rst macerating thejellysh tissue in distilled water and then separating them usinglaser microdissection and pressure catapulting (LMPC). Matrix-assisted laser desorption/ionization-time of ight mass spec-trometry (MALDI-TOF MS) of the resulting collections of iso-lated nematocysts (70–80 cells each) resulted in different massspectra. While the authors were unable to determine the iden-tity of compounds indicated by themass spectra, they suggestedthat this was evidence that different types of nematocystscontain different toxins.

Another recent study on toxins associated with nematocystsrevealed that in some sea anemones the potent neurotoxinspreviously thought to be localized within nematocysts wereactually found in ectodermal gland cells.130 The starlet seaanemone Nematostella vectensis produces a potent Type 1neurotoxin, Nv1. Using immunolocalization techniques it wasdiscovered that Nv1 is conned to glandular ectodermal cellsand is not found within the nematocysts. Furthermore, thetoxin was secreted from the gland cells when the anemone cameinto contact with crustacean prey, and both sh and crusta-ceans were susceptible to the toxin when it was deliveredexternally in the seawater surrounding the prey, without thepuncture of a nematocyst. Two other anemones examined alsopossessed toxin-containing ectodermal gland cells. Type 1neurotoxins were found exclusively in ectodermal gland cells inAnthopleura elegantissima but were found in both ectodermalgland cells and nematocysts in Anemonia viridis. The authorssuggested that the location of Type 1 neurotoxins may provideinformation about the evolution of venom delivery mechanismswithin sea anemones.

Fire corals contain a number of unknown toxins that havebeen shown to have various toxicological and pharmacologicalactivities. A recent study investigated the importance of thecoral's symbiotic algae (zooxanthellae) in the production ofsuch toxins in an attempt to understand how periods of coralbleaching might affect coral toxins.131 Aqueous extracts frombleached hydrocorals (Millepora alcicornis and Millepora com-planata) retained the same vasoconstrictor activity as healthycolonies containing zooxanthellae. The authors suggested thatthis indicates that the compounds responsible for eliciting this

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effect are produced by the corals themselves and not theirsymbiotic algae. However, the active compounds could beproduced by the zooxanthellae and stored by the corals duringbleaching events. Boiling the aqueous extracts did notcompletely eliminate their activity suggesting that there areboth heat-stable and heat-labile compounds involved in thevasoconstrictor activity. The heat-stable compounds are likely tobe secondary metabolites other than peptides, which typicallybreak down under high temperatures (>60 �C). Interestingly,hemolytic activity was reduced in extracts from bleached coralscompared to non-bleached controls indicating that either thehemolysins are being produced by the zooxanthellae or that theproduction of hemolysins by the coral requires contributionsfrom the symbiotic algae. Bleaching signicantly reduced butdid not eliminate phospholipase A (PLA2) activity in M. com-planata but did not affect PLA2 in M. alicornis, suggesting thatcompounds responsible for PLA2 activity are being produced bythe corals and not their algal symbionts. Overall, this studyprovided evidence that toxicologically and pharmacologicallyactive compounds found in aqueous extracts of hydrocorals inthe genus Millepora are being produced by the coral itself andthe production of these compounds is not dependent on thepresence of the symbiotic algae. Therefore, it is likely that thesehydrocorals maintain the ability to produce toxins necessary fordefense and feeding during bleaching events.

Hines and Pawlik132 recently examined the prevalence ofnematocyst and chemical defenses in non-scleractinian zoan-tharians (sea anemones, zoanthids and mushroom polyps) todetermine whether there are trade-offs between defensivestrategies in these animals. Chemical extracts of 5 of the 18species tested were deterrent against feeding by the blueheadwrasse, Thalassoma bifasciatum, when extracts were incorpo-rated into feeding pellets. Interestingly all ve chemicallydefended species came from reef habitats. None of the speciesfrom mangroves, seagrass beds, or intertidal zones werechemically defended. Twelve of the 18 species tested possessednematocyst defenses, measured by the intensity of the tentacleresponse to prey, duration of tentacle contact with prey, andreaction of prey to contact with the tentacle. The authorsconcluded that there was limited evidence for a trade-offbetween defensive strategies because 65% of the species testedhad either chemical or nematocyst defenses, but not both. Theother 35% of species tested had either both chemical andnematocyst defense or neither type of defense. The nutritionalquality of the zoantharians was also assessed to determine ifpoor nutritional quality could also be functioning as a defensivestrategy for these species. While all species tested had totalenergy content comparable to other benthic invertebrates, thesoluble protein content was notably lower. There was noevidence for a relationship between other defensive strategiesand nutritional content.

So corals continue to be investigated for their ability tochemically defend themselves against predation and fouling.Only ethyl acetate extracts from the gorgonian coral Subergorgiareticulata were tested, and they showed antifouling activityagainst a suite of common macrofoulers dominant in the SouthChina Sea.133 Extracts coated onto Petri dishes completely


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inhibited settlement of acorn barnacle (Balanus reticulatus)larvae at concentrations above 1 mg cm�2, but not at concen-trations between 0.01 and 0.1 mg cm�2. Extracts exhibitedinhibitory activity at concentrations as low as 0.01 mg cm-2 andcompletely inhibited settlement of pearl oyster (Pinctada mar-tensii) larvae at concentrations above 1 mg cm-2. Germination ofspores from the green algae Ulva lactuca and Ulva linza wasreduced compared to controls in Petri dishes coated withextracts at a concentrations > 0.01 mg cm�2 and completelyinhibited at 100 mg cm�2. Extracts signicantly reduced sporegermination in the red alga Gracilaria tenuistipitata at concen-trations above 0.01 mg cm�2 and completely inhibited germi-nation at 100 mg cm�2.

Two new fatty acids named renillenoic acids 1 65 and 2 66were discovered in the Patagonian sea pen Renilla octo-dentata.134 These compounds are analogs of eicosapentaenoicacid (EPA) and arachadonic acid (AA) and made up approxi-mately 20% of the organic extract from R. octodentata. Thecompounds were distributed uniformly throughout the octo-coral tissue, occurring at equal concentrations in the rachis(main axis) and peduncle (lower unbranched part of the stalk).Tissue from the rachis of R. octodentata, crude extracts, andsclerites at natural concentrations all deterred feeding by thegeneralist sh Pagrus pagrus. The individual renillenoic acidscould not be tested directly because of their instability. Acetoneand ether extracts of R. octodentata and a mixture of 65 and 66inhibited settlement of barnacle (Amphibalanus amphitrite)larvae by 50% at concentrations estimated between 1 and 10 mgl�1, but had no effect on larval mortality in toxicity assays.

Gorgonian corals produce compounds that affected thegrowth and behavior of bacteria, demonstrating the potential toregulate the composition of their associated bacterial ora.135

Ethanol extracts from eight gorgonian coral species were testedfor their antimicrobial activity against a panel of marine path-ogens and non-marine human pathogens. All eight speciesinhibited bacterial growth; however, which bacterial strainswere inhibited by each gorgonian species was highly variable.Overall, gorgonian extracts had a greater effect on non-marinebacteria than marine bacteria. Extracts of several speciesinhibited and/or stimulated quorum sensing activity. Plexauraexuosa extract inhibited the quorum sensing reporter for longchain N-acyl-homoserine lactones (AHL) signaling molecules.Pseudopterogorgia americana and P. acerosa inhibited the shortchain AHL reporter. P. porosa inhibited the short chain AHLreporter, but strongly stimulated the long chain AHL reporter.Gorgonia ventalina showed moderate inhibitory activity towardslong and short chain AHLs, but also had moderate stimulatoryactivity toward the long chain AHL reporter. The authors sug-gested that this species may contain multiple compounds that


act in different ways on the quorum sensing pathway. Thesestudies indicate that instead of limiting growth of marinebacteria directly, the gorgonians tested here control the bacteriathrough the induction or inhibition of quorum sensing signals.

Crude extracts, pseudopterosins and seco-pseudopterosinsfrom the gorgonian Pseudopterogorgia elisabethae collected inthe SW Caribbean selectively inhibited the growth and biolmformation of Gram-positive marine bacteria isolated fromheavily biolmed surfaces.136 These compounds had minimaleffects on the growth of Gram-negative bacteria isolated fromthe same source and enhanced biolm formation in thesestrains. The selective inhibition of Gram-positive bacteria andenhancement of Gram-negative bacteria was reected in thecomposition of the bacterial community (92% Gram-negativeProteobacteria) found on the surface of P. elisabethae as deter-mined by uorescence in situ hybridization (FISH). The fractionof the crude extract that contained pseudopterosins and seco-pseudopterosins also exhibited selective antibacterial activityagainst bacterial strains isolated from the surface of P. eli-sabethae, inhibiting the growth of 11 Firmicutes strains and 8Actinobacteria strains but having no effect on the growth of 8Proteobacteria strains tested.

The success of invasive species can sometimes be attributedto the production of potent secondary metabolites. The chem-ical composition of two species of cup corals, Tubastrea tagu-sensis and T. coccinea, that have successfully invaded the coastof Brazil were investigated by GC-MS, and a series of experi-ments were performed to determine if these corals alter theirchemical make-up in response to wounding or competition withnative organisms. Colonies of both corals contained a diversityof hydrocarbons, fatty esters, and sterols.137 In order to deter-mine if these corals release compounds into the surroundingseawater upon wounding, compounds were extracted fromseawater near articially injured T. tagusensis and bleached T.tagusensis skeleton controls using in situ Sep-Paks. Five hydro-carbons were identied from seawater surrounding both thecontrol and the injured T. tagusensis colonies. Of these, three (1-hexadecene, n-hexadecane, and n-eicosane) were found insignicantly higher concentrations in seawater near injuredT. tagusensis.137 Proximity to two potential competitors, acommon native coral Mussimilia hispida and an importantspace occupier, the sponge Desmapsamma achorata, causedchanges in the chemical composition of both species of cupcorals.138 For T. tagusensis there was no signicant difference inthe overall chemical make-up of the coral exposed to competi-tors versus controls, but there was a signicant decrease in theconcentration of 5-bromoindole-3-carbaldehyde 67 in coralsexposed to the sponge and a signicant increase in theconcentration of stigmasta-5,24(28)-dien-3b-ol 68 in coralsexposed to M. hispida. There was an effect of treatment on theoverall chemical composition of T. coccinea; however, there wereno signicant differences in concentrations between controlsand treatments for any individual chemical compounds.T. tagusensis and T. coccinea placed in close proximity ofM. hispida colonies caused necrosis on 45.6% and 39.5% of theTubastrea colonies, respectively. They had no effect on thesponge, D. achorata, which overgrew the cup corals in the eld.

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Whether this constitutes an allelopathic interaction is unclearbecause the authors did not test the effects of cup coral extractson the health of M. hispida or D. achorata. However, in anotherexperiment, crude extracts from both coral species were testedfor feeding deterrent and antifouling activity.139 Crude extractsfrom T. tagusensis deterred feeding by generalist shes in a eldassay, and crude extracts from both cup corals altered thecomposition of macrofouling communities when integratedinto Phytagel plates at natural concentrations. Plates containingcoral extracts had signicantly less of the red alga Lithophyllumsp. and the green alga Cladophora sp. in terms of percent coverand signicantly more hydrozoa compared to controls.139

7 Ascidians (tunicates)

There have been a few studies of the chemical ecology ofascidians since our last report. Chemical and structuraldefenses of the solitary ascidiansMolgula occidentalis and Styelaplicata, and the colonial ascidian Aplidium stellatum, Botryl-loides nigrum and Distaplia bermudensis found in sub-tropicalseagrass meadows were evaluated using the pinsh Lagodonrhomboides in feeding assay experiments. When offered smallpieces of fresh tissues of the ascidians, the tissues from S. pli-cata, M. occidentalis and D. bermudensis were unpalatable to thesh. The lipophilic and hydrophilic extracts of these species didnot deter sh feeding, suggesting that chemical defenses do notplay a role in deterring L. rhomboides. The authors suggestedthat the toughness of the tunic is a plausible explanation for thelack of palatability observed for S. plicata and M. occidentalisbecause tunics required a force of 34 N to tear.140

Another study investigated the microbial and antifoulingchemical defenses of Antarctic ascidians.141 The lipophilic andhydrophilic extracts and their respective seawater-soluble frac-tions from four solitary and ten colonial tunicates collected inthe Western Antarctic Peninsula were assayed for antibacterialand antifouling activity. The bacterial screening panel consistedof 16 strains of gamma proteobacteria, one strain of Fla-vobacterium and three unidentied strains isolated from thesurface of invertebrates from the same habitat. Growth inhibi-tion was measured using standard disk diffusion assays.Mortality of the diatom Syndroposis sp. was used as a measure ofantifouling activity. Overall, only the extracts of the colonialascidian Distaplia colligans exhibited signicant antibacterialactivity, while extracts from thirteen of the fourteen of thetunicates caused diatom mortality at 3� natural concentration.The lipophilic extract from D. colligans inhibited the growth of

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all strains in the panel at 3� the natural volumetric concen-tration. Psychrobacter fazii was inhibited at natural concentra-tion. The hydrophilic extract of D. colligans exhibited limitedantibacterial activity against two strains of P. fazii and one strainof Alteromonas elyakovii. In the diatommortality assays, seven ofthe fourteen lipophilic extracts were active at 0.3� the naturalconcentration. The hydrophilic extracts of only four speciescaused diatom mortality at 3� natural concentration. Two werestill active at 0.3� natural concentration. The seawater solubleextracts from nine of the fourteen extracts caused diatommortality at 3� the natural concentration, while the seawatersoluble extract from D. colligans was active at 0.3� the naturalconcentration. The authors suggested that it is likely that theascidians in the study use chemical defenses to prevent diatomfouling.141

Recent studies of tunicates have been concerned with theintra- and inter-specic variation in chemical defenses. In therst of two investigations of the chemical defenses of Antarctictunicates, the known indole alkaloids meridians A-G 69–75isolated from the colonial ascidians Aplidium meridianum andAplidium falklandicum chemically defend the tunicates frompredation by the sea star Odontaster validus. A. meridianum andA. falklandicum were collected from the Weddell Sea fromdepths between 280–340 m using Bottom and Agassiz Trawls.The tunicates were dissected to separate the external tunic fromthe visceral tissues and then extracted. LC-MS analysis of theextracts showed that A. falklandicum tissues contained varyingconcentrations of the meridians A–C and E, while meridians Fand G were only found in the extracts of some individuals. Therewere few qualitative differences between the tunic and visceraltissues. The extracts from A. meridianum contained all of themeridians isolated in the study. Crude extracts from the tunicand the visceral tissues deterred feeding by O. validus. Inaddition, the sea stars avoided shrimp coated with differentcombinations of the aromatic alkaloids. The authors concludedthat 69–75 chemically defend the tunicate from predation byO. validus.142

In the second study of colonial Antarctic ascidians from theWeddell Sea, the chemical defenses of tunicates in the generaAplidium and Synoicum were investigated against the starshOdontaster validus, the amphipod Cheirimedon femoratus and asympatric bacterium. Similar to the previous study of Aplidiumspp., the tunicates were dissected to separate the tunic andvisceral tissues before extraction. Chemical investigation of


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some of the extracts yielded the indole alkaloids meridianins A–G 69–75 from A. meridianum and A. falklandicum, and rossinoneB 76, 2,3-epoxy-rossinone B 77, 3-epi-rossinone B 78, and 5,6-epoxy-rossinone B 79 from Aplydium fuegiense as the majormetabolites. There was little intra-specic variation of metab-olites in A. meridianum and A. falklandicum; LC-MS analysisshowed that the tunic of one sample of A. fuegiense contained alow concentration of rossinone B while the visceral tissue con-tained all four of the rossinone derivatives. Crude extracts fromall ve species of tunicates in this study were unpalatable to O.validus; however, in the case of A. millari, the extract preparedfrom the tunic did not deter predation. Twelve of the thirteencrude extracts were unpalatable to the amphipod C. femoratusi.In addition, 76 and a mixture of 69–75 deterred feeding againstboth predators. The mixture of 69–75 inhibited the growth of anunidentied sympatric marine bacterium. Meridianins A–G 69–75 may serve multiple roles in the chemical defense oftunicates.143

Many species of tropical tunicates are host to the pinno-therid crab Tunicotheres moseri; however, the cues associatedwith host recognition are not well understood. In a recent studyof Western Florida populations of T. moseri and its host tuni-cates Styela plicata, Molgula occidentalis and Phallusia nigra,sensory cues affecting host recognition use and mate acquisi-tion were investigated. Specimens for the chemoreceptionexperiments were collected from Tampa Bay and conditionedfor one of the three host tunicates by placing the crab with thetunicate for two weeks. The control group was maintained in aseparate tank without a host. Three sets of experiments wereconducted with T. moseri. Groups of crabs were then condi-tioned for one of the three tunicates. Groups were then exposedto waterborne cues from one of the three tunicates, waterbornecues from conspecics, or placed in the tank with two differenttunicate hosts. While the crabs responded with search behaviorto waterborne cues from all of the tunicates, stronger responseswere observed when the tunicates were placed directly in thetank with the tunicates, suggesting that T. moseri uses multiplewaterborne cues as well as tactile cues to locate an appropriatehost. In addition, male crabs responded to cues from nongravidfemales.144 The authors suggested that T. moseri is a generalist,


but prefers Styela plicata, showing a distinct preference for thistunicate even aer conditioning with Molgula occidentalis andPhallusia nigra.

8 Crustaceans

A recent book “Chemical Communication in Crustaceans”provides a broad overview of crustacean chemical ecology145 anddiscusses many topics including detecting prey, predators andpotential mates. In addition, Breithaupt and Thiel145 solicitedchapters on understudied chemical ecology topics such asmultimodal cues and the disruption of sensory ecology byanthropogenic pollutants. This book brings together manyexperts from different disciplines to deliver a comprehensiveresource for any marine chemical ecologist.

Much of the recent work in crustacean chemical ecology hasfocused on testing behavioral responses to chemical cues invariable ow environments. A recent study tested different typesof ows, continuous, meandering and pulsed, to determine howchemical cues affected blue crab (Callinectes sapidus) trackingbehavior in a ume.146 The chemical cue of potential prey wasmade by soaking 2.21 g of shrimp in one l of seawater for onehour. This was mixed with a uorescent dye, rhodamine 6G, sothat laments of cue could be imaged by three dimensionallaser-induced uorescence. Blue crabs were outtted with a LEDbackpack so that their behavior in response to different types ofow and cue laments could be imaged. Crabs tracked towardsthe source of prey chemical cues more quickly and stopped lesswhen the cues were offered in a continuous ow. The crabsresponded to the presence or absence of cue laments but didnot track along a concentration gradient. A second experimentused similar methods to determine how blue crabs orientatedtheir bodies in response to chemical cues from potential prey.147

The crabs would orientate their body upstream and wouldcorrect their orientation towards the source of the prey chemicalcue. This orientation allowed the crabs to rapidly respond tominor changes in the location of the odor. The crabs main-tained a body angle that reduced drag but also increasedchemical cue reception along their leg chemoreceptors. Both ofthese studies show that crustaceans have behavioral adaptionsthat maximize their ability to nd potential prey using chemicalcues.

Flow and turbulence can affect the ability of blue crabs tond their food. Different hydrodynamic features were measuredin different estuarian streams in Wassaw Sound, Georgia, USAto determine their effect on the ability of blue crabs to nd theclams Mercenaria mercenaria.148 Skidaway River had the lowestow rates and turbulence and it had less crab predation onclams than the rivers with intermediate ow rates and turbu-lence (Wilmington and Herb Rivers). Oyster shells were addedto the test plots to increase turbulence, which increasedpredation on clams in the two slowest ow rivers (Skidaway andWilmington Rivers) but decreased predation in the two fastestowing rivers (Herb and Moon Rivers). This experiment illus-trates the importance of ow and turbulence on a crab's abilityto detect prey in the eld.

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Higher ow rates also decreased the ability of the green crabCarcinus maenas to nd mussels, Mytilus edulis.149 At six loca-tions with different ow regimes within the Damariscotta River,Maine, USA, traps were baited with 6 crushed mussels. Morecrabs were attracted to the traps at low ow sites even thoughcrab densities were higher at sites with high ow rates. Crabbehavior in response to a crushed mussel was tested in labo-ratory umes. Crabs had slower walking speeds and greaterforaging times in a high ow environment (19 cm s�1)compared to 15 cm s�1. Handling time of a dogwhelk (Nucellalapillus) placed 1.0 cm from the crab's mouthparts increased inows of 15 and 19 cm s�1 when compared to 3 cm s�1. Theseexperiments show that high ow can interfere with these crab'sability to nd and consume their prey.

Different sources of chemical cues can send different signalsto crabs. Further work was done on blue crabs to describe theirbehavior in response to an attractive and an aversive cue.150 Anattractive cue was created by conditioning one l of seawater with7.5 g of shrimp for one hour, and an aversive cue was created bysoaking one injured blue crab in three liters of seawater for 3.5hours. Flume experiments consisted of the attractive cue aloneand a conicting treatment in which both the attractive andaversive cues were delivered at the same time. The crabs trackedtowards the attractive and the conicting cue in the owwithout turbulence. When a cylinder was used to increaseturbulence the crabs were still attracted to the food cues butthere was signicantly less tracking towards the conictingcues, due to increased mixing of the two types of cues. The timeit took for the crab to reach the end of the ume was less in theattractive cue than the conicting cue regardless of increasedturbulence. The tracking path was straightest in the attractivetreatment, but crabs exposed to the conicting treatment alsohad relatively straight tracks. To test which sensory organs thecrabs used to distinguish these cues their antennules and legs/claws were deafferented (nerves were disconnected). Crabswithout antennule nerves tracked the conicting plume at ahigher frequency and had higher tracking speeds whencompared to intact crabs. Crabs without their leg nerves did notdemonstrate different tracking behavior from intact crabs.These experiments show that blue crabs can track towards foodcues and discriminate between food and aversive cues.Furthermore, they demonstrate that chemosensory organsresponsible for detecting aversive cues are located on theantennules of the crabs.

To better understand the sensory feeding ecology of bluecrabs, the oesophageal chemoreceptors of C. sapidus werestudied when they were exposed to different deterrentcompounds.151 Initial experiments showed that crabs withblocked antennular or pereiopod chemoreceptors had the samesearch time and grasped their food the same amount regardlessof whether shrimp were offered alone or combined with inkfrom the sea hare Aplysia californica. When either the inner orouter mouthparts were removed there was a reduction in themanipulation time of the shrimp with the ink treatment. Puri-ed compounds, denatonium, nicotinamide, quinine, cinna-mal and caffeine, which are known feeding deterrents fromother systems were tested for their deterrence to blue crabs.

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Denatonium and quinine increased the handling times ofshrimp in intact crabs, and this effect was not changed whencrab mouthparts were removed. These compounds were thentested for their effect on oesophageal dilation, a knownresponse to unpalatable compounds. Sea hare ink inducedoesophageal dilation in a dose dependent manner with higherdoses inducing more dilation. Quinine, denatonium, cinna-maldehyde and caffeine also induced oesophageal dilation.When oesophageal receptors were blocked with silicone thenumber of crabs that had oesophageal dilation in response todenatonium and quinine was reduced. The oesophagealreceptors are critical for crustaceans to discriminate amongcompounds that are feeding deterrents.

Parasites also use chemical cues to nd their host species.Experiments were conducted with parasitic pea crabs from twodifferent echinoids (Meoma ventricosa and Plagiobrissus grandis)to test whether they used chemical cues to distinguish betweenhost species.152 Tests conducted in a y-maze showed that thecrab Cissocactylus primitivus was attracted to its host, the hearturchin M. ventricosa, more than seawater or the non-hostechinoid Clypeaster rosaceus. Crabs collected fromM. ventricosawere attracted to this species more than P. grandis, but whencrabs were collected from P. grandis they had no preferencebetween these two echinoid species. Symbiotic shrimp, Gna-thophylloides mineri, were also tested for their attraction towardstheir host, the sea urchin Tripneustes gratilla.153 With just visualcues 42 of 50 shrimp moved towards T. gratilla more thanltered seawater (FSW), 37 of 50 moved to T. gratilla more thanthe non-host urchinHeliocidaris tuberculata and 30 of 50 shrimpmoved to H. tuberculatamore than FSW. In a Y-maze system setup to deliver only chemical cues (no visual cues) signicantlymore shrimp moved towards T. gratilla than FSW or either ofthe non-host urchins, H. tuberculata and Pseudoboletia indiana.More shrimp moved towards H. tuberculata than FSW but therewas no preference for P. indiana over FSW. Both of these studiesshow that parasitic crustaceans can use chemical cues to ndhost species.

Crustacean parasites also use chemical cues to evaluate theirresources. The host use behavior of sea lice Lepeophtheirus sal-monis was studied in response to the presence of other sea liceon Atlantic salmon, Salmo salar.154 Ten l of water was condi-tioned with salmon with different parasite loads: no lice, 10juvenile females, 10 adult males and a combined treatment ofve juvenile females with ve males. Fewer male lice le thehost salmon aer seven days of incubation in the conditionedwater treatment with no lice and the water conditioned withonly juvenile female lice. The most male lice le salmon incu-bated in water conditioned with 10 other male lice, and thetreatment with both female and male lice was not signicantlydifferent from all the other treatments. In the presence of water-soluble cues from potential competitors more of the parasiticcrustaceans le their host sh than those exposed to waterconditioned without male competitors.

Hermit crabs also use chemical cues to evaluate potentialresources. One study focused on the chemical cues that hermitcrabs used to evaluate empty shells.155 Empty, well-tting shellswere provided to hermit crabs in the presence of ve different


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treatments, seawater, seawater conditioned with live and deadsnails, seawater conditioned with live and dead conspecics,predator-conditioned seawater and food-conditioned seawater.The amount of time it took the hermit crabs Clibanarius eryth-ropus and Pagurus bernhardus to contact the shell and theduration of shell investigation was compared. The time to rstcontact was less for P. bernhardus in the control, food, deadconspecic and live snail treatments compared to C. erythropus.There was no difference between the two species for the livepredator and live conspecic treatment. P. bernhardus had alonger time until rst contact than C. erythropus in the deadsnail treatment. The time spent investigating the potential shellwas greater for P. bernhardus in every treatment except for thedead snail treatment in which C. erythropus investigated theshells for a longer duration. These hermit crabs had differentbehaviors in response to water soluble cues associated withpotential resources.

A recent study examined the effects of reduced pH condi-tions on hermit crab shell selection behavior.156 Hermit crabs,Pagurus bernhardus, were kept in articial seawater aspiratedwith CO2-enriched air so that it was maintained at a pH of 6.8for 5 days. When they were offered an empty shell they showedincreased time until they changed shells, lower rates of anten-nular icking, and less time spent moving compared to crabsincubated at a control pH of 8.2. However, when this experi-ment was repeated there was no difference between the crabsincubated at a pH of 6.8 compared to controls. Further workshowed that P. bernhardus response to food cues is alsoinhibited at lower pH.156 Hermit crabs incubated at a pH of 6.8for 5 days spent less time in the side of the chamber with foodcues (prepared by crushing sh, Pollachius virens, at a ratio ofone gram of sh per 50 ml of articial seawater and thenltering the slurry). Crabs incubated at a lower pH took longerto nd the food cue and also spent less time in contact with thecue. The hermit crabs conditioned at pH of 6.8 had lessantennular icking and spent less timemoving compared to thecontrol group conditioned at a pH of 8.2. Hermit crab behaviordid not change when the cue was exposed to lower pH, showingthat the lower pH was not affecting the chemical properties ofthe cues, but instead it was decreasing the ability of the crabs todetect and respond to these potential chemical cues.

Chemical cues are also important in nding a potentialmate. Recently, uridine diphosphate (UDP) 80 was character-ized as a sex pheromone for the green crab Carcinus maenas.157

Through bioassay guided HPLC fractionation, 80 was found tobe a sex pheromone released in female conditioned seawaterand in female urine. Both male guarding of females and initi-ation of mating behaviors were observed at concentrations of 80as low as 10�5 M in seawater. Mate guarding behavior wasobserved more oen in male crabs aer exposure to syntheticUDP compared to seawater controls. In the summer months(mating season), male crabs spent more time in water with UDPthan a food stimulus, but in winter they chose both stimuliequally. UDP was also present in male urine during the spring(their molting season), but UDP concentrations were less than5% of that found in the females. Through extensive bioassayguided fractionation, Hardege et al., show that UDP is found in


females during their mating season and that males show thesame mating behavior to synthetic UDP as to female urine orfemale conditioned water.

Contact sex pheromones are important for shrimp, andfurther work was done to characterize the pheromonesproduced by the shrimp Lysmata boggessi.158 These shrimp areonly receptive females when they molt, so hexane extracts weretaken from molting shrimp and those extracts were comparedto intermolt and newly molted individuals by GC-MS. Thechromatograms showed many similar compounds among theseshrimp but (z)-9-octadecenamide 81 was unique to the moltingindividuals. When coated on a small piece of plastic tubing,synthetically produced (z)-9-octadecanamide induced matingbehavior in almost half of the shrimp, which was greater thanthe behavior observed in the control of just hexane (3 of 30shrimp). However, there was a greater response (22 of 30shrimp) in response to molting shrimp so the authors alsotested a blend of compounds that produced variable responses:(z)-9-octadecanamide with squalene caused mating behavior in16 of 30 shrimp, and (z)-9-octadecenamide with hex-adecanamide 82 and methyl linoleate 83 induced matingbehavior in 19 of 30 individuals. While (z)-9-octadecanamidemay serve as an important contact pheromone for these shrimp,a complex blend of compounds probably drives matingbehavior.

There continues to be important progress testing and char-acterizing larval settlement cues for crustaceans. A recent reviewdiscussed the limits and advantages of different laboratorymethods to characterize the chemical cues responsible forbarnacle settlement.159 Thiyagarajan159 discussed proteomicsand the potential of gene regulation to better understandbarnacle settlement. The response of barnacles to settlementcues has been attributed to regulation of a series of six barnacle

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cyprid specic genes expressed in the cyprid larvae at thesettlement stage, bcs genes 1–6. A recent paper showed therewas a down regulation in bcs genes (1–5) but an upregulation ofthe bcs-6 gene aer settlement in response to a natural biolmor conspecic settlement cues.160 Further integration ofmolecular and chemical ecology would improve our under-standing of the process of settlement and metamorphosis formany taxa.

Barnacle larvae can respond to conspecic water solublecues in addition to the bound protein complex (SIPC) that isknown to induce settlement. Elbourne and Clare161 conductedexperiments to better understand how conspecic cues mightaffect larval swimming behavior and settlement in Balanusamphitrite. Water (1.5 to 5 l) was conditioned with adultconspecics (ranging from 44 to approximately 475 individ-uals) or B. balanoides (approximately 500 individuals in 0.5 l)for 18 hours, and then larvae were exposed to a variety oftreatments of this water. Settlement of larvae of B. amphitritein conspecic treated water was increased compared to arti-cial seawater (ASW) aer one and three days, but this activitywas lost aer ve and seven days. Serial dilutions of thisconspecic conditioned water increased settlement atconcentrations as low as 0.01% of full strength in one trial, butonly at full strength in a second trial (compared to ASW). If thelarvae of B. amphitrite were exposed to conspecic conditionedwater for three minutes there was no change in the amount ofsettlement, but when the larvae were exposed for 15 minutes,one hour or three hours there was increased settlement. Watersamples were taken from the eld adjacent to a piling withB. amphitrite, and water collected one cm away from the pilinginduced higher rates of settlement compared to watercollected 19 cm and 38 cm from the piling. B. balanoides larvaespent more time probing the bottom of a Petri dish inconspecic treated water than in FSW, and this was true evenwhen the larvae were 11 days old. Barnacle larvae are capableof detecting conspecic water soluble cues, and these cuescaused behavioral changes that increased barnaclesettlement.

Due to the potential impact of the invasive Asian shore crabHemigrapsus sanguineus on native marine species along the eastcoast of the United States, some researchers have focused on thechemical cues that might affect its larval settlement. Larvae ofthis crab have shorter larval durations in response to watersoluble cues from conspecic adults, and recent research hastried to characterize this water soluble cue.162 Water wasconditioned with adult and juvenile H. sanguineus at aconcentration of three g of live crab in one l of ltered seawaterfor 48 hours. Water conditioned with either adults or juvenilesinduced metamorphosis sooner than the seawater controls.Conditioned water that was used immediately aer it was madeinduced metamorphosis sooner than water that was stored fortwo days or longer, but in this experiment the conditioned waterdid not induce metamorphosis sooner than the seawatercontrol. The full strength conditioned water induced meta-morphosis sooner than the 0.01 dilution and the seawatercontrol, but the 0.1 dilution was not different from the fullstrength treatment or the seawater control. When the adult

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conditioned water was treated with trichloracetic acid, trypsinand proteinase K its ability to induce earlier metamorphosiswas reduced. The authors concluded that a water-solubleprotein or peptide from adults reduced the planktonic durationof H. sanguineus larvae.

Laboratory experiments provide researchers with acontrolled environment, but eld experiments expose larvalcrustaceans to natural characteristics found in their habitat.One study examined the settlement of H. sanguineus larvae intwo different intertidal habitats, a salt marsh and a cobblehabitat that contained adult crabs.163 This study used two typesof containers to test time until larval metamorphosis, cylin-drical cages made with 500 mmmesh and glass jars with 200 mlof ltered seawater. The time until metamorphosis was reducedfor more megalopae in the cage treatment on cobble than in thejar container consistently in three different experiments. In adirect comparison of habitats using the cage containers therewere similar rates of molting between the cobble and marshhabitat, even though themarsh habitat does not usually containadult H. sanguineus. These larval crabs had greater rates ofmetamorphosis in response to habitat cues, but the larvae donot require specic cues associated with a cobble habitat.

Multiple crustacean postlarvae were tested for their settle-ment preferences in laboratory habitats.164 The postlarvae of 9different crustacean species demonstrated habitat preferenceswhen given a choice between live coral, dead coral, macroalgaeand sand. Within their preferred habitat, they showed nopreference between treatments with and without conspecicjuveniles, demonstrating that these crustaceans are moreattracted by habitat than conspecics. To understand thesensory modalities involved in choosing a settlement habitat,the crustaceans were presented with either visual cues in whichthe treatment was presented in an isolated but visibleaquarium or chemical cues in which the postlarvae were pre-sented with treatment conditioned seawater. Given a choicebetween conspecic and heterospecic cues, 7 of the 9 specieshad no preference. Xanthidae postlarvae used chemical cues toselect between conspecics and heterospecics. Palaemonidaeused both visual and chemical cues to settle with its conspe-cics. The sensory modality driving settlement in response todifferent habitats was also tested. Xanthidae larvae selectedmacroalgae using both visual and chemical cues. Larvae ofPachygrapusus planifrons and 2 Lysiosquillina spp. selected theirhabitat with just visual cues and 5 other crustacean speciesshowed no preferences for settlement habitat. Water solublecompounds from each habitat and a few of the adult crusta-cean species were compared by HPLC and each habitat con-tained unique compounds, but there was no further bioassayguided characterization or structural characterization of thosecompounds. By testing different crustacean larvae for theirsettlement patterns this research illustrated how some but notall species can use chemical cues to distinguish appropriatesettlement habitats.

Detection of potential predators is also important for larvalsurvival. Work was conducted to determine whether megalopaeof H. sanguineus could detect water-soluble cues of commonshes from the East Coast of the United States.165 Feeding


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experiments showed that Tautogolabrus adspersus, Fundulusheteroclitus (medium and large sizes), Stenotomus chrysops,Myoxocephalus aeneus, and Tautoga onitis would all prey onH. sanguineus larvae. Water was conditioned with adult femaleH. sanguineus and each species of sh at a volume ratio of 50 mlorganism to 1950 ml of articial seawater for 24 hours. Whenmegalopae were exposed to water conditioned with these shthere was no change in the number that molted compared tothe articial seawater control, but conspecic water-solublecues increased the number of megalopae that molted. Thebehavior of megalopae was tested in response to chemical cuesthat were swabbed from shes. These swabs were placed in anaquarium and le with a single megalopa for 8 minutes, aerwhich the larvae were assessed to determine if they avoided anyof the swabs. Larvae swam away from cues swabbed fromT. adspersus and M. aeneus. In owing water assays, chemicalcues from T. onitis caused a “pushing” behavior that increasedlarval distance from the conditioned seawater. Megalopae ofH. sanguineus were capable of detecting water-soluble cues frompotential predators in the laboratory (although the larvae neverresponded to F. heteroclitus), suggesting that they can usechemical cues to avoid predation.

Embryos can also produce chemical cues that control thebehavior of their parents to increase their dispersal success. Arecent study showed that pheromones induced abdominalpumping and swimming behavior of ovigerous C. sapidus.166

Blue crab eggs were crushed in seawater and the supernatantwas collected and used in experiments at an approximateconcentration of 19 000 eggs per ml of seawater. Blue crabfemales increased their abdominal pumping activity inresponse to the egg extracts at volumes above 250 ml (equivalentto approximately 4750 hatched eggs) during both ebb and oodtides. Trypsin, which is known to stimulate pumping in othercrustaceans, also induced more abdominal pumping at aconcentration of 17 000 units per ml than either the control ofseawater or 0.1 mM HCl in a NaCl solution with seawater, buttrypsin at 44 000 units per ml was not different from the HClcontrol. The addition of bradykinin, which is active in a varietyof crustacean signaling systems, increased abdominal pumpingcompared to the seawater control at a concentration of 10�7 Mbut not at a concentration of 10�8 M or 10�6 M. Blue crabs willswim up in the water column when releasing their larvae so thisbehavior was also assessed in response to these same treat-ments. Egg extract caused increased swimming behavior atconcentrations greater than 250 ml but trypsin and bradykinindid not affect crab swimming behavior. Blue crab hatching eggsrelease chemical cues to stimulate adult behavior that increaseslarval dispersal.

9 Molluscs

A recent review on the chemistry and chemical ecology ofmolluscs discussed the chemical diversity of molluscs with afocus on the taxonomical distribution of secondary metaboliteswithin the phylum as well as the medicinal properties of thesecompounds.167 As part of a broader review on the neuroecologyof chemical defense in aquatic environments, Derby and Aggio


described what is currently known about the use of ink as adefense by marine molluscs.10 Several studies on this topic werepublished between 2010–2012 and are included here.

The sea hare Aplysia californica releases an ink secretionwhen threatened. The secretion is a mixture of two compo-nents, ink and opaline. Both components of this secretioninhibited food seeking behavior and feeding in blue crabs(Callinectes sapidus) although ink consistently had a strongereffect than opaline.168 Neither attracted feeding when coatedonto sponges in the absence of any other food odor, showingthat the ink does not act as a phagomimic. Ink inhibited foodseeking behavior in blindfolded crabs indicating that crabsdetect the ink through chemical, not visual cues. Throughbioassay guided fractionation the authors determined that themain compound responsible for feeding deterrence was aply-siaviolin (APV) 84, which is derived from a photosyntheticprotein found in the red algal diet of these sea hares. Theconcentration of APV in A. californica ink was 27 mg ml�1, wellabove the active concentration of 6.25 mg ml�1. APV was alsofound in the ink of the conspecic sea hare, A. dactylomela butat a much lower concentration (2.4 mg ml�1). Phycoery-throbilin (PEB) 85 was found in the ink of both sea hares andinhibited feeding, but at higher concentrations (6.25 mg ml�1)than what was found naturally occurring in either sea hare(3 mg ml�1 and 0.7 mg ml�1, respectively). Escapin is anothercompound found in the ink of A. californica. When ink issecreted, escapin from the ink reacts with L-lysine from theopaline and creates a series of escapin intermediate products(EIP) and releases hydrogen peroxide. While EIP alone had noeffect, hydrogen peroxide and EIP + hydrogen peroxideinhibited feeding by C. sapidus indicating that the hydrogenperoxide produced by this reaction may be an additional anti-feeding defense.

Ink secretions also protected A. californica against shpredators. Feeding pellets containing A. californica ink or ink +opaline were rejected by four out of ve sh species tested(bluehead wrasses Thalassoma bifasciatum, senorita wrassesOxyjulis californica, pinsh Lagodon rhomboides, andmummichogs Fundulus heteroclitus).169 The sh did not rejecttreatments containing just opaline indicating the feedingdeterrence is from the ink. The h sh species (bonnetheadshark Sphyrna tiburo) showed a similar trend, but the resultswere not signicant. Escapin products only moderately

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deterred feeding in two of the sh species, bluehead wrasseand senorita wrasse, and hydrogen peroxide did not deterfeeding in any sh. The authors concluded that EIP are notlikely to be the main feeding deterrent compounds becausethe concentrations of EIP tested were near the upper limits ofthose found naturally within A. californica ink secretions andhad only moderate effects.

Nusnbaum and Derby170 demonstrated that feeding deter-rent activity of A. californica ink secretion against predation bythe bluehead wrasse was due to reduction in prey capturesuccess and lower prey acceptance rates. Ink secretion by liveA. californica signicantly reduced the number of strikes bybluehead wrasses in laboratory assays when compared to indi-viduals that had the ink previously removed. An ink cloud (inkalone), produced by injecting ink between the wrasse and apiece of food, reduced the number of sh reaching food,increased the time it took to reach the food, and caused sh toswim around the cloud to reach food. Inhibiting olfactorydetection by occluding the sh nares with petroleum jelly didnot affect the total percentage of sh that reached and capturedshrimp in the presence of an ink cloud, but did reduce the timeit took to nd and capture shrimp compared to sh withoutoccluded nares. This suggests that the reduction in capturesuccess caused by ink secretions is related to olfactory proper-ties of the ink cloud. In the absence of an ink cloud, sh rejectedshrimp-alginate pellets containing ink at concentrations as lowas 0.01% natural concentration. Fish with and without naresoccluded rejected ink containing food pellets equally, demon-strating that the rejection response is not olfactory. Neither thecapture nor acceptance response was affected by the opalineportion of the ink secretion. The possibility that A. californicaink secretions act via phagomimicry was again rejected becausesh did not accept unavored pellets containing ink, opaline orink + opaline.

Sea catsh (Ariopisis felis) feeding was also deterred byA. californica ink secretion.171 As in assays with blue crabs(C. sapidus), feeding deterrence was attributed to the

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compounds aplysiaviolin (APV) 84 and phycoerythrobilin(PEB) 85. Shrimp avored pellets with APV + PEB wererejected as frequently as pellets with whole ink. Ink � (APV+ PEB) was also deterrent, but signicantly less than wholeink. Catsh were able to detect APV and PEB with theirbarbels. Fish turned away when barbels were touched withfreeze dried shrimp soaked in ink, APV or PEB but eatenwhen soaked in articial seawater (ASW). Electrophysio-logical recordings of the facial-trigeminal nerve complexthat innervates the barbels demonstrated an electrophysi-ological response when barbels were stimulated with APVand PEB. Cross adaptation experiments determined thatboth compounds were detected by the same gustatorypathways in the facial-trigeminal nerve complex and wereequally effective as deterrents. These pathways were inde-pendent from those excited by other stimuli tested (aminoacids and bile salts). Sea catsh also have gustatory path-ways that are excited by only amino acids and bile salts andones that are activated by APV, PEB, amino acids, and bilesalts.

In addition to directly deterring feeding by both sh andcrabs, ink secretions also send an alarm signal to nearbyconspecics, causing them to move away from the source ofthe secretion. This response can be elicited by uracil, uridine,and cytadine in the ink component of the secretion,172 butKicklighter et al.173 recently demonstrated that the opalinecomponent also elicits an alarm response in juvenile conspe-cics. Using bioassay guided fractionation, three alarm cueswere identied within the opaline. The three cues weremycrosporine-like amino acids (MAAs); asterina-330 (N-ethanol palythine) 86, N-isopropanol palythine (aplysiapaly-thine A) 87 and N-ethyl palythine (aplysiapalythine B) 88. Thelatter two were previously unknown compounds. In combi-nation, these three cues elicited the same response as the totalopaline secretion at concentrations ranging from full strengthnaturally occurring within the opaline to 1/500th of the fullstrength concentration. Opaline secretion with these three


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cues removed no longer acted as an alarm cue. Two otherMAAs, palythine 89 and N-methyl palythine (aplysiapalythineC) 90, were present in the opaline but did not act as alarmcues. The authors suggest that A. californica sequester theMAAs from their red algal diet. A. californica raised in thelaboratory on the red algae Gracilaria ferox and Agardhiellasubulata had three of the ve MAAs found in wild caughtA. californica (86, 89 and 90) and two additional unidentiedMAAs (MAA-1 and MAA-2). All ve major MAAs were also foundin the algal tissue of G. ferox and A. subulata, but not a brownalga (Egregia menziesii) that lives in the same environment asA. californica but is not eaten by the sea hare. MAAs were foundin the highest concentrations in the opaline, but were alsofound in high concentrations in the mantel and dorsal skin.Other body parts had signicantly lower concentrations ofMAAs. MAAs are known to act as sunscreens, and the authorssuggest that MAAs are probably being used as sunscreen in themantle and dorsal skin.

The body tissues of A. californica are also unpalatable topredators.174 Food pellets made from the skin and body walltissue of A. californica were signicantly less palatable to hermitcrabs (Pagurus samuelis) compared to two other sea hares,Phyllaplysia taylori and Dolabrifera dolabrifera.

Other molluscs also use ink secretions to defend them-selves from predation. Ink from the Caribbean reef squid,Sepioteuthis sepioidea, deterred feeding by the predatoryFrench grunt, Haemulon avolineatum.175 Squid ink, carbox-ymethyl cellulose (a control that mimics the consistency ofsquid ink) and carboxymethyl cellulose + food coloring weresquirted between the sh and an alginate-shrimp food pelletto determine if the presence of the ink affects sh behavior.The presence of squid ink and carboxymethyl cellulose coloredto resemble squid ink increased the length of time it took shto reach and attack food compared to controls of uncoloredcarboxymethyl cellulose. Fish responded to ink and coloredcarboxymethyl cellulose most oen by avoiding it, pausing, orbiting it. They most oen had no response to uncolored car-boxymethyl cellulose controls. Ink decreased but did notsignicantly deter sh feeding on disks soaked in squid meat;however the addition of the same food coloring used above didcause a signicant reduction in feeding. To demonstrate theability of French grunts to be deterred by potent chemicals,they were also presented and rejected disks soaked in meatand sea hare ink. However, when the amount of time sh helddisks in their mouth was used as a measure of palatability, shheld disks containing meat alone signicantly longer thanthose with ink or food coloring added indicating that bothwere unpalatable. Disks soaked in ink alone were also rejectedby sh, showing that ink does not protect squid throughphagomimicry.

The behavioral, physiological and morphological responsesof molluscs to exudates from co-occurring competitors and/orpredators continue to interest researchers in the eld ofchemical ecology. Dogwhelk (Nucella lapillus) moved signi-cantly less (a predator avoidance response) in the presence oftethered green crabs (Carcinus meanas) or crushed conspe-cics, but not other common predators (Jonah crabs, Cancer


borealis and rock crabs, Cancer irroratus) or crushed hetero-specics (mussels, Mytilus edulis and Littorina littoria snails)in ume experiments.176 The authors suggest that N. lapilluseither are unable to detect Jonah crabs and rock crabs or donot respond to them as a threat, possibly because they rarelyco-occur in nature. However, the same group later found thatN. lapillus movement was signicantly reduced compared tocontrols when exposed to both rock crabs and green crabs, butthat the effect of green crabs was signicantly higher than rockcrabs.177 Green crabs fed N. lapillus or mussels caused asignicant reduction in movement, while starved green crabsdid not.176 Green crab exudates elicited the same reduction inmovement as intact, live green crabs indicating the N. lapillusdetected green crabs via chemical cues.176 The presence ofgreen crabs and rock crabs that had been fed N. lapillus causeda signicant reduction in foraging, shell mass, and body mass,while those that had been fed a heterospecic snail, L. littoria,had no effect. That movement is affected by the presence of thepredator alone and foraging, shell mass, and body mass areonly altered in the presence of predators that have eatenconspecics indicates that prey respond to a variety of cues indifferent ways.176,177 Additionally, ow rate affected the move-ment response of N. lapillus in the presence of green crabs.178

At no ow (0 cm s�1) and high ow (12 and 20 cm s�1)N. lapillusmovement was not affected by the presence of crabs.At low ow (4 cm s�1) N. lapillusmoved signicantly less in thepresence of green crabs tethered 0.5 m (high risk) and 1.0 m(low risk) upstream compared to no crab controls. There wasno difference in the response to high and low risk at this owlevel. However, at a ow rate of 8 cm s�1 the high risk treat-ment caused the highest reduction in movement, causingsignicantly less movement compared to the low risk treat-ment, and movement in response to both high and low risktreatments was signicantly lower than the no predatorcontrols.

Invasive oyster drills, Urosalpinx cinerea and Ocinebrinainornata, displayed predator avoidance behavior in response tocues from both native crabs (Cancer productus) and injuredconspecics.179 Both species exhibited increased hiding anddecreased feeding in the presence of cues from caged C. pro-ductus feeding on conspecic drills. To pinpoint the source ofthe cue, U. cinerea were subjected to various combinations ofpredator and injured conspecic cues. Injured conspecics hadthe greatest impact on U. cinerea behavior; however, they alsoresponded to the presence of C. productus regardless of whetheror not the crabs had digested conspecic drills. The response ofU. cinerea and O. inornata to predator cues was not affected byprey density. The ability of these invasive molluscs to recognizeand respond to native threats may be important in invasionsuccess.

Bivalve clams (Mercenaria mercenaria) also changed theirbehavior in response to predator (blue crab) cues.180 Clamsdownstream from a caged blue crab increased uctuations intheir pumping behavior resulting in an increased standarddeviation and randomness within their excurrent verticalvelocity values. They also changed pumping patterns inresponse to changes in hydrodynamic conditions regardless

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of the presence of predator cues by increasing the jet-to-crossow velocity ratio under low crossow conditions. Theauthors suggest that uctuations in chemical signal releasedby the clams in the presence of a predator may result in lesspredictable chemical information for downstreampredators.

Gastropods with high predation susceptibility (Littorina lit-torea), using high aspect ratio as a measure of susceptibility,responded more strongly to predator cues (Carcinus maenas fedon conspecics) compared to gastropods with low predationsusceptibility (Gibbula umbilicalis).181 L. littorea spent more timeat the water line or out of the water, less time feeding andconsumed less algae compared to G. umbilicalis when presentedwith predator cues. The presence of predator cues also alteredinteractions between L. littorea and G. umbilicalis in competi-tion trials. In the absence of predator cues, L. littorea exhibited acompetitive advantage over G. umbilicalus and this was reversedin the presence of predator cues which caused increase eeingand decreased feeding by L. littorea.

Marine snails (Nucella lamellosa) changed morphologicaltraits aer 48 days of exposure to cues from predators andpredators eating conspecics.182 Snails exposed to cues from acrab predator (Cancer productus) had increased shell thicknessand decreased body mass compared to controls. Snails exposedto cues from crabs eating conspecics had thickened aperturallips and teeth, while those exposed to cues from crabs eatingsh or heterospecic snails (Littorina sitkana) exhibited nochange in thickness. Exposure to cues from injured conspe-cics, injured heterospecics, or injured sh alone (in theabsence of the predator) had no effect on any of the morpho-logical traits examined. That the snails responded to cues fromthe predator eating the conspecic, but not the injuredconspecic alone indicates that these snails may have differentmorphological defense strategies dependent on what type ofthreat is present and not solely on the presence of distressedconspecics.

In eld assays, scavenging whelks (Buccinanops globulosum)fed signicantly less on crab carcasses that had injuredconspecics placed next to them compared to crab carcassesalone.183 The presence of injured heterospecic snails (Tegulasp.) also reduced feeding by B. globulosum, but chitons(Chaetopleura isabellei), limpets (Fisurella radiosa) and slip-persnails (Crepidula aculeate) had no effect. The were nodifferences in the morphological traits (sex ratio, shell length,shell width, total weight, shell weight, so tissue weight, andbody condition) of whelks found feeding on crabs with andwithout injured conspecics, except for shell hardness.Whelks feeding on crabs in the presence of injured conspe-cics had signicantly lower values for shell hardness. Thismay indicate an ability of lighter whelks to ee predators fasterand therefore to be more likely to feed in the presence ofpredator risk. Whelks that were fed immediately prior to theassay fed less than those that were starved regardless of thepresence of crushed conspecics.

Mussels (Mytilus edulis) were exposed to seawater that hadbeen conditioned with one of ve naturally co-occurring foulingspecies to determine the effects of excretion-secretion products

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(ESP) on lysosomal enzyme activity.184 Activity of lysosomalenzymes within gills and margin mantle tissue varied widelyacross the 24 hour experiment in control mussels that were notexposed to ESP. The patterns of lysosomal activity were differentin the gills and the mantle. Exposure to conspecic ESP affectedDNase activity in both the mantle and gill tissues and affectedcathepsin B activity only in the gill tissues. ESP from the ascdianStyela rustica, the sponge Halichondria panacea and the sea starAsterias rubens all activated DNase, RNase, b-glucosidase, andb-galactosidase activity in the gill tissue. In addition, S. rusticaactivated cathepsin D activity in the gills. The authors suggestthat the activation of these enzymes may be related to changesin metabolism that occur when the mussel shell closes in thepresence of a predator and/or to the production of mucus,which can act as a defense against allelopathic secondarymetabolites.

Nudibranch molluscs are well known for their use of potentchemical defenses. A group of chromodorid nudibranchswithin the genus Hypselodoris found in the Mediterranean andnorthern Atlantic form a Mullerian mimic complex. Thesenudibranchs have distinctive blue, white and yellow colorationand are chemically defended. The species H. fontandraui lacksthe mantle dermal formations (repugnatorial glands) that actas repositories for feeding deterrent compounds in otherHypselodoris species and it was hypothesized that this speciesmight lack chemical defense and therefore represent a case ofBatesian rather than Mullerian mimicry.79 Despite the lack ofrepugnatorial glands, H. fontandraui contained the fur-anosesquiterpenoid tavacpallescensin 91, mainly in themantel rim. This compound deterred feeding by the generalistshrimp Palaemon elegans at concentrations as low as 1.0 mgml�1, much lower than the concentration found within themantle tissue (25.98 � 1.41 mg ml�1). The presence ofdefensive metabolites within H. fontandraui suggests that thisspecies is part of the Mullerian mimic complex with itscongeners.

Another dorid nudibranch, Hypselodoris cantabrica, hadhigher natural concentrations of crude extracts compared to itsprey sponge, Dysidea fragilis.185 The crude extracts of the nudi-branch were also more potent than those from the sponge inpalatability assays with Palaemon elegans. Both the nudibranchand sponge extracts contained the furanosesquiterpene naka-furane-9 92 as the major compound within the crude extractand this pure compound decreased palatability at the sameconcentrations as the crude extract from the nudibranch. Theauthors suggest that the enhanced defense in the nudibranchcompared to its sponge prey, from which it sequesters itsdefensive compounds, indicates a defensive escalation in theco-evolutionary arms race.


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Two new molecules, 9-chloro-phorbazole D 93 and N1-methyl-phorbazole A 94 were isolated from another doridnudibranch Aldisa andersoni.186 These compounds, along withthe previously known phorbazoles A 95, B 96, and D 97 werefound mainly in the external tissues of the nudibranch. The twonew compounds and phorbazole A were tested for their feedingdeterrence activity against shrimp, Palaemon elegans. All threecompounds signicantly reduced feeding at a concentration of1.0 mg ml�1; however, the natural concentrations of thesecompounds within the nudibranch were not discussed.

The role of chemical cues in mate seeking behavior wasdemonstrated in recent studies of cuttlesh and littorinidsnails. Sexually mature cuttlesh (Sepia officianalis) detectedand were attracted to chemical extracts from reproductivestructures of conspecic females.187 Cuttlesh increased venti-lation, indicating that the animal was detecting a chemical cue,in response to extracts of new eggs (<1 h), ovaries, and accessorynidamental glands, but not old eggs (36–48 h) or nidamentalglands. In a Y-maze, sexually mature male and female adultswere attracted to extracts of old eggs (36–48 h). These animalsform spawning aggregations and the authors suggest that cuesfrom eggs may be acting as pheromones that aid in the coor-dination of reproductive behavior.

In laboratory assays, male littorinid snails (Littorariaardouiniana and L. melanostoma) were able to track conspecicfemales during the mating season.188 During the mating season,males followed mucus trails laid down by conspecic females ata higher intensity than any other pairing (male followingconspecic male, female following conspecic male, or anyheterospecic combination) regardless of trail complexity.Tracker snails followed trails approximately twice as fast duringmating season as during non-mating season. There were nodifferences in the tracking intensity of various pairing duringthe non-mating season. For both species, most of the trackersfollowed in the same direction that the females laid the trailwhenmales were placed at a 90 degree angle to trail inmiddle oftrail and allowed to choose to go le or right. Littorariaardouiniana exhibited more intense tracking (males followingfemales) than L. melanostoma during mating season andtracked faster than L. melanostoma in both seasons.


Understanding the role of sperm chemotaxis in fertilizationsuccess is dependent on the development of tools that allow forinvestigation of sperm–egg interactions under natural condi-tions. Eggs of the red abalone (Haliotis rufescens) releaseL-tryptophan, which elicits a chemotactic response by conspe-cic sperm. Himes et al.189 developed a synthetic egg mimic thatreleases L-tryptophan 98 at rates equivalent to that known forred abalone eggs. They found that sperm responded similarly tolive eggs and the mimic, but did not respond to a placebosynthetic egg with no compound released, showing that thechemotactic response of red abalone sperm can be completelyattributed to the release of L-tryptophan.

The effectiveness of L-tryptophan 98 as a chemoattractantwas dependent on the hydrodynamic conditions surroundingH. rufescens eggs in laboratory experiments. Theoreticalconcentrations and distributions of the L-tryptophan plumesurrounding eggs under a variety of ow conditions weremodeled and it was determined that surface areas and volumesof plumes were greatest in still water or water with weakshears.190 These conditions most closely match to those foundin the natural spawning environment of H. rufescens. Underexperimental conditions in a Taylor–Couette ow chamber,sperm swam faster and moved toward eggs within the theoret-ical plume areas predicted. This led to higher fertilization ratesunder weak sheer conditions. Deactivation of L-tryptophan withtryptophanase eliminated the sperm swimming responsewithin the plume and decreased fertilization success, furthersupporting the presence of L-tryptophan as a chemoattractantwithin the plume areas. Both uid shear and sperm chemo-attraction impacted fertilization dynamics. Using a series ofstepwise multiple regressions, the authors determined thatunder low sperm concentrations, uid shear explained most ofthe variation in fertilization success; however, at high spermdensities, chemoattraction had a greater impact.

In addition to using chemoattractants as a way to locate eggs,sperm from the broadcast spawning mussel, Mytilus gallopro-vincialis, used chemical cues to differentiate between the eggs ofdifferent females within the same species.191 When given achoice between the eggs of two conspecic females, there was asignicant interaction between the male being tested and thepreference for an individual females eggs. This preferentialresponse based on chemical cues provides a possible mecha-nism for the selection of genetically compatible partners.

Female blue-ringed octopuses (Hapalochlaena lunulata andH. fasciata) appeared to invest tetrodotoxin (TTX) 99 in theiroffspring.192 TTX was found in the posterior salivary gland offemales where this toxin has previously been shown to be stored

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in blue-ringed octopuses. TTX was also detected in the ovaryand oviducal gland of H. fasciata as well as within hatchlings.No TTX was detected in the oviducal gland of H. lunulata butTTX was found within the eggs and paralarvae at all develop-mental stages. The amount of TTX within eggs and paralarvaewas positively correlated with the levels of TTX within thebrooding female. TTX levels within eggs/paralarvae alsoincreased with developmental age, but the trend was onlysignicant between stages 6 (2 weeks aer egg deposition) and 8(paralarvae hatched). The investment of TTX in offspring didnot reduce TTX levels within females, providing support for thepossibility of bacterial symbionts as TTX producers.

Chemical cues are oen responsible for inducing settlementbehaviors in invertebrate larvae, including molluscs. Mono-specic biolms and exudates of two marine bacteria, Macro-coccus sp. AMGM1 and Bacillus sp. AMGB1 (isolated fromseawater and the guts of the green-lipped mussel, Perna cana-liculus, respectively) induced settlement of the P. canaliculuslarvae.193 A variety of physical and chemical separation tech-niques were used to characterize the active metabolites from thebacterial exudates.194 Fractions from Macrococcus sp. AMGM1exudates that induced larval settlement were identied as polarproteins and non-polar lipoproteins. Bacillus sp. AMGB1exudates contained one active fraction consisting of glycolipidmolecules. A third bacterium tested (Pseudoalteormonas sp.AMGP1, isolated from the green alga Ulva lactuca) was toxic tothe larvae and did not induce settlement.193 Extracts of eightalgal species (5 red and 3 brown) and one hydrozoan that areknown settlement substrata for P. canaliculus did not signi-cantly increase P. canaliculus larval settlement.195 However,substratum texture had a signicant effect on settlement, with agreater number of larvae settling on the rough side of a cable tiecompared to the smooth side regardless of the presence ofextracts.

Dibromomethane (DBM) 100, naturally produced by coral-line algae, induced larval metamorphosis within three hours ofexposure in the common slipper shell Crepidula fornicata atconcentrations of 5000 ppm and 500 ppm, but concentrationsof 50 ppm or below did not induce metamorphosis.196 Theactivity of DBM at 5000 ppmwas not diminished by allowing thesolution to stand for up to 5 hours prior to use indicating thatwithin the testing period there was no loss of activity due to thevolatility of the compound. Larvae were responsive to DBM at5000 ppm 10 days aer release, 5 days earlier than thoseexposed to KCl, a known articial inducer. However, juvenilemortality was signicantly higher in larvae exposed to DBMcompared to those exposed to KCl, regardless of the age atmetamorphosis.

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In eld experiments limpets (Siphonaria pectinata, Patellacaerulea, and Cymbula nigra) colonized tiles treated withg-amino butyric acid (GABA) 101 earlier than control tileswithout GABA.197 Additionally, a signicantly higher number ofindividuals were found in the area surrounding GABA treatedtiles compared to control tiles even two months aer GABAapplication. Within individual limpet species, there weresignicantly more P. ferruginea and C. nigra recruits andP. caerulea adults surrounding GABA treated tiles thanuntreated controls at the end of the two-month GABA applica-tion period. This difference in abundance persisted for onemonth aer the end of the GABA application period for P. fer-ruginea, but not the other two species. There was no signicantdifference in the abundance of recruits or adults for two otherspecies P. rustica and S. pectinata at any time period or for any ofthe species two months aer GABA application.

Larvae of the Hawaiian bobtail squid (Euprymna scolopes)moved toward seawater that had been treated with conspecicswhen given a choice between a chamber containing waterconditioned with conspecics and water conditioned with het-erospecic crabs (Calappa calappa). The authors suggest thatE. scolopes are attracted to chemical cues released by conspecics;however, the possibility that the squid are being repelled by thecrabs cannot be eliminated based on the data presented.198

The ability of molluscs, especially nudibranchs, to tolerateand sequester potent chemical toxins is well established.However, the mechanisms by which these organisms are able totolerate such compounds are still an active area of investigation.The gastropod Cyphoma gibbosum and the nudibranch Tritoniahamnerorum were examined for the presence of multixenobiotictransporters.199 These molluscs feed exclusively on chemicallydefended gorgonians and may be able to tolerate toxins in theirdiet by exuding them or sequestering them in specializedstructures. Multixenobiotic transporters are well known fortheir ability to reduce the toxicity of pharmacological agentsand may also aid predators in tolerating dietary toxins. Diges-tive glands of C. gibbosum and whole tissues of T. hamnerorumcontained cDNA sequences that were homologous to knownpermeability glycoproteins (P-gp), members of a subclass ofmultixenobiotic transporters. Western blot analysis demon-strated that P-gp were present in whole tissues of T. hamnero-rum, but not in C. gibbosum digestive glands. The authorssuggest that this could reect the differences in foragingbehavior between the two species. T. hamnerorum is a specialistpredator while C. gibbosum is a generalist.

Immunochemical analyses revealed that P-gp are found inthe apical epithelial of the midgut and epidermis of T. ham-nerorum which is consistent with the proposed role in toleratingpotent chemicals produced by the gorgonian prey. Inhibition of


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P-gp and another type of multixenobiotic transporter (multi-drug resistance protein; MRP) in vivo led to an increase in cal-cein-AM concentration within T. hamnerorum cells compared tocontrols lacking inhibitors, demonstrating that P-gp and MRPare present and capable of transporting toxins out of cells.Although P-gp were not found in the digestive glands ofC. gibbosum, transcripts of a MRP homologous gene were foundconstitutively expressed in individuals fed on gorgonians withvarying defensive compounds. The expression of the MRP genewas not affected by the species of prey gorgonian or thecompounds produced by the gorgonians. The authors suggestthat C. gibbosum may rely more on MRP transporters becausethey are involved in transporting glutathione. Based on the highamount of glutathione S-transferases found in the digestivegland of C. gibbosum it has been proposed that these molluscsconjugate prostaglandins and other lipophilic compounds withglutathione and then transport them out of their cells usingMRP.

10 Echinoderms

Holothuroids are known to produce a large number of diversetriterpene glycosides (saponins), and while they have beenshown to selectively bind to cell wall proteins their role inchemical defense for the host organism has not been wellstudied. A study aimed at the investigation of the chemicaldefenses of Holothuria forskali attempted to localize the knownsaponins in the tissues, determine the saponin concentrations,and assess the response of the holothuroid when exposed tomechanical stimulation or the sh Coris julis and Symphodusocellatus. H. forskali was collected at the Biological Station ofBanyuls-sur-Mer, France and specimens were either anes-thetized (relaxed), placed in an apparatus to repetitively hit theanimals (stressed) or maintained in tanks (non-stressed).MALDI imaging experiments detected differences in theconcentration and location of eight saponins in the body wallthat were dependent upon the state of the animal. For example,two saponins present in the non-stressed animals appeared to


be absent in the stressed animals. Seawater samples taken fromthe tanks of stressed versus non-stressed animals containedsaponins present in the epidermis. Holothurinoside G wasdetected in the seawater from non-stressed holothuroids, whileholothurinosides C and F, and desholothurin A appeared to besecreted under stress. Two new holothurinosides, L 102 and M103 detected at m/z 1301 and 1317, were present in the seawaterfrom the stressed holothuroids. Because these new compoundswere not detected in the epidermis the authors suggest that theycould come from an internal organ. Comparatively, theconcentrations of saponins secreted in the seawater were verylow compared with the body wall and Cuvierian tubules. Incu-bation of C. julis and S. ocellatus with high concentrations ofsaponins (30% of the total saponin content) invoked the sh toswim quickly around the tank, appear to increase the respira-tion rate and then die within a few minutes; however, while thelethal effect was not observed at the natural concentration thesh still appeared to swim at an increased rate and increasetheir respiration when rst detecting the compounds. Theauthors concluded that the location and the ability of the sh todetect the presence of the saponins suggest that thesecompounds act as an aposematic signal warning predatorsaway.200

Chemical cues for metamorphosis and defense have beenthe subject of several recent studies of echinoderms. One studyconcerning chemical cues affecting the metamorphosis of seaurchin larvae tested the hypothesis that histamine, previouslyreported as a metamorphosis cue of the sea urchinHolopneustespurpurascens, is a common metamorphic cue for sea urchinlarvae. Sea urchins were collected during their reproductiveperiod from Botany Bay and Coffs Harbor including Hol-opneustes purpurescens, H. inatus, Heliocardis erythrogramma,H. tuberculata, Tripneustes gratilla, and Centrostaphanus rogersii.Histamine induced metamorphosis in 35–40% the lecitho-trophic larvae from H. purpurascens and H. inatus at concen-trations as low as 1 mM aer one hour while the 10 mMconcentration of histamine induced metamorphosis in Helio-cidaris erythrogramma larvae aer 24 h. Thirty percent of

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planktotrophic larvae from Centrostephanus rodgersii alsometamorphosed when exposed to 10 mM of histamine aer 24hours. Larvae of Tripneustes gratilla and Heliocidaris tuberculatadid not metamorphose in response to histamine. To determineif the larvae of the sea urchins were viable they were exposed tothe coralline alga Amphiroa anceps or Corallina species. In theseexperiments, larvae from all six species settled within 24 hoursof exposure to one of the coralline algae species. Additionalexperiments with seagrasses and algae, the host plants ofH. inatus and C. rodgersii, respectively, showed that exposure tothe seagrasses induced rapid metamorphosis in larvae of thetwo Holopneustes species, and several algae induced meta-morphosis in C. rodgersii larvae. The authors concluded that thehistamine from algae and seagrasses may act as a generalchemical cue for habitat recognition and metamorphosis in seaurchin larvae.201

Inducible defenses have been described in a number ofmarine invertebrates and algae as a way to avoid predation. Ina study of the sand dollar Dendraster excentricus the larvae(plutei) have been shown to exhibit a unique response to thechemical cues in predatory sh mucus by cloning via anterior-posterior ssion.202 Recently, a more detailed study addressedthe potential benets cloning provides as a defense mecha-nism for plutei in D. excentricus against three planktivoroussh, the Dover sole Microstomus pacicus, the three-spinesickleback Gasterosteus aculeatus and the Pacic sand lanceAmmodytes hexapterus. Larvae from D. excentricus, collectedfrom East Sound, Orcas Island, were exposed 0.01 mg ml�1

external sh mucus swabbed from the surface of M. pacicusfor nine days during early development. Increased larvaldensity and active budding of larvae were measured in 3 dayintervals as indirect and direct measurements of cloning. Byday 6, cloning was evident in all of the treatment tanks.Measurements of the midline body length, length of thepostoral arms and the diameter of the echinus rudimentshowed that the cloned plutei were smaller than their sisterplutei not exposed to the sh mucus. Feeding experiments ofcloned versus non-cloned plutei against G. aculatus andA. hexapterus demonstrated more uncloned sibling plutei wereeaten compared to the smaller clones. The authors suggestthat the smaller size of the clones would put visual predatorsat a disadvantage and that their results support the idea thatasexual reproduction resulting in rapid size reduction is aviable defense against planktivores.203

The ability of marine organisms to detect cues from pred-ators can be vital to survival. A study of the black sea urchinEchinometra lucunter investigated the hypothesis thatE. lucunter would detect echinivorous predator odors andchemical cues from the green sea urchin Lytechinus variegatusand injured conspecics. E. lucunter, L. variegatus and thecushion sea star Oreaster reticulatus were collected from SaoSebastiao city in Sao Paulo state to conduct laboratory exper-iments that evaluated the sea urchin behavior in response topredator odor and alarm cues released by the black and greensea urchins. E. lucunter were exposed to articial seawater inwhich O. reticulatus were fed a diet of green or black sea urchinor brown mussels. Black sea urchins responded strongest to

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the water of predators fed the conspecics by moving theirtube feet and spines, while they showed a weaker response topredators fed on green sea urchins. In contrast, E. lucunter didnot exhibit a response to the seawater of O. reticulatus fed on adiet of green mussels. In the second set of experiments inwhich the black sea urchins were exposed to extracts preparedby crushing sea urchins in articial seawater, the urchinsexhibited similar behaviors with a strong response to theconspecic and a weaker response to heterospecic urchin.The authors suggest that black sea urchins use chemical cuesfrom conspecics and heterospecic to recognize predatorycushion sea stars and the failure to detect sea stars that havenot recently feed on sea urchin will likely increase their risk ofbeing eaten.204

11 Other invertebrates

A broad study of chemical defenses among Antarctic benthicinvertebrates and algae used a new method for assessingfeeding deterrence that involves incorporating lipophilicextracts into caviar-textured alginate food pearls. The majorityof the species tested contained lipophilic compounds thatwere deterrent against the circumpolar omnivorousamphipod Cherimedon femoratus.143 Thirty-one species ofinvertebrates were collected including sponges, cnidarians,ascidians, a bryozoan, an echinoderm, a hemichordate andalgae. For four species, multiple morphotypes were collectedso that there were a total of 40 samples. Some of these sampleswere further divided into different body sections to determineif defenses were sequestered in specic parts of the animal.Extracts from 26 of the 31 species tested were unpalatable toC. femoratus. The incidence of feeding deterrence was highestamong ascidians with 91.7% of the extracts exhibiting deter-rent activity. This was followed by sponges (86.7%), cnidar-ians (85.7%) and algae (75%). The bryozoan, hemichordateand echinoderm extracts were not deterrent. There wasevidence for localization of defenses within certain bodyregions for two of the dissected samples. Extract from theaxial body region of the pennatulacean Umbellula antarcticawere unpalatable while those from the polyparium werereadily consumed. Basal-external and visceral extracts of theascidian Synoicum adareanum were deterrent, while the apicalextract was not.

Sessile marine organisms from the south-east coast of Chinawere tested for the production of antifouling compounds. Fourextracts (hexane, ethyl acetate, ethanol and distilled water) from11 species of marine invertebrates, seagrasses and algae weretested in larval settlement assays using the barnacle Balanusalbicostatus. Most of the antifouling activity was found in thenonpolar (hexane and ethyl acetate) extracts. Hexane extracts ofthe hydroid Tubularia mesembryanthemum and the tunicateStyela conopus, hexane and ethyl acetate extracts of the sea hareNotarcus leachii cirrosus, ethyl acetate and ethanol extracts of thebryozoan Bugula neritina, and ethanol extracts of the anemoneAnthopleura sp. strongly inhibited settlement of B. albicostatus(EC50 < 50 mg ml�1). At least one extract from each species tested


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inhibited the settlement of B. albicostatus with an EC50 of<500 mg ml�1.

12 Vertebrates

Research continues to test how sh use chemical cues to detectprey, predators, and appropriate settlement habitat. Findingfood in the marine environment is necessary for survival. Arecent review highlights the role of dimethyl sulde as achemical cue, especially for marine organisms.12 Juvenile jacksswim towards dimethylsulfoniopropionate (DMSP), which maybe a feeding cue for these sh.205 In laboratory ume experi-ments both bluen and crevalle jacks swammore when exposedto a concentration of 10�9 M DMSP when compared to articialseawater. The authors suggest this response could be an effec-tive mechanism of nding prey for these predatory sh.

A eld study in New Zealand showed that sh that usedchemical cues were more likely to nd baited traps.206 Under-water video cameras were placed over a bait bucket with 300grams of pilchard (Sardinops neopilchardus) to quantify shabundance at the traps. Fish surveys were also conducted byresearchers using SCUBA in the same habitats, which showedthat the video traps were more effective at detecting southernbastard cod, northern conger eel and yellow moray eel. All ofthese species use olfactory cues for hunting and they found thetraps faster than non-olfactory sh even though non-olfactoryspecies were more common on the SCUBA surveys. Theseexperiments show that some sh detect prey using olfactorycues.

Some buttery sh feed on epibionts that live on clam shells,and how ve species of Chaetodon detect their prey was tested inFrench Polynesia.207 Fish attraction was tested in a laboratory y-maze that had a control of lagoon water on one side comparedto a treatment of water-soluble cues. Water was conditionedwith ve live oysters that had been cleaned, and the epibiontscollected from ve oysters were soaked in 15 liters of seawaterfor 3 hours. Both C. aurga and C. lunula spent more time in theume channel with the water conditioned with clean oysters.C. aurga, C. lunula and C. citrinellus were attracted to seawaterconditioned with epibioints. When epibiont and live oysterwater were directly compared C. aurga and C. citrinellus spentmore time swimming in the epibioint water and C. lunula spentmore time in the live oyster conditioned water. Water condi-tioned with oysters and epibionts were analyzed with highperformance liquid chromatography and both containedunique peaks; however, no further work was done to charac-terize those compounds. The buttery sh detected potentialprey using water-soluble chemical cues.

Some marine predators can detect prey and prey qualityusing chemical cues. Predatory dottybacks (Pseudochromis fus-cus) prey on newly recruited damselsh and were attracted toskin extracts of the damselsh Pomacentrus amboinensis.208 Skinextracts of the damselsh were created by making 6 scalpelincisions in each sh and rinsing the damaged sh with 15 mlof seawater. This seawater extract was ltered and then used ina y-maze aquarium where P. fuscus consistently chose skinextracts of damselsh that were well fed compared to extracts


from sh that were poorly-fed. The predators also distinguishedthe size of the damselsh and spent more time in the tube withskin extracts from juvenile damselsh compared to skinextracts of adults. In the eld similar results were found wherepredatory sh were attracted to and spent more time in closeproximity to the site of release of juvenile skin extractscompared to extracts of adult damselsh. These sh werespecically attracted to chemical cues released from potentialprey showing that these predators discriminate among cuesfrom the same species of prey.

Some of the recent work on chemical ecology of marine shhas also focused on how sh detect potential predators. Juveniledamselsh, Pomacentrus amboinensis were studied for theirbehavior in response to chemical cues from damaged conspe-cics.209 Newly settled P. amboinensis were assessed for theirbehavior in response to three different concentrations of skinextracts created as described in the research above. Feedingstrikes were counted as a behavioral trade off thought todecrease in the presence of predators, and the new recruits werealso assessed for the distance they swam from shelter, and thetime they spent in their shelter. Of the skin extracts from newrecruits the least amount of feeding strikes were caused by thehighest concentration of skin extracts, but all of the skinextracts from the new recruits and the juveniles had less feedingstrikes than the skin extracts from adults which induced asimilar number of feeding strikes as a heterospecic (Apogoncyanosoma) skin extract and seawater alone. Only the high andmedium concentration of recruit skin extracts reduced thedistance the sh traveled away from their shelter, reduced thenumber of times they exited the shelter and increased theamount of time they spent in the shelter. These sh usechemical alarm cues from specic ontogenetic stages ofconspecics to modify their behavior to avoid potentialpredators.

Potential predator cues were also examined to determine ifthe feces of a predator induced antipredator behavior in the reefgoby Asterropteryx semipunctatus.210 In the laboratory the pred-ator Cephalopholis boenak was fed A. semipunctatus or the het-erospecic sh Xiphonphorus helleri for three days. The fecesfrom the predators were collected in 300 ml of seawater, whichwas ltered and then used as the predator cue. In additionalarm cues were prepared from conspecics and X. helleri bycutting themwith a razor blade and rinsing the wounds in 15mlof seawater. The seawater conditioned with X. helleri fedA. semipunctatus and seawater conditioned with damagedA. semipunctatus both induced less movement and less feedingstrikes in conspecics, but both heterospecic treatments andthe seawater control did not change A. semipunctatus behavior.These gobies responded to chemical cues from conspecicseven aer a predator had digested their body suggesting thatthese alarm cues could be reliable indicators of recent preda-tion events on conspecics.

Detecting predators using chemical cues is critical to survivaland a few experiments tested how conditioning and learningcould inuence the ability of sh to detect potential prey. Onestudy manipulated sh health and prior exposure to chemicaland visual predator cues to test the survival of the damselsh

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Pomacentrus wardi.211 Larval P. wardi were captured and broughtinto the laboratory and they were given a high (2500 Artemia perliter) or a low (320 Artemia per liter) amount of food for 6–8 days.Then these sh were released in the eld into different coralheads and their behavior was recorded. The sh in the lowfeeding treatment traveled away from their coral head more,ventured a longer distance away and had a higher boldnessvalue (a subjective behavioral category that measured acombination of the movement of a sh in and out of its shelterand its willingness to feed). When these sh were exposed tovisual and chemical cues of a predator there was no change inthe total distance they moved or in their boldness values but thesh exposed to either cue or both together did not venture as farfrom their coral home as the control shes exposed to none ofthe predator cues. Survivorship of these new recruits weremonitored in the eld for the next 3–4 days and the sh thatwere only exposed to seawater (no predator cues) or poorly fedand exposed to the chemical cue had higher rates of mortalitythan all the other treatments. These sh had higher survivalwhen they were better fed and exposed to cues from a predatorsuggesting that learning is an important component of survivalfor newly recruited sh.

Another study tested whether Pomacentrus moluccensis(lemon damselsh) can learn predator chemical cues atdifferent times during the day.212 P. moluccensis were treated for6 days with water conditioned with damaged conspecics(scalpel wounds from 3 individuals rinsed with 20 ml ofseawater) and predator water (2 Cephalopholis cyanostigma in 30l of seawater for 56 hours) either in the morning or the evening.When tested in the morning those sh that had been condi-tioned to morning chemical cues had less feeding strikes thanthe sh conditioned in the evening when offered water withpredator odor alone. Conversely, those sh conditioned in theevening had less feeding strikes when exposed to predatorconditioned water in the evening. Pomacentrus moluccensislearned to respond to predator odors and was conditioned torespond to those chemical cues at different times during the daysuggesting these sh can discriminate among times to heightentheir response to predation risk.

Social learning in response to predator cues was also testedusing the damselsh Acanthochromis polyacanthus.213 Thisdamselsh was shown to reduce its movement in the presenceof conspecic alarm cues (made by rinsing damaged sh with15 ml of seawater), but they did not change their swimmingbehavior in the presence of water conditioned with the preda-tory rock cod Cephalopholis boenak (one sh in 15 liters ofseawater for 24 hours). To test social learning some sh wereexposed to both the conspecic alarm cue and the waterconditioned with C. boenak at the same time and measured forthe distance they traveled from shelter. These sh were thenincubated with naıve sh that did not previously respond to thepredator conditioned water and both sh were exposed to thewater conditioned with C. boenak. Aer the sh were separatedwhen the naıve individuals were exposed to water conditionedwith the predators they traveled from their shelter less than thecontrols but the same amount as the sh that were offered thealarm cues in combination with the predator conditioned

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water. Social learning is known from other animals but this isthe rst study to show that sh can learn to respond to apredator cue from another individual.

As climate change continues to change the oceans, increasedresearch is being conducted on how ocean acidication willimpact sh sensory ecology. A recent review highlights theeffects of climate change on coral reef sh and discusses thepotential for acclimation and adaptation in sh.214 Anotherreview discusses how sh communication will respond toenvironmental changes and stresses the importance of amultimodal approach to better understand how differentsensory modalities might compensate for each other in stressfulenvironments.215

An experiment tested whether ocean acidication wouldinhibit learning ability in the damselsh Ponacentrus amboi-nensis in response to the predator, Pseudochromis fuscus.216 Toteach damselsh, they were exposed to water conditioned withP. fuscus (2 adult sh held in 70 liters of seawater for at least 24hours) at the same time as conspecic alarm cues (made byrinsing a damaged P. amboinensis with 15 ml of seawater). As alearning control some of the sh were pseudo-conditioned,where the sh were exposed to water conditioned with P. fuscuswith only seawater added (no conspecic alarm cue). TheP. amboinensis were conditioned in CO2 treatments (naturalseawater control and CO2 concentration of 850 matm) for 4 days.Fish that were in the pseudo-conditioning treatment showed noresponse to the water conditioned with predators regardless ofthe CO2 treatment. Fish that were conditioned with alarm cuesshowed a decrease in feeding strikes, amount of swimming andthe range of area covered when they were exposed to waterconditioned with predators. Fish that were conditioned withalarm cues but incubated at elevated concentrations of CO2 didnot display these anti-predator behaviors. In the same study,elevated CO2 concentrations were tested for their effects onsocial learning (as described in Manassa and McCormick213).Fish that were previously conditioned or not were held with shthat were naıve to predator odors. When sh were conditionedat a CO2 concentration of 850 matm they no longer showedavoidance behavior that was learned by the sh at control CO2

concentrations. To determine if sh were losing their ability tolearn or simply less active in CO2 treatments an experiment wasconducted testing shes behavior one and 5 days aer CO2

treatment, allowing the CO2 effects to wear off. Fish conditionedto predator odor reduced their feeding strikes, activity andspace utilized when exposed to water conditioned with preda-tors, but when they were incubated at a CO2 concentration of700 matm their behaviors did not change in response to pred-ator water. Five days aer CO2 treatment the sh treated with aCO2 concentration of 700 matm still did not respond to predatorodor but they would respond to conspecic alarm cues. Thisseries of experiments shows that in future concentrations ofCO2 the ability of sh to detect predators could be seriouslyinhibited and their ability to learn from conspecics could alsobe inhibited, potentially reducing their survival in the wild.

To better understand the physiological effects of oceanacidication on sh sensory ecology one recent study looked atthe effect of elevated CO2 on GABA receptors.217 These authors


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show that in high CO2 environments GABA-A receptors aredisrupted, causing changes in sh behavior. Larval Amphiprionpercula were raised in high CO2 (900 matm) conditions for 11days and then tested in a two channel ume for their avoidanceof water soluble cues from the rockcod predator. Larval shtreated with high CO2 did not respond to predator cues, butlarval sh exposed to high CO2 and exposed to the GABA-Areceptor antagonist gabazine had similar avoidance behavior asthe control sh. This experiment showed that the GABA-Areceptor is one critical neurological mechanism involved indetecting predator chemical cues and that ocean acidicationcould greatly interfere with the GABA-A receptor. These resultsare an important mechanistic explanation of how sh mightloose their ability to sense predators in future ocean conditions.

The effects of ocean acidication on the ability of sh todetect prey were studied using the brown dottyback, Pseudo-chromis fuscus.218 P. fuscus held in natural seawater at ambientCO2 levels were attracted to skin extracts of Pomacentrus mouc-censis (damaged sh rinsed with 15 ml of seawater). However,when P. fuscus were held in seawater enriched with CO2 (both apH of 8.0 and 7.9) they no longer responded to the waterconditioned with potential prey. P. fuscus in the 7.9 treatmentshowed increased activity compared to the control sh but thesh in 8.0 did not change their swimming activity. The responsetime of P. fuscus to food in the tank increased and feedingstrikes were signicantly reduced in the 8.0 treatment but therewas no change in the response time or feeding strikes for thesh in the 7.9 treatment. Elevated CO2 modied the behavior ofthese predatory sh and it reduced their ability to detectchemical cues that might be critical for nding potential prey.

Other research focused on how water quality might modifythe response of sh to chemical cues. The damselsh A. poly-acanthus was studied to determine how it responds to chemicalcues in the presence of increased turbidity.219 These sh wereexposed to conspecic alarm cues (damaged A. polyacanthusrinsed with 15 ml of seawater), heterospecic alarm cues(damaged Xiphophorus helleri rinsed with 15 ml of seawater) orthe seawater control under three turbidity levels, clear water,low turbidity (9 mg l�1 of Eckalite kaolin clay) and high turbidity(41 mg l�1 of clay). A. polyacanthus swam less in the presence ofthe conspecic cues (the only type of cue to have a behavioraleffect) in the high turbidity treatment when compared to theclean water or the low turbidity treatment. However, the timeactive and the number of lines that were crossed were notsignicantly different among the turbidity treatments. Thisstudy suggests that A. polyacanthus behavior is modied in lowvisibility situations with a greater reliance on their olfactorysense.

Chemical cues are known to be important sexual phero-mones for some sh. The compound petromyzestrosterol 104was recently isolated frommale sea lamprey.220 Through variouschromatography techniques 104 was isolated from freshwaterconditioned with adult male sea lamprey. When female sealamprey were exposed to 104 it caused electro-olfactogram(EOG) responses similar to the known pheromone 3-keto pet-romyzonol sulfate (3k PZS) at concentrations of 10�10 M. But 3kPZS illicited more EOG response at concentrations of 10�9 to


10�6 M than 104 did. Organic chemistry techniques werefurther improved to increase detection of this compound in theenvironment.221 Through solid phase extraction using cation-exchange and reversed-phase mixed-mode and ultra-highperformance liquid chromatography the detection limit of3kPZS in freshwater was below 0.1 ng per liter. Stream water wasmeasured from natural spawning populations of sea lampreyand the concentration of 3kPZS was found to be between 0.15and 2.85 ng per liter in seven different streams, but was notdetected in stream segments that did not contain sea lampreys.These advances in chemistry techniques should increase theability to quantify this pheromone in natural streams and willenable managers to identify streams that contain spawningpopulations of these invasive sh.

Chemical cues are also important for sh to detect appro-priate habitat. Building on a large body of work, petromyzon-amine disulfate was further studied for its potential role as amigratory pheromone for the sea lamprey.222 The release rate ofpetromyzonamine disulfate (PADS), petromyzosterol disulfate(PSDS) and petromyzonol sulfate (PS) from larval sea lampreywere measured from both fed and unfed larvae. Fed larvaereleased 10–25 ng per larvae per hour of all three steroids withPSDS released more than the other two steroids. Unfed larvaereleased signicantly less of all the steroids and PADS was themost concentrated of the three steroids. In ume experimentsadult sea lamprey were attracted to both fed and unfed larvaeshowing that they both produce enough steroids to elicitresponse. All three steroids degraded in stream water at asimilar rate with approximately 50% remaining aer 3 days.Natural organic matter was tested to see if it would reduce thebioavailability of these three steroids. The presence of organicmatter did not change steroid concentration in streamwaterand the sh had the same EOG responses to the steroids aloneor the steroids in combination with natural organic matter.These compounds are produced at ecologically relevantconcentrations that should persist in natural stream water longenough for them to be effective migratory compounds for sealamprey.

Further work on the importance of lamprey swimming andhoming behavior in response to natural chemical cues wasstudied in the eld.223 Lamprey olfactory organs were blockedwith inert dental material or they were blocked with gelatin(a control that dissolves away), or not blocked at all. Treated shwere tagged and released at river mouths to see if they wouldtrack to baited traps. In three different rivers sh that had theirolfactory organs blocked were trapped less than the control shaer 31 or 55 days, even when the sh were released 3.6 kmoffshore (in Lake Huron) from the river mouth. Control lampreytracked to the traps regardless of the stream they were collected

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from more than the treated sh with blocked olfactory organs.Further ne scale swimming behavior of sea lamprey wasstudied using acoustic transmitters.224 Lamprey swam at similarspeeds regardless of being released in the open water or in ariver plume. The lamprey released in the river plume swam incircular movements, which allowed them to nd the rivermouth. The swimming linearity and turning angles increasedfor sh that were in the river plume. When sea lamprey'solfactory organs were blocked with dental material, they nolonger exhibited this swimming behavior in a river plume andswam straight with few turns at the same rate as sh that wereobserved outside of a river plume. None of the sh with blockedolfactory organs entered the stream that contained the homingcues. Chemical cues were necessary for sea lamprey to nd theirspawning habitats.

The Pacic lamprey, Lampetra tridentata were also studiedfor their migratory behavior in response to chemical cues tobetter understand how to increase lamprey populations in theirnative streams.225 In y-maze experiments Pacic lamprey wereattracted to larval chemical cues that included PADS, PSDS andPS. Using electro-olfactogram each of these compounds causedan olfactory response showing that these compounds aredetected by these sh. Pacic lamprey responded to some of thesame compounds as sea lamprey to nd their spawningstreams.

Much like the disruptions of detecting predators in oceanacidication experiments, the homing ability of sh exposed toocean acidication was recently tested. Cardinalsh, Cheilo-dipterus quinquelineatus were maintained in four differentconcentrations of CO2 (ambient, 550, 700, and 950 ppm) forfour days and then tested for their ability to nd conspecicscollected from their home site or a novel site.226 Fish treatedwith CO2 concentrations were released into a 300 liter roundaquarium with two opaque chambers, one with a conspecicfound on the same reef from which the cardinalsh was collect(native reef) and one with a conspecic collected from a novelreef. At all the elevated CO2 concentrations the cardinalsh didnot discriminate between native conspecics more than novelconspecics even though those at ambient CO2 concentrationspreferred native conspecics. When treated sh were taggedand released 200 meters from their native habitat in the eld,more of the sh at ambient CO2 found their native habitat 3days aer release compared to those that were treated withelevated CO2. Showing that shes will not be able to home intonative sites in elevated CO2 environment is another aspect ofhow sh sensory ecology might be disrupted by oceanacidication.

Chemical cues are known to be important settlement cuesfor sh larvae. A recent review discussed the current state ofknowledge for auditory, chemical, visual and environmentalcues that might aid sh in nding appropriate recruitmenthabitat.227 The relative roles of auditory, chemical and visualcues from multiple habitats were tested for their role in settle-ment of early-juvenile French grunts (Haemulon a-volineatum).228 H. avolineatum were maintained in cages in theeld and exposed to recordings from different habitats. In thesecages the sh swam towards coral reef sounds, but were not

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attracted to sounds frommangrove, seagrass or rubble habitats.This result was signicant for sh that were <20 mm long butwas not signicant for those that were 20–30 mm long sug-gesting that juvenile sh are more likely to use auditory cues toswim towards coral habitat. Visual cues were tested by exposingsh in wire cages to small sealed Plexiglas boxes with differenthabitat types including reef, mangrove, seagrass and rubble ineach corner of the sh cage. There was no difference in theamount of time the sh spent in front of each habitat using justvisual cues. However, both the three conspecics and theconspecics with rubble treatments increased the time spent infront of those habitats more than the sh exposed to rubblealone or rubble with heterospecics (Acanthurus bahianus).Chemical cues were tested in a modied y-maze and H. a-volineatum spent more time in the water from the bay (bothmangrove and seagrass habitats) when compared to reef water.These sh responded to multiple types of cues including audi-tory, visual and chemical to nd their preferred habitat.

Two anemone sh were studied to see how their preferencesfor habitat changed every two days since hatching until theirlarvae were 11 days old.229 Both Amphiprion percula and A. mel-anopus were tested in a two channel ume with articialseawater as a control compared to water conditioned with theirhost anemone or coral (Porites cylindrica). A. percula was alsoexposed to water conditioned with leaves from the Xanthoste-mon chrysanthus, Melaleuca nervosa and Panicum maximumbecause this species was previously shown to respond to leaflitter chemical cues. 20 g of leaves were conditioned in 10 litersof seawater for 2 hours to produce the chemical cues for each ofthe plant species. Larvae of A. percula did not distinguish anycue from the seawater control when the larvae were 1, 3 and 5days old. Older larvae consistently swam to the ume thatcontained the water conditioned with their host anemone, coralor the plant X. chrysanthus, but the other two plants were notdifferent than the seawater. For A. melanopus more time wasspent in the ume containing the anemone and the coralconditioned water when compared to seawater aer the larvaewere 7, 9 or 11 days aer hatching. This research suggests thatas the larvae age they are more likely to track to settlementolfactory cues.

A novel experiment tested water-soluble cues from Acroporaspp. coral colonies on their ability to attract larvae of Chromisviridis.230 Coral colonies in the eld were identied as settle-ment inducers or inhibitors by the amount of C. viridis theycontained. Water was pumped from one inducing colony to aninhibiting colony and vice versa to determine if water-solublecues were responsible for the settlement patterns observed.When inducer water was pumped to an inhibiting coral colony,recruitment of C. viridis signicantly increased. For Acroporahyacintus coral colonies that previously increased settlement,water cues from an inhibitory colony inhibited settlement, butwater from inducer colonies did not increase settlement toinhibitory colonies. For A. eurystoma colonies that inhibitedsettlement, water from inducer colonies increased settlement,but water from inhibitory colonies had no effect on inducercolonies. When water was no longer pumped between coloniesthe sh le the colonies that were inhibitory and settlers were


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observed on inductive colonies that had been treated withinhibitory water. This study shows newly recruited sh can usewater soluble compounds to detect some unknown quality oftheir recruitment habitat that allows them to distinguishamong coral colonies of the same species.

Some sh can distinguish among settlement cues fromdifferent coral colonies, but a recent study tested how shwould recruit to Pocillopora damicornis coral colonies that werehealthy, bleached or dead.231 A eld experiment was created byputting P. damicornis colonies (live, bleached, dead) in threedifferent patches on a sand benthos. Aer ten days the mostspecies and a higher abundance of shes were found on healthyP. damicornis colonies compared to the bleached colonies, andthe dead colonies were not signicantly different than eithertreatment. A laboratory settlement experiment with recentlycaught Pomacentrus spp. larvae tested how the larval shdistinguished healthy from bleached corals. Initial trials withlive, bleached and dead coral showed that larvae of all threeshes species that were tested (P. moluccensis, P. amboinensisand Dischistodus sp.) settled on live P. damicornis more than onbleached, dead, and sand habitats. When each of these habitattypes were displayed in sealed clear Plexiglas cages (visual cues),the sh continued to choose live coral over the other habitatchoices, except for P. amboinensis which also settled on deadcoral more than would be expected at random. When the shwere exposed to fake coral habitat with water pumped from live,bleached, dead corals (olfactory cues) all three sh specieschose dead coral more than would be expected by random.P. amboinensis was the only species that also settled at greaterfrequency in response to chemical cues from live corals. Habitatselection among coral colonies of different health was driven byboth visual and olfactory cues for these sh species.

Detecting potential predators using chemical cues is animportant adaptation for survival that was tested in the eld bymeasuring sh recruitment to articial reefs.232 A series of vetreatments were added to articial reefs (80% rubble and 20%live P. damicornis colonies) to test settlement behavior ofdamselsh (Pomacentridae) in a sand habitat, which is not anatural settlement habitat for these sh. One patch reef washeld as a control and the rest had opaque 3.5 liter cylindersplaced on them with a treatment; an empty cylinder as a devicecontrol, a cylinder with a herbivorous sh (Acanthurus nigro-fuscus), a cylinder with the predator Cephalopholis microprionfed squid, and a cylinder of C. microprion fed juvenile damsel-sh. Both predator treatments had less damselsh settlementthan the two controls or the herbivore treatment. The ability todetect predator cues is probably a critical ability to increase thesurvival of newly recruited sh.

Larvae of Pomacentrus amboinensis were tested for theirability to detect a gradient of predation cue concentrations.233

The newly settled P. amboinensis had more bobs in theirresponse to visual cues of potential predators compared to non-predator or control visual cues. There was no change in shfeeding strikes, horizontal movements and height in watercolumn in response to visual cues from predators. Whendifferent concentrations of conspecic alarm cues (1, 2 or 4damaged sh rinsed with 15 ml of seawater) were given to the


sh, the largest decrease in feeding strikes and decrease inmovement away from shelter was in response to 4 damaged sh.This study shows that sh increase their behavioral modica-tions in response to higher concentrations of predator chemicalcues.

The ability of larvae of P. amboinensis to detect potentialpredators can be modied by their growth and feedinghistory.234 The presence of conspecic alarm cues (made bydamaging two P. amboinensis and rinsing them with 15 ml ofseawater) reduced feeding strikes, reduced the distance swam,and increased the time and frequency of shelter use. WhenP. amboinensis had a range of growth history and body condi-tion, better body condition decreased P. amboinensis feedingstrikes and increased their shelter time aer exposure toconspecic alarm cues. P. amboinensis that were well andmoderately fed consistently showed more avoidance behavior(less feeding strikes, less distance swam, increased shelter useand more time spent in the shelter) in response to a conspecicalarm cue than sh that were poorly fed or sh exposed toheterospecic chemical cues. Larvae of P. amboinensis respon-ded to conspecic chemical alarm cues appropriately when theywere more healthy, suggesting that healthy sh are morecapable of avoiding predation.

Another study showed that Amphiprion percula larvae avoi-ded water conditioned with sh fed different diets.235 Larvae ofA. percula avoided water conditioned (one sh held in 70 litersof standing seawater for 2 hours) with the predatory shesPseudochromis fuscus and Cephalopholis argus that had been fedarticial sh food diets of NRD pellets (66% sh products) orFrozen Marine Dinners (46% sh products), respectively. Whenwater conditioned with non-predatory shes (Acanthurus pyro-ferus, Rhinecanthus lunula, and Signus coralinus) was introducedinto the ume the A. percula larvae did not show any avoidancebehavior. However, when the diet of the non-predatory sh waschanged to the NRD pellets all three species were avoided bylarvae of A. percula. A eld experiment was conducted (with asimilar design as that described above by Vail and McCor-mick232) which showed that there was less settlement of larvaldamselsh to the articial reefs with chemical cues from aherbivore (Ctenochaetus striatus) and an invertivore (Rhine-canthus aculeatus) fed NRD pellets and a natural predator (Epi-nephelus hexagonatus) compared to the sh fed their naturaldiet or the two controls of no chamber or an empty chamber. A.percula were capable of detecting potential predators usingchemical cues from their previous diet, probably increasingtheir ability to avoid predation when they rst settle to a newhabitat.

Further work with larvae of Amphiprion percula showed thatocean acidiciationmight inhibit their ability to detect predatorchemical cues.236 Larvae were tested in a laboratory ume whenthey had just hatched and 11 days aer hatching in response towater conditioned with one sh in 70 liters of seawater for 2hours. Water was conditioned with predatory (Cephalopholiscyanostigma and Pseudochromis fuscus) and non-predatory sh(Acanthurus pyroferus and Siganus corallinus). In the choice trialsthe newly hatched A. percula always chose the seawater controlover any of the water conditioned with sh but when they were

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directly compared they spent more time in the water condi-tioned with non-predator sh over the two species of predators.At 11 days aer hatching this was still true except the larvae didnot choose between seawater and the water conditioned witheither non-predator species. When the larvae had been incu-bated in acidied seawater (pH 7.8) the newly hatched larvaebehaved the same as those raised in natural seawater. However,the 11 days old larvae preferred water conditioned with predatorand non-predator sh over the seawater control. Also they didnot distinguish between the predatory and non-predatory shwhen they were directly compared. Ocean acidication ruinedthe ability of these larvae to swim away from predatory shusing chemical cues, suggesting that future levels of oceanacidication could threaten sh survival by disrupting theirolfactory abilities.

Sedimentation was also studied to determine how it mightdisrupt the ability of larval Pomacentrus amboinensis and P.moluccensis to detect appropriate settlement habitat.237 Aer 90minutes, both P. amboinensis and P. moluccensis larvae selectedlive coral more than partially dead coral or dead coral in thecontrol treatment with no clay (bentonite was used to simulatesediment). At low, medium and high concentrations of sedi-ment both of these sh species did not distinguish among thethree types of coral. Chemical cues were made by soaking coraland natural sediment (collected from the reefs) in 10 liters ofwater for 2 hours. Recently settled juveniles of P. moluccensisspent more time on the side of a ume in water conditionedwith live coral than clean seawater and they also spent moretime in water conditioned with live coral than in water condi-tioned with dead coral. With the addition of water with sedi-ment added, they no longer distinguished between waterconditioned with live or dead coral. Increased concentrations ofsediment inhibited these shes ability to detect chemical cuesthat allow them to nd appropriate habitat.

13 Conclusions

Research in the eld of marine chemical ecology continues tohighlight the importance of marine natural products inecological interactions. It has been well established that naturalproducts play important roles in predator–prey interactions,symbioses, competition, reproduction and larval settlement. Asresearch on the roles of natural products in these interactionsadvances we are gaining greater insight into not only the pres-ence of chemical-mediated interactions, but also thecompounds involved and mechanisms by which they are func-tioning. Advances in microscopy and molecular techniqueshave led to a greater understanding of the ecological impor-tance of the microbiomes of many organisms, including thenatural products produced by these microbes. There has alsobeen increasing interest in the ecological implications ofchemically-mediated alterations in the natural microbiome ofsome organisms.

Studies on the effects of ocean acidication on chemically-mediated interactions are growing in light of increasingconcern over the ecological impacts of global climate change.Several studies on sh behavior have demonstrated that ocean

1548 | Nat. Prod. Rep., 2014, 31, 1510–1553

acidication interrupts chemical signaling, hindering theability of sh to detect predators, prey and habitats. There isalso evidence that ocean acidication can alter microbialcommunities that may produce chemical cues for coral larvalsettlement. Understanding how environmental changes,including ocean acidication, will impact chemically-mediatedinteractions is crucial for assessing and addressing the overallimpact of climate change on marine ecosystems. Given itswidely recognized importance, we expect to see more researchon the production, detection and stability of natural products ina high CO2 environment in the near future.

As the eld of marine chemical ecology continues to prog-ress, it is essential that researchers from multiple disciplinesincluding ecologists, natural product chemists, physiologists,biochemists, molecular biologists, microbiologists and, giventhe new concerns over ocean acidication, chemical and phys-ical oceanographers collaborate to understand the roles thatnatural products are playing within ecological interactions, themechanisms by which they are working, and the impacts ofenvironmental changes on these interactions.

14 Acknowledgements

We acknowledge the staff of the Smithsonian Marine Station atFort Pierce for their kind assistance. Support was provided bythe Smithsonian Hunterdon Oceanographic Endowment andthe Mote Protect our Reefs License Plate Program. Assistancewith literature review was provided by students in the EckerdCollege Marine Science Chemical Ecology Senior Seminar. Wethank Yartiza Lopez, Chicago State University, for her assistancein preparing the manuscript. This is contribution #957 of theSmithsonian Marine Station.

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141 G. Koplovitz, J. B. McClintock, C. D. Amsler and B. J. Baker,Mar. Biol., 2011, 158, 2661–2671.

142 L. Nunez-Pons, R. Forestieri, R. M. Nieto, M. Varela,M. Nappo, J. Rodrıguez, C. Jimenez, F. Castelluccio,M. Carbone, A. Ramos-Espla, M. Gavagnin and C. Avila,Polar Biol., 2010, 33, 1319–1329.

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165 J. A. Rasch and N. J. O'Connor, J. Exp. Mar. Biol. Ecol., 2012,416–417, 196–201.

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215 I. van der Sluijs, S. M. Gray, M. C. P. Amorim, I. Barber,U. Candolin, A. P. Hendry, R. Krahe, M. E. Maan,A. C. Utne-Palm, H.-J. Wagner and B. B. M. Wong,Evolutionary Ecology, 2011, 25, 623–640.

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225 S.-S. Yun, A. J. Wildbill, M. J. Siees, M. L. Moser,A. H. Dittman, S. C. Corbett, W. Li, D. A. Close and


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226 B. M. Devine, P. L. Munday and G. P. Jones, Oecologia, 2012,168, 269–276.

227 J. M. Leis, U. Siebeck and D. L. Dixson, Integr. Comp. Biol.,2011, 51, 826–843.

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229 D. L. Dixson, P. L. Munday, M. Pratchett and G. P. Jones,Coral Reefs, 2011, 30, 903–910.

230 O. Ben-Tzvi, D. Tchernov and M. Kiawi, Mar. Ecol.: Prog.Ser., 2010, 409, 181–187.

231 M. I. McCormick, J. A. Y. Moore and P. L. Munday, CoralReefs, 2010, 29, 537–546.

232 A. L. Vail and M. I. McCormick, Biol. Lett., 2011, 7, 921–924.

233 T. H. Holmes and M. I. McCormick, Anim. Behav., 2011, 81,543–550.

234 O. M. Lonnstedt and M. I. McCormick, Coral Reefs, 2011,30, 805–812.

235 D. L. Dixson, M. S. Pratchett and P. L. Munday, Anim.Behav., 2012, 84, 45–51.

236 D. L. Dixson, P. L. Munday and G. P. Jones, Ecology Letters,2010, 13, 68–75.

237 A. S. Wenger, J. L. Johansen and G. P. Jones, Coral Reefs,2011, 30, 879–887.

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Marine chemical ecology in benthic environments

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