REVIEW www.rsc.org/npr | Natural Product Reports
Marine chemical ecology
Valerie J. Paul,* Melany P. Puglisi and Raphael Ritson-Williams
Received (in Cambridge, UK) 6th January 2006, Accepted 1st February 2006
First published as an Advance Article on the web 23rd February 2006
DOI: 10.1039/b404735b
Covering: 2003?2005
This review covers the recent marine chemical ecology literature for phytoplankton, macroalgae,
sponges and other benthic invertebrates; 249 references are cited.
1 Introduction
2 Microorganisms and phytoplankton
2.1 Bacteria
2.2 Cyanobacteria
2.3 Eukaryotic marine microalgae
3 Macroalgae
4 Seagrasses
5 Sponges
6 Cnidarians
7 Ascidians (tunicates)
8 Bryozoans
9 Crustaceans
10 Molluscs
11 Polychaetes
12 Echinoderms
13 Other invertebrates
14 Vertebrates
14.1 Fish
14.2 Birds
15 Conclusions
16 Acknowledgements
17 References
1 Introduction
In this report, we review progress over the past two years in the ?eld
of marine chemical ecology. Research in this ?eld has continued
at a rapid pace since we last reviewed this topic.1 As the ?eld of
chemical ecology matures, research shows that natural products
drive complex ecological interactions at all stages of marine plant
and animal life histories. Research in marine chemical ecology
continues to focus on predator?prey and competitive interac-
tions, settlement cues, and potential defenses against infection
by microorganisms. There are many new studies that address
chemically mediated interactions between macroorganisms and
microorganisms associated on surfaces, adjacent substrates, or in
seawater. In addition, a few studies examine chemically mediated
interactions between different microorganisms. This review also
includes several examples demonstrating that symbionts chemi-
cally defend their host against predation.
Smithsonian Marine Station at Fort Pierce, 701 Seaway Drive, Fort Pierce,
FL, 34949
Several review articles dealing with various aspects of marine
chemical ecology have been published since our last overview of
this topic.1 Pohnert2 reviewed chemical defense strategies in marine
organisms with an emphasis on chemical defense in the plankton
and dynamic defenses in benthic organisms, including examples
of induced and activated defenses in marine algae. Allelopathic
interactions of primary producers in all aquatic ecosystems
have been recently reviewed by Gross.3 For marine systems she
discusses allelopathy (including antimicrobial and antifouling
activity) in planktonic species and benthic species, including
seagrasses, macroalgae, microalgae, and corals.3 Marine micro-
and macroalgae have also been studied as promising resources for
antifouling natural products. A recent review of marine natural
products that are implicated in antifouling and larval settlement
and metamorphosis was published by Fusetani.4 Another review
covers much of the algal antifouling literature and focuses on
the published activity of extracts and algal secondary metabolites
tested in antibacterial, antifungal, and antialgal assays.5 Here we
report the research in marine chemical ecology that has been
published since these reviews.
2 Microorganisms and phytoplankton
2.1 Bacteria
While chemically mediated microbial interactions in the terrestrial
environment are well studied, the chemical ecology of marine
bacteria is an emerging ?eld. Miao and Qian6 examined the
antibacterial and antifungal activities of 46 strains of fungi isolated
from seawater and 19 strains of bacteria isolated from natural
bio?lms in Hong Kong. Antibacterial activity was prominent in
the fungal extracts (32 active extracts), and six strains showed
broad spectrum antibacterial activity, inhibiting the growth of
at least 15 bacterial strains. Antifungal activities were observed
for 11 bacterial cultures. The result of this and other studies of
chemically mediated microorganism?microorganism interactions
demonstrate that the interactions in the microbial communities
are complex and warrant further studies.
Even though bio?lms are recognized as important in the
establishment of fouling communities, there has been little research
on antifouling compounds isolated from the associated microbial
community. Burgess et al.7 conducted a screening of bacteria
sampled from the surfaces of 30 benthic organisms. Six hundred
and ?fty bacterial strains were isolated, and forty two of these had
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Valerie J. Paul is currently Head Scientist at the Smithsonian Marine Station at Fort Pierce, Florida. She received her BA from the University
of California, San Diego in 1979 with majors in Biology and Studies in Chemical Ecology and her PhD in Marine Biology in 1985 from
the University of California San Diego, Scripps Institution of Oceanography. Dr Paul joined the faculty of the University of Guam Marine
Laboratory in 1985, served as Director of the Laboratory from 1991?1994, and as full Professor from 1993?2002. She was elected a Fellow
of the American Association for the Advancement of Science in 1996 and was elected and served as chairperson of the Marine Natural
Products Gordon Research Conference in 2000 (vice-chair in 1998). She currently serves on the editorial boards of the journals Coral Reefs
and Journal of Natural Products. Valerie?s research interests include marine chemical ecology, marine plant?herbivore interactions, coral
reef ecology, and marine natural products. She is the author or co-author of over 150 research papers and review articles.
Melany P. Puglisi is currently a postdoctoral fellow at the Shannon Point Marine Center, Western Washington University. She received her
BS from Southampton College, Long Island University in 1991 in Chemistry, her MS from the University of Guam Marine Laboratory
in Biology in 1995, and her PhD in Pharmacognosy specializing in the ?eld of Marine Chemical Ecology in 2001 from the University
of Mississippi, School of Pharmacy. Melany?s postdoctoral research at the University of California at San Diego Scripps Institution of
Oceanography and Smithsonian Marine Station at Fort Pierce focused on marine microbial chemical defenses of macroalgae. Her current
research interest is in chemical defenses of marine plants against co-occurring microorganisms.
Raphael Ritson-Williams is currently working as a technician in the ?eld of marine chemical ecology at the Smithsonian Marine Station at
Fort Pierce. He received his BS in Biology from The Evergreen State College in 1998, and his MS in marine biology from the University of
Guam in 2002. After Guam he moved to Fort Pierce, Florida to continue his ecological studies of marine natural products with Dr Valerie
Paul. His research interests include coral reef biodiversity and natural product diversity especially those compounds that in?uence evolution
and ecology. Recent research projects include the feeding ecology of coral reef herbivores and predators, chemical and aposematic defenses
in nudibranchs, the function of tetrodotoxin in marine ?atworms, and chemical cues for larval settlement and metamorphosis.
Valerie J. Paul Melany P. Puglisi Raphael Ritson-Williams
antibacterial activity against at least one strain of the nine fouling
bacteria used in antimicrobial assays. The most potent bacterial
isolate was a Pseudomonas sp. isolated from the nudibranch
Archidoris pseudoargus. This bacterium contained ?ve compounds
1?5 that had high antimicrobial activity against the nine bacterial
strains, inhibited settlement of barnacle larvae, and reduced
Ulva lactuca spore settlement and percent cover of germlings.
When tested independently, phenazine-1-carboxylic acid 1 had
the highest activity against the bacterial strains. This study and
similar research with Vibrio sp., isolated from the surface of the
green alga Ulva reticulata,8 showed that epibiont microorganisms
may protect their hosts from fouling organisms.
Research on the bacterium Pseudoalteromonas tunicata found
that its yellow pigment reduced settlement of potential fouling
organisms.9 Using transposon mutagenesis the authors were able
to create strains of P. tunicata with different combinations of two
pigments and showed that only the yellow strain, like the wild
type strain, reduced settlement of Balanus amphitrite, Hydroides
elegans, and Ulva lactuca and Polysiphonia sp. propagules. The
use of differential expression allowed manipulation of the natural
products without destroying the bacterium of interest.
2.2 Cyanobacteria
Cyanobacterial blooms are common types of harmful algal
blooms that appear to be increasing in frequency worldwide.10,11
While many cyanobacterial blooms occur in freshwater and
will not be discussed here, recent studies have also focused
on planktonic blooms in the Baltic Sea. During bloom events,
concentrations of cyanobacterial hepatotoxins such as nodularin-
R (nodularin) 6, demethylnodularin-R 7, and even microcystin-
LR 8 have been detected.12 Karjalainen et al. examined the
accumulation of nodularin in natural assemblages of zooplankton
during blooms of Nodularia spumigena.13 Nodularin was present
in relatively low concentrations in common zooplankton species
collected during cyanobacterial blooms, although variation in
concentrations occurred between species and years. Nodularin
concentrations could be detected in copepods as well as in
carnivorous zooplankton species, which are major prey items for
planktivorous ?shes. Not surprisingly, the highest concentrations
in zooplankton were observed during cyanobacterial blooms.13
Nodularin 6 has been found in mussels, ?sh and seabirds (the
common eider Somateria mollissima) in the Baltic Sea.14
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Benthic cyanobacteria such as Lyngbya spp. continue to in-
trigue natural products chemists as a rich source of nitroge-
nous secondary metabolites. To examine genetic, chemical, and
morphological diversity, Thacker and Paul15 compared partial
16S ribosomal DNA sequences among Paci?c collections of
Lyngbya spp. and Symploca spp. Genetic divergence did not
correlate well with chemical and morphological differences. The
authors concluded that chemical variability among species of
Lyngbya cannot be predicted by 16S rDNA sequence analyses
and that other factors including interactions between genotypes
and environmental conditions may be important to explain the
high degree of chemical variation found in these cyanobacteria.15
Interactions between generalist and specialist grazers and the
benthic cyanobacterium Lyngbya majuscula have recently been
reviewed.16 Rabbit?sh and sea hares are the grazers most often ob-
served to feed on Lyngbya. Capper et al.17 examined the chemical
defenses of a bloom of L. majuscula from Moreton Bay, Australia,
against a variety of consumers that co-occur in Moreton Bay and
other allopatric consumers from Guam. Crude extracts containing
mainly lyngbyatoxin A, which were incorporated into arti?cial
diets, deterred feeding by all consumers tested in Guam except for
the specialist sea hare Stylocheilus striatus, which was stimulated
to feed by the extract. Australian S. striatus showed no preference
for control or treated foods. Wild-caught rabbit?sh (Siganus
fuscescens) avoided the L. majuscula extract, but captive bred
S. fuscescens showed no preference between control and treated
food.17 Nuisance blooms of L. majuscula in reef habitats can also
have negative effects on the settlement of coral larvae,18 but it is
not yet known whether this inhibition is chemically mediated.
2.3 Eukaryotic marine microalgae
During the past few years some excellent studies on the ecology
and chemical ecology of toxin-producing marine phytoplankton
and other microalgae have been published. These studies have un-
doubtedly been fueled by interest in harmful algal blooms (HABs)
and particularly in understanding bloom dynamics, toxicity, and
effects on consumers and competitors. The chemical ecology of
eukaryotic marine microalgae was the subject of a recent review by
Cembella,19 which provides a comprehensive overview of marine
microalgal toxins. The review includes allelopathic effects and
inhibition of grazers by saxitoxins, domoic acid, polyether toxins
such as brevetoxins, ciguatoxin and maitotoxin, okadaic acid, and
prymnesins and their analogues. Pohnert2 also covered the topic
of chemical defense in the plankton, discussing the few examples
that demonstrate a role for microalgal toxins in chemical defense or
allelopathic interactions. Both Cembella19 and Pohnert2 concluded
that we know little about functional roles of secondary metabolites
produced by microalgae, especially given the variable results with
different phytoplankton and grazer species, or about genetic
regulation of the production of these compounds. The topic of
allelopathy in phytoplankton, with an emphasis on HAB-forming
species, was reviewed by Legrand et al.20 In addition to a thorough
review of the literature on this topic, their paper focused on
chemical interactions, especially growth inhibition, among com-
peting algae and bacteria and methods of determining allelopathy
in phytoplankton. Heil et al.21 reviewed the literature about the
dino?agellate Prorocentrum minimum, the cause of harmful algal
blooms in estuarine and coastal environments worldwide. This
HAB species has expanded its range geographically in past decades
and has been increasingly observed to cause deleterious effects.
The detrimental effects of toxins produced by P. minimum to
other organisms and the environment seem to be variable, and
the toxins remain to be characterized (see also Wikfors22). Heil
et al.21 reviewed evidence for toxicity and allelopathic activity of
P. minimum, and Wikfors22 reviewed evidence for negative impacts
of this microalga on bivalves (clams, oysters, scallops).
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Studies of the chemical ecology of marine microalgae generally
focus on allelopathic effects against competitors or consumers.
Microalgal toxins can potentially play a role in these interactions
but have rarely been experimentally demonstrated to do so.
Several recent studies have examined the allelopathic activities
of microalgae and the roles of microalgal toxins in competition
among phytoplankton species.
Kubanek et al.23 tested whether the red tide dino?agellate
Karenia brevis uses allelopathy to outgrow 12 species of co-
occurring phytoplankton in laboratory experiments. Nine of the
12 species were suppressed when grown with K. brevis at bloom
concentrations. Extra-cellular ?ltrates and extracts of ?ltrates
inhibited six of the nine species; however, the effects could not
be attributed to brevetoxins, which at ecologically relevant con-
centrations only inhibited one competitor, Skeletonema costatum.
Allelopathic effects observed in the ?ltrate experiments may have
been due to highly water soluble, volatile, or unstable compounds
that were not extracted into the lipophilic extracts and were not
identi?ed. K. brevis was out-competed by several microalgae in this
study, including the dino?agellate Prorocentrum minimum and the
diatom Odontella aurita, both of which can be abundant during
K. brevis blooms.23
Dino?agellates of the genus Alexandrium were shown to have
toxic effects on other dino?agellates in culture24 and on natural
plankton communities.25 The effects were not related to levels
of paralytic shell?sh poisoning (PSP) toxins in Alexandrium
strains because both toxic and non-PSP producing strains of
Alexandrium had negative effects on other phytoplankton. A.
tamarense affected the whole natural phytoplankton community
by decreasing overall growth rates (measured by chlorophyll a
concentrations) for all phytoplankton organisms except the small
(<30 lm) dino?agellates.25 Extracellular substances appeared
to be responsible for the allelopathic effects,24 which included
immobilization and lysis of other microalgal cells. It is noteworthy
that toxins such as the brevetoxins,23 PSP toxins,24,25 and domoic
acid26 did not have a negative effect on other microalgal cells in
culture. Recently, a role for domoic acid together with copper
in iron acquisition in diatoms has been demonstrated, and iron
limitation is a likely trigger for toxin production and release.27
The bloom-forming planktonic alga Prymnesium parvum also
uses excreted toxins for food uptake and allelopathy. The toxic
properties are due to prymnesins and possibly other compounds,
which display haemolytic, ichthyotoxic, and cytotoxic activities. P.
parvum is photosynthetic but also feeds on microorganisms such
as bacteria and protists. Excretion of the toxins in dense cultures
of P. parvum caused immobilization and lysis of prey cells such
as the dino?agellate Heterocapsa rotundata.28 Cell-free ?ltrates of
P. parvum added to different size fractions of a natural Baltic
Sea plankton community negatively affected the whole plankton
community. Cyanobacteria and dino?agellates were more resistant
than diatoms and ciliates to the toxins, however, all phytoplankton
size classes showed reduced chlorophyll a levels and decreased
carbon uptake when exposed to the ?ltrates.29
Several studies have evaluated the effects of microalgal toxins
on consumers. The negative effects of polyunsaturated aldehydes
(PUAs) produced by some species of marine diatoms on copepod
reproduction are now well documented.30,31 These aldehydes,
cleaved from fatty acid precursors when diatom cells are damaged,
caused developmental arrest and deformed copepod larvae when
mothers and their larvae were fed diatom diets that contained the
compounds.31 Several recent reviews provide excellent background
on this wound-activated defense and its implications for copepods
and marine food webs.30,32,33 PUAs may also have impacts on
the larvae of benthic organisms. The aldehyde 2,4-decadienal
reduced survival of the larvae of two benthic polychaetes and two
echinoderms depending on the concentration of exposure.34 The
production of PUAs can be strain-speci?c in diatoms, and thus
only certain diatoms might be chemically defended in nature. GC-
MS methods have been developed to quantify various polyunsat-
urated aldehydes in diatom cultures and in natural phytoplankton
assemblages.35 These methods were applied to screen 50 species
(71 isolates) of diatoms for polyunsaturated aldehydes.36 Twenty
seven PUA-producers were identi?ed, including major spring-
bloom forming diatoms of the genus Thalassiosira.36 In addition
to effects on copepods, these polyunsaturated aldehydes may have
allelopathic effects on microalgae. Decadienal, one of the common
PUAs from diatoms, reduced growth of the diatom Thalassiosira
weiss?ogii in a dose- and time-dependent manner. It negatively
affected cell membrane integrity, decreased photosynthetic ef?-
ciency, and caused cell death.37 PUAs may be responsible for chem-
ical signaling within phytoplankton communities37 in addition to
their negative effects on invertebrate larvae and copepod grazers.
Another activated defense system in phytoplankton, which
is also found in some green macroalgae, is the conversion of
dimethylsulfoniopropionate (DMSP) to dimethyl sul?de (DMS)
and acrylate.2 Microzooplankton herbivory is considered to be the
key process causing this transformation to occur, however, there
have been few studies in natural systems. Archer et al.38 used direct
measurements of microzooplankton herbivory on Phaeocystis sp.
in the North Sea to account for DMS production rates. Estimates
of DMSP ingested accounted for the DMS produced by different
concentrations of microzooplankton grazing on Phaeocystis sp. in
bottles.38 Strom et al.39,40 studied feeding and growth by protists
on the DMS-producing microalga Emiliania huxleyi. Different
strains of E. huxleyi had different levels of DMSP-lyase activity,
the enzyme responsible for the conversion. Five out of six
protist grazer species showed lower feeding rates on strains with
high DMSP-lyase activity than on low-lyase strains.39 DMSP,
DMS, and acrylate dissolved in seawater were tested as chemical
defenses against protist grazers that were offered E. huxleyi.40
Interestingly, DMSP rather than the activation products DMS and
acrylate reduced grazing on E. huxleyi by four different species of
dino?agellate grazers.40
The red tide dino?agellate Karenia brevis regularly blooms in
late summer and fall months. It produces the brevetoxins, which
are neurotoxic to mammals and have been implicated in shell?sh
poisonings and die-offs of ?n?sh, invertebrates, sea turtles and
marine mammals.41 Prince et al.42 studied the copepod Acartia
tonsa and the ?tness effects of feeding on different microalgal
diets, including diets rich in Karenia brevis. Copepods did not
avoid feeding on K. brevis, and actually ate more K. brevis
than the more preferred microalga Rhodomonas lens when these
were offered as unialgal diets, suggesting possible compensatory
feeding. On diets of 93?100% K. brevis, copepods experienced
decreased survivorship and fecundity per female, but egg hatching
rates were the same for all diet regimens. Survivorship of starved
copepods was only marginally lower than for copepods fed only
K. brevis, which was lower than survivorship on all other diets.
156 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
Extra-cellular extracts of K. brevis did not directly affect A. tonsa
survivorship or consumption rates when copepods were exposed to
extracts while feeding on Rhodomonas lens. The authors attributed
the negative effects of Karenia brevis on the copepods to nutritional
inadequacy rather than overt toxicity of the dino?agellate.42 A
potential mechanism for the nutritional limitation of K. brevis
was suggested by Giner et al.43 who studied sterol composition of
this dino?agellate. Four novel and two rare sterols were isolated,
including gymnodinosterol and brevesterol as the major sterols.
The authors suggested these novel sterols might offer protection
from predation, but this was not directly tested. They hypothesized
that these modi?ed sterols might be of poor nutritional value
to marine invertebrates, thus reducing grazing on K. brevis and
contributing to bloom formation.43
A similar study of copepod feeding was conducted with Acartia
tonsa feeding on the dino?agellate Prorocentrum minimum,44
which varies in toxicity depending on location and strain.21,41
A. tonsa readily ingested P. minimum cells, and egg production
rate was positively correlated with ingestion rate. Egg hatching
rate was high and not affected by consumption of P. minimum.
Ingestion, egg production and egg hatching success of females
were measured for mixed diets consisting of P. minimum and the
diatom Thalossiosira weiss?ogii. Diets containing T. weiss?ogii
increased egg production, and the authors concluded that P.
minimum was nutritionally inadequate but that there was no
evidence of toxicity to A. tonsa.44 The dino?agellate Gyrodinium
corsicum had signi?cant adverse effects on survival of the copepod
Acartia grani but not on another copepod Euterpina acutifrons.
PSP toxins were not present in the dino?agellate; however, aqueous
and methanol extracts showed evidence of toxicity in mouse
assays, but they were not tested on the copepods.45 Females of the
copepod Temora stylifera demonstrated reduced egg production
and hatching success when fed uni-algal diets of the dino?agellate
Alexandrium tamarense.46 This strain of A. tamarense was not
neurotoxic, contained low levels of saxitoxins, and also did not
contain polyunsaturated aldehydes; however, extracts blocked
fertilization success in sea urchin oocytes, indicating that other
unidenti?ed compounds were responsible for the reduced egg
production and hatching success observed.46
Toxin accumulation by zooplankton grazers was also studied for
zooplankton feeding on the dino?agellate Alexandrium fundyense,
which contains the paralytic shell?sh poisoning (PSP) toxins such
as saxitoxin and its analogues.47 The three studied species of
copepods accumulated measurable levels of toxin, but some of
the PSP toxins ingested were excreted in fecal pellets, and most of
the toxins present in A. fundyense (?90%) were not recovered
and could have been released by Alexandrium cell breakage
during feeding. The copepods Acartia hudsonica and Eurytemora
herdmani consumed different relative amounts of A. fundyense
cells when offered as mixed diets with Heterocapsa triquetra,
displaying avoidance of A. fundyense cells at higher concentrations.
Centropages hamatus did not change its feeding behavior and
also retained the highest concentrations of toxin during feeding.
The authors concluded that copepod grazing primarily disperses
PSP toxins into the environment, but even the small amounts
accumulated could be passed onto higher trophic levels.47
Excellent examples of how PSP toxins (saxitoxin and analogues)
can in?uence consumer?prey interactions with community level
implications have been recently reported.48,49 In these studies of
tritrophic level interactions, microalgal toxins that accumulated in
prey items in?uenced feeding behavior of predators such as sea
otters48 and shorebirds.49 Variable concentrations of PSP toxins
in shell?sh in?uenced sea otter foraging behavior and diet as
well as prey abundance at a range of sites in the Inside Passage
of southeast Alaska. Toxin concentrations in?uenced when and
how otters fed on their preferred prey, the butter clam Saxidomus
giganteus. At sites where bivalve toxicity was high (>500 lg STX
eq 100 g?1), sea otters rarely ate the toxic prey and fed primarily
on sea urchins, small clams, and other less toxic and less abundant
prey items. At sites with intermediate levels of toxicity (200?500 lg
STX eq 100 g?1), sea otters fed selectively, often discarding some
prey items, or parts of prey such as clam siphons, which contained
higher levels of toxins. Sea otters did not avoid feeding at sites with
high prey toxicity, but they did avoid prey items with the highest
levels of toxicity; thus, butter clams were larger and more abundant
at toxic sites.48 Kvitek and Bretz49 also showed that shorebirds
avoided foraging on toxic prey. Black oystercatchers (Haematopus
bachmani) forage on mussels (Mytilus californianus) on rocky
shores in California. During harmful algal bloom (HAB) events in
the late summer months, mussels accumulated PSP toxins rapidly
and showed highest levels of toxicity in September. Oystercatchers
rejected mussel tissue and switched to less toxic prey such as
limpets when PSP toxin levels in mussels were high. On sandy
shores, shorebirds such as godwits, sanderlings, whimbrels and
willets primarily consumed sand crabs (Emerita analoga). When
sand crab toxin levels became high during algal bloom events,
shorebird abundance decreased on sandy shores and rejection rates
of sand crabs increased. Shorebirds were able to detect PSP toxins
and avoided lethal concentrations through changes in foraging
behavior.49 Studies with shorebirds and otters demonstrate that
HAB toxins can alter upper-level trophic interactions in marine
food webs and that PSP toxins can provide prey items with
chemical defense against predators.
The transfer of microalgal toxins to upper trophic levels can
have unexpected consequences. For example, mass mortalities of
marine mammals have been caused by exposure to brevetoxins
after a Karenia brevis bloom subsided.50 Manatees were probably
exposed by eating seagrass that had high levels of brevetoxins
(through active uptake or passive adsorption onto the seagrass),
and dolphins died after consumption of contaminated ?sh, espe-
cially menhaden, that had high levels of brevetoxins.50 Relatively
few studies have addressed the fate and effects of natural products
through marine food webs.
3 Macroalgae
Several useful reviews of natural products and chemical ecology
of macroalgae have been recently published. While not directly
related to chemical ecology, Smit51 provided an excellent review of
medicinal and pharmaceutical uses of seaweed natural products,
which demonstrates the broad range of bioactivities of natural
products from macroalgae. La Barre et al.52 discussed modern
analytical techniques that might be useful for characterizing and
sometimes quantifying chemical defenses in macroalgae. They
provide examples in which high ?eld multinuclear and solid-state
NMR and mass spectrometry (MS) have been used in metabolic
studies of marine plants. The diterpenes in Dictyotacean brown
algae from the tropical Atlantic region have been well studied, and
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the structures and geographical distributions of these compounds
were recently reviewed.53 The authors discuss chemotaxonomy,
biogeography, and chemical differentiation of brown algal
diterpenes. In addition, they postulate that biosynthetic pathways
diverged as Dictyotacean algae moved into different regions of
the world?s oceans. Amsler and Fairhead54 present an overview
of chemical defenses and other chemically mediated ecological
interactions in the brown algae. The authors provide a discussion
of the brown algal phlorotannins and colorimetric methods used
to measure them, the functions of phlorotannins as both primary
and secondary metabolites, and the high spatial and temporal
variability observed for concentrations of phlorotannins. They
also review non-phlorotannin defenses in brown algae, including
terpenes, acetogenins, and related mixed-biosynthetic compounds
in Dictyotacean brown algae. Amsler and Fairhead54 discuss
how brown algae can serve as model organisms to test chemical
defense theories (induced defenses, carbon?nutrient balance,
resource allocation models, and optimal defense theory). This
review also encompassed the chemosensory aspects of brown
algae from chemo-attraction of brown algal gametes in response
to pheromones to chemosensory behaviors of brown algal spores
that are attracted by nutrients and other environmental cues.54
Several broad surveys of the bioactivity of crude extracts from
seaweeds against marine microorganisms and potential fouling
organisms have been recently conducted. Twenty one species of
algae from the coast of Yucatan, Mexico, were surveyed for
antimicrobial activity against pathogenic microbes.55 Eighteen
species were active against the gram-positive bacteria, and almost
all extracts inhibited Bacillus subtilus. Extracts of Caulerpa spp.,
which are abundant algae along the Yucatan coast, were highly
active in the surveys. Extracts of three different parts of the algae
(apical, basal, and stolon regions) were evaluated in antimicrobial
assays for C. ashmeadii, C. paspaloides, and C. prolifera. In general,
extracts from the stolon region had the highest antibacterial
activity.55 In a broad survey of Antarctic macroalgae from three
phyla, crude lipophilic and hydrophilic extracts were assayed for
their toxicity to diatom cells as a test for possible antifouling
activity.56 Extracts of 16 of the 25 species of algae tested caused
signi?cant diatom mortality at natural concentrations. Another
study tested the antifouling potential of nine macroalgae species
from the west coast of France. Crude extracts were tested against
three species of marine fungi, six species of marine bacteria, three
species of diatoms, three species of macroalgae, the mussel Mytilus
edulis, and the barnacle Balanus amphitrite.57 Some of the extracts
were seasonally variable with 52% inactive, 31% seasonally active
(with highest activity from May to July), and 17% active all
year.
Toxicity of crude extracts of 32 species of Mediterranean sea-
weeds was examined by Microtox R? assay, which analyzes toxicity
against the marine bacterium Vibrio ?sheri.58 Intra- and inter-
speci?c variation in toxicity was observed, with approximately
half of the species demonstrating toxicity. Seasonal variation
was found between samples collected in June and November.
Overall, toxicity was higher in November, but some species, such as
Lobophora variegata and Falkenbergia rufolanosa, showed higher
toxicity in June. Results of the Microtox R? assay correlated well
with a sea urchin assay, which tested cytotoxic and antimitotic
effects of extracts on Paracentrotus lividus embryos.58 While it is
dif?cult to relate these toxicity assays to ecological properties of the
seaweeds, the results demonstrate a broad distribution of bioactive
compounds in the algae surveyed.
Chemical defense against consumers continues to be an im-
portant area of research in chemical ecology. In a recent study,
common subtidal macroalgae from the Antarctic Peninsula were
evaluated for chemical defenses against the sea star Odontaster
validus, the rock?sh Notothenia coriiceps, and the herbivorous
amphipod Gondogeneia antarctica.59 Pieces of thalli of 35 species
were assessed for palatability in assays with the sea star and
rock?sh, and those that were unpalatable (over 60% of species
tested) were then extracted. Natural concentrations of lipophilic
and hydrophilic extracts were tested in arti?cial diets, which
showed that many of these unpalatable algae were chemically
defended. At least one extract type of all the ecologically dominant
brown algae was unpalatable, except for Desmerestia antarctica.59
Thallus toughness and nutritional quality did not seem to relate
to palatability. Another survey of macroalgae from the Antarctic
Peninsula examined the nutritional composition (protein, carbon
and nitrogen content) of 40 species of algae.60 Overall protein
content was high, and C : N ratio was low relative to temperate
and tropical macroalgae, which is probably related to the nutrient-
rich waters where these algae reside.60 Despite nutrient-rich waters,
natural products from these unpalatable Antarctic seaweeds
generally lack nitrogen.61 The red alga Delisea pulchra yielded new
dimeric halogenated furanones, pulchralides A?C 9?11, and the
previously reported furanones ?mbrolide 12, acetoxy?mbrolide
13, and hydroxy?mbrolide 14. The red alga Plocamium carti-
lagineum yielded halogenated monoterpenes including anverene
15, epi-plocamene D 16, and pyranoid 17. Anverene 15 and epi-
plocamene D 16 deterred feeding by the amphipod G. antarctica,
but compound 17 did not; whereas, none of these halogenated
monoterpenes inhibited feeding by the sea star O. validus.61
The red alga Myriogramme smithii contained simple aromatic
compounds such a p-hydroxybenzaldehyde and p-methoxyphenol,
which might be responsible for the unpalatability of this alga.
The brown alga Desmerestia menziesii contained diterpenes of
mixed biogenesis including menzoquinone 18 and hydroquinone
19. Menzoquinone 18 deterred feeding by the sea star O. validus
at a concentration three times the isolated concentration.61 The
endemic brown alga Cystosphaera jacquinotii contained the novel
sterol cystosphaerol 20. Many of these metabolites were bioactive
in antimicrobial assays, but only the few previously mentioned
were tested for feeding deterrence against putative consumers.61
Two non-acidic Antarctic species of Desmerestia (D. anceps and
D. menziesii) that were found to be unpalatable59 were investigated
for within-thallus variation in chemical defenses and toughness.62
Lipophilic and hydrophilic extracts from three different thallus
parts (holdfast, primary stem, lateral branches) were incorporated
into arti?cial foods and offered to the sympatric, herbivorous
amphipod Gondogeneia antarctica. For D. anceps, the primary
stem extracts were most deterrent, and the primary stem and
holdfast were the toughest parts of the thallus. These results
were consistent with predictions of optimal defense theory, with
the most valuable thallus parts being the most chemically and
physically defended. No differences were observed in palatability
of extracts among the different thallus parts in D. menziesii;
however, primary stem and holdfast were once again tougher than
lateral branches.62 The natural products responsible for chemical
defense were not explored in this study, but another study looked
158 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
for phlorotannin concentrations in these algae.63 Phlorotannin
concentrations were high in both species of Desmerestia; they
were also highly variable within individual thalli and at different
locations and depths. Phlorotannins were consistently higher in
shallow D. anceps than in individuals from deeper sites, but
this same pattern was not observed for D. menziesii.63 The
phlorotannins from Desmerestia spp. were not directly tested for
their effects on consumers.
Among the unusual chemical defenses found in marine algae,
some brown algae of the orders Desmarestiales and Dictyotales
are known to contain high concentrations of sulfuric acid within
cell vacuoles (pH < 1) (reviewed by Amsler and Fairhead54).
Pelletreau and Muller-Parker64 investigated the role of sulfuric
acid in Desmerestia munda as a defense against herbivory. They
found that D. munda was a low preference food for the sea
urchin Strongylocentrotus droebachiensis and that sulfuric acid
concentrations added to arti?cial diets at or below 3.5 pH units
inhibited feeding by the sea urchin. A recent survey of 61 species of
algae by pH measurements and ion chromatography65 identi?ed
three species within the Desmarestiales and seven species in
the Dictyotales that contained high sulfuric acid concentrations.
Additionally, some dictyotalean species were found that contain
high concentrations of SO42? balanced by high concentrations of
Mg2+; these were not acidic.65
Several brown algal terpenoid compounds function as chemical
defenses against herbivores. The crude extract and major diterpene
21 from Dictyota pfaf?i deterred feeding by the sea urchin
Lytechinus variegatus and generalist herbivore ?shes in ?eld assays,
but did not in?uence feeding by the crab Pachygrapsus transver-
sus.66,67 The brown alga Stypopodium zonale produces diterpenes
of mixed biogenesis (meroditerpenes) that vary qualitatively and
quantitatively depending on where the samples were collected.68
In an investigation of variation in this alga collected from areas of
the Brazilian coast, Soares et al.68 found two distinct chemotypes.
Collections from more northern locations contained stypoldione
22 (air-oxidation product of stypotriol 23) as the major metabolite,
and collections from southern locations contained atomaric acid
24 as the major metabolite. Studies of chemical defense by extracts
and major metabolites from these two chemotypes showed that
This journal is ? The Royal Society of Chemistry 2006 Nat. Prod. Rep., 2006, 23, 153?180 | 159
extracts from both locations inhibited feeding by the sea urchin
Lytechinus variegatus and the crab Pachygrapsus transversus, but
the crude extract containing atomaric acid 24 deterred feeding
more than the extract containing stypoldione.69 Tests with the pure
compounds con?rmed these results; atomaric acid was a more
potent feeding deterrent than stypoldione 22 even though it was
present in S. zonale at lower concentrations. The deterrent effect
of the natural product stypotriol 23 was not tested and compared
in these assays.
In an excellent study of geographic and genetic variation in feed-
ing preference of the amphipod Ampithoe longimana for species
of Dictyota, Sotka et al.70 studied eight populations of amphipods
from North Carolina to Maine. The authors assayed the feeding
preferences of the amphipod for two species of Dictyota and two
species of red algae (Hypneamusciformis and Gracilaria tikvahiae),
tested the genetic potential for evolution of feeding preferences
among full-sib families of one population, and determined the
?tness consequences of these feeding preferences. Mitochondrial
cytochrome oxidase I (COI) and nuclear ITS-1 genes showed
a strong historical separation between northern populations of
A. longimana in Connecticut and Rhode Island and the other
populations further south. Feeding assays showed that amphipod
populations sympatric with Dictyota had a higher preference for
Dictyota and lower preference for Hypnea relative to other popu-
lations. A previous study had demonstrated that a North Carolina
population of A. longimana sympatric with Dictyota menstrualis
was more tolerant of Dictyota diterpenes than a Connecticut pop-
ulation from outside of the geographic range of Dictyota spp.71 An
experiment with full-sib families of amphipods raised in the labo-
ratory on Enteromorpha sp. detected signi?cant heritable variation
in preferences for Dictyota.72 To determine ?tness consequences,
juveniles from seven populations were raised for 30 days on D.
menstrualis, D. ciliolata and Enteromorpha sp. Reproduction of
females from northern populations that did not co-occur with Dic-
tyota was lower on the Dictyota diet relative to the Enteromorpha
diet, but this was not the case for the sympatric populations.70 This
is one of very few studies of marine plant?herbivore interactions to
investigate the genetic basis for feeding preferences of consumers,
although a recent review suggests that other marine invertebrates
may also display local adaptation in host use.72
Chemical variation in levels of brown algal phlorotannins has
been the subject of numerous studies in the past two years. The ease
of quantifying this class of compounds by colorimetric assays such
as the Folin?Denis or Folin?Ciocalteu assay probably explains
why there are so many studies on phlorotannins compared to
other macroalgal natural products. Patterns of variation have been
explored among different species of brown algae, within species
collected at different locations, in different thallus parts, and over
different seasons.63,73?77 In many of these studies, no accompanying
measurements of herbivory or feeding deterrence were made.
Other researchers examined the palatability of brown algae to
herbivores but did not relate that to changes in phlorotannins or
other secondary metabolites. For example, several investigations of
inducible defenses in brown algae looked at changes in palatability
in grazed versus ungrazed algae to different consumers.78,79 One
study examined genetic variation for growth and phlorotannin
concentrations under different environmental conditions for the
brown alga Fucus vesiculosus.80 Growth and phlorotannin concen-
trations showed both genetic variation and phenotypic plasticity.
Heritability for phlorotannin concentration was high and did not
differ signi?cantly between environments.80
Connan et al.81 surveyed polyphenolic content in eight domi-
nant species of brown algae along the northern coast of Brittany
(France) monthly over a 14 month period. Phenolic content varied
among species with Ascophyllum nodosum and Fucus vesiculosus
showing highest levels (>5% dry mass). Not unexpectedly, all
Fucalean algae showed higher levels than Laminaria digitata
(<0.5% dry mass), the only member of the Laminariales studied.
Seasonal patterns in polyphenolic content were observed, with a
summer maximum for the Fucales and a winter maximum for
Laminaria digitata (although levels were very low for L. digitata
overall). Stiger et al.74 examined phenolic content in Turbinaria
ornata and Sargassum mangarevense, which are abundant on reef
?ats around Tahiti. Overall, polyphenolic content in these algae
was low, but T. ornata had higher levels (approximately 1.2% dry
mass) than S. mangarevense, and levels were slightly higher in
summer than in winter. There was considerable site to site variation
in polyphenolic concentrations. Juveniles and recruits had lower
concentrations of phenolics than mature thalli.74
Several studies examined the effects of solar radiation or UV
radiation on phlorotannin concentrations in macroalgae. The
effects of UV radiation on phlorotannins and growth in Fucus
gardneri embryos and juveniles were examined by growing these
life history stages of the alga under different UV radiation ?lter
treatments for three weeks.76 UV-B treatments inhibited and UV-
A treatments enhanced the growth of embryos but had no effect
on F. gardneri juveniles. Phlorotannin concentrations were not
in?uenced by the various UV treatments in either embryos or
juveniles. The authors suggested that embryos are negatively
affected by UV light but that F. gardneri develops a tolerance
to UV as the alga matures. Phlorotannin concentrations were
not induced by UV light over the time course of the study.76
A study of the effects of solar radiation on annual and diurnal
variation in photosynthetic activity and polyphenolic content
of the brown alga Cystoseira tamariscifolia indicated that pho-
tosynthetic activity and phlorotannin concentrations were very
dynamic in this alga.75 Optimum quantum yield was determined
with a portable pulse amplitude modulated (PAM) ?uorometer,
and this was measured monthly during one year as well as every
two hours during the course of one day. There was seasonal
variation in phlorotannin concentrations, with highest levels in the
summer months. Optimum quantum yield decreased as irradiance
increased at noon, possibly indicating photoinhibition. A negative
correlation between phlorotannin concentration and irradiance
in the summer months was observed, but no relationship was
observed in the fall?winter months. Phenolic compounds were
exuded from apical portions of the thalli at high irradiances,
suggesting that short-term variation in phenolic compounds is
controlled by exudation under high irradiances.75
Desiccation stress and herbivory levels on the brown alga
Fucus gardneri were manipulated in ?eld and outdoor mesocosm
experiments during spring and summer months to determine their
effects individually and in combination.82 Field studies used screen
awnings to shade the intertidal, which reduced temperatures on
sunny days and generally increased relative humidity. Herbivory
was manipulated with limpet removal. F. gardneri kept under
awnings showed enhanced rates of net photosynthesis and survival
and grew larger than algae in control areas without awnings.
160 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
Phlorotannin concentrations were higher in thalli kept under
awnings, but there was no effect of limpet removal. Loss of
tissue, increased reproductive output, and increased stress all
correlated with lower phlorotannin levels.82 Similar manipulations
of desiccation and herbivory by the isopod Idotea wosnesenskii
were conducted in mesocosm experiments. The amount of algal
tissue lost and growth rates in this experiment were related to
levels of herbivory; there was no effect of stress. Concentrations
of phlorotannins were again lower in stressed thalli and were not
affected by herbivory.82 In these experiments stress and herbivory
seemed to act independently rather than interactively to in?uence
plant growth and chemical defense parameters.
Koivikko et al.83 developed methods to separately examine cell-
wall bound phlorotannins and soluble phlorotannins in Fucus
vesiculosus. They used alkaline degradation of cell-wall bound
phlorotannins to release the phenolics for subsequent analysis
by the Folin?Ciocalteu assay. Concentrations of cell-wall bound
phlorotannins (<1% dry mass) were much lower than soluble
phlorotannins (approximately 8% dry mass) in F. vesiculosus.
Concentrations of both were lower in growing tissue than in non-
growing tissue. Nutrient enrichment experiments reduced the con-
centration of soluble phlorotannins. Phlorotannins were actively
exuded into seawater under all nutrient treatments, and exudation
rates more than doubled when the alga was grazed by the isopod
Idotea baltica.83 Herbivory did not induce increased phlorotannin
concentrations in the alga, suggesting that increased exudation was
related to compound release rather than compound biosynthesis.
Shibata et al.84 conducted a study examining the localization of
phlorotannins in Japanese brown algae (Eisenia bicyclis, Ecklonia
cava, and E. kurome). Vanillin?HCl staining and light microscopy
were used to observe phlorotannins accumulated in vegetative cells
of the outer cortical layer of the thalli. The authors also identi?ed
some of the phlorotannin components of the algae by thin layer
chromatography and HPLC based on comparisons to standards
obtained from prior studies.85,86 Three species of brown algae
contained similar types and amounts of phlorotannins including
phloroglucinol 25, eckol 26, dieckol 27, phlorofucofuroeckol
A 28, and 8,8?-bieckol 29. E. bicyclis also had larger amounts of an
unidenti?ed tetramer (MW 478). There were no clear seasonal
patterns in concentrations of phlorotannins among samples.
A prior study had shown that phlorotannin mixtures as well as the
polymers dieckol 27, phlorofucofuroeckol A 28, and 8,8?-bieckol
29 strongly inhibited digestive enzymes (glycosidases) partially
puri?ed from the viscera of the herbivorous turban snail Turbo
cornutus.85
Lu?der and Clayton87 microscopically examined the cellular
changes during wound healing after Ecklonia radiata was sub-
jected to simulated herbivory. In E. radiata, physodes containing
polyphenolics are located predominately in the outer epidermal
cell layer. The alga was mechanically wounded with a cork borer
that created a hole 3 mm in diameter in the center of a piece of
a blade. The healing response, including structural changes and
distribution of polyphenolics, was examined up to 9 days after
wounding. Immediately after wounding new medullary cells
formed around the wound site, and within three days physodes
containing phlorotannins accumulated around the wound site lo-
calized to medullary cells. The authors propose that polyphenolics
are needed for wound sealing and wound healing following damage
and might also be important in preventing both microbial and
herbivore attack on the area of the wound.87
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Induced defenses in both terrestrial and marine plants can
in?uence the feeding behavior of herbivores. Changes in plant
chemistry and palatability can cause herbivores to be more mobile,
and thus, damage from grazing would be more dispersed on
plants. Borell et al.88 tested these predictions, which they termed
the herbivore mobility model, with the brown alga Ascophyllum
nodosum and the herbivorous snail Littorina obtusata. A. nodosum
plants of similar size were subjected to three treatments (grazing
by L. obtusata, simulated grazing with a ?le, and control) for two
weeks. L. obtusata individuals were then assayed for their mobility
on algae from the three treatments, and dispersal of feeding
damage and amount of grazing on algae were also assessed. Snails
feeding on algae subjected to prior herbivory moved more than
snails on algae subjected to simulated herbivory or control algae.
Algae exposed to prior herbivory had less tissue consumed by
snails in the feeding assays. Phlorotannin concentrations were
highest in previously grazed plants and did not increase in algae
in the simulated grazing treatment relative to controls; however,
phlorotannins were not tested directly for their deterrent effects
on L. obtusata. The herbivore mobility model?s predictions were
supported in this study.88
In another study of Ascophyllum nodosum and Littorina ob-
tusata, Toth et al.89 examined whether inducible defenses (assumed
to be phlorotannins) in A. nodosum would differ between thallus
parts (basal stipes and annual shoots) with different ?tness values.
Phlorotannin content is highly variable in this alga;73 basal por-
tions have the highest ?tness value and phlorotannin concentration
compared to annual shoots and reproductive tissues.90 Phlorotan-
nin concentrations increased after grazing by L. obtusata, and the
effect was ?ve times larger in basal stipes than in apical shoots.
While juvenile snails grew well on all portions of the alga, whether
grazed or ungrazed, adult females produced fewer viable eggs
when fed basal compared to apical shoots. Also, the number of
non-viable eggs increased when snails fed on previously grazed
tissues.89 The study shows that induced resistance and its variation
among algal parts ?ts the predictions of optimal defense theory
and can negatively in?uence herbivore ?tness.
Polyphenolics have been shown to be inducible defenses in the
brown algae,78,89 but not in all cases. Jormalainen et al.91 tested
the effects of nutrient enrichment and feeding by two snails on
phlorotannin production by Fucus vesiculosus. Nutrient enrich-
ment alone increased fouling and decreased phlorotannin levels.
Presence of the snail Theodoxus ?uviatilis induced phlorotannin
production; however, this snail does not graze F. vesiculosus. The
researchers suggested that rather than interpreting this as an
induced response, it might instead be the result of altered nutrient
uptake because the snails graze hyaline hairs off the surface of
the alga, and hyaline hairs are involved in nutrient acquisition.91
Hemmi et al.77 found no in?uence of nutrients or herbivory by
the herbivorous isopod Idotea baltica on phlorotannin levels
in F. vesiculosus in the northern Baltic Sea. In another study
with F. vesiculosus, parts of thalli were subjected to simulated
grazing (arti?cial clipping) and enhanced nutrients.92 Two, ten
and thirty eight days after simulated grazing the algal parts were
offered to the isopod I. baltica to determine preference between
clipped and unclipped portions of the thalli. Within two days
after clipping an induced resistance to the isopod was observed
in both fertilized and unfertilized algae that disappeared after 10?
38 days; however, this induced resistance did not relate to increased
concentrations of phlorotannins. Nutrient enrichment negatively
affected phlorotannin concentrations. The authors concluded that
I. baltica was not deterred by phlorotannins; but there might be
some rapidly induced compounds in F. vesiculosus that function
as feeding deterrents.92
Care must be taken in interpreting the results of the many studies
that show a correlation between phlorotannin concentrations
and bioactivity in brown algae. Several studies have now shown
that compounds other than phlorotannins in brown algae can
be responsible for feeding deterrence and other bioactivity.92?94
In studies of Fucus vesiculosus, a polar galactolipid as well
as uncharacterized water-soluble compounds were shown to
be responsible for deterring consumers.93,94 Through bioassay-
guided fractionation Kubanek et al.94 tried to isolate herbivore
deterrent compounds from the aqueous extracts of F. vesiculosus,
but activity was lost during the isolation process. There was
no relationship between phlorotannin decomposition and loss
of feeding deterrence. Semi-puri?ed mixtures of phlorotannins
did not deter three different herbivores at 3 or 6 times the
isolated concentration, but the amphipod Ampithoe valida was
deterred at 12 times the isolated concentration. Furthermore,
juvenile A. valida raised for 64 days on arti?cial diets containing
phlorotannins demonstrated enhanced survivorship and growth
relative to diets without phlorotannins and ovulated only on
the phlorotannin-rich diet. The authors suggest that methods
other than measurements of total phlorotannins are needed for
understanding chemical defenses of brown algae, because polar,
unstable compounds other than phlorotannins may confound
interpretation of chemical defenses in this group of algae.94
Hemmi and Jormalainen95 compared geographic variation in
nutritional quality (phlorotannin, nitrogen, protein, and sugars)
in Fucus vesiculosus and compared this with among-population
variation in size and fecundity of the herbivorous isopod Idotea
baltica. Collections of the alga and isopods were made at 12
sites that were 0.5?40 kilometres apart. Algal populations differed
in concentrations of phlorotannins, nitrogen, protein and sugars
(fucose, mannitol, and melibiose); number of eggs, total egg mass,
and egg size of I. baltica also differed among collection sites. Size
of females positively covaried with phlorotannin concentration
in F. vesiculosus, and egg size positively covaried with mannitol
concentrations. While host plant chemistry was related to variation
in herbivore size and reproduction, phlorotannins had a positive
rather than negative relationship with herbivore ?tness.95
Fucus evanescens and F. vesiculosus were found to be dif-
ferentially fouled in the ?eld, and aqueous methanol extracts
from both species decreased settlement of barnacle cyprid larvae
(Balanus improvisus) in the laboratory.96 When treated with
polyvinylpolypyrrolidone, which binds and removes phlorotan-
nins, the F. vesiculosus extract lost its activity, but the F. evanescens
extract continued to inhibit settlement, suggesting compounds
other than phlorotannins were active. Fucus evanescens also
inhibited post settlement survival of barnacles when compared
to F. vesiculosus.96 While Fucus phlorotannins may have the
potential to inhibit barnacle settlement in laboratory assays, this
did not explain the lower barnacle settlement on F. evanescens
observed in the ?eld. Another study of fouling in the brown
alga F. vesiculosus examined genetic variation in tolerance and
resistance to fouling organisms.97 Thirty algal genotypes grown in
the ?eld showed highly variable levels of fouling after 44 days.
162 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
Phlorotannin content and biomass of fouling organisms were not
correlated. In an aquarium experiment where snails were used
to control fouling, algae grew less and phlorotannin content was
lower in the fouling (no snails) than in the non-fouling treatment;97
however, the possible effects of snail grazing on these factors made
these results dif?cult to interpret. Phlorotannins did not seem
to explain antifouling activity of brown algae in either of these
studies.
Inducible defenses have been observed for other brown algae
that are probably not related to concentrations of phlorotannins.
A study of the effects of amphipod (Parhyalella ruffoi) grazing and
UV radiation on two Chilean brown algae, Glossophora kunthii
and Macrocystis integrifolia, demonstrated evidence of inducible
defenses in G. kunthii but not M. integrifolia and no effect of
UV radiation on palatability of these two algae.79 Experiments
included acclimation for 12 days without grazers, and non-polar
extracts of G. kunthii increased in palatability during that time.
Addition of grazers for 12 days caused a decrease in palatability
of non-polar extracts relative to controls, and then these induced
effects disappeared after another 12 days without grazers. Treat-
ments that included the presence of grazers without direct grazing
on the alga also induced chemical defense in G. kunthii, suggesting
a waterborne cue served as a signal for induced defenses. Defenses
of different parts of the thalli were compared and apical and
basal portions of G. kunthii were chemically defended against
the amphipod herbivory. Herbivores also avoided the stipes of
M. integrifolia but this preference was not chemically mediated.79
A study of inducible defenses in two brown and one red alga
from the Kenyan coast also found evidence of induced chemical
defenses in response to grazing by the amphipod Cymadusa
?losa. Nonpolar extracts of grazed Sargassum asperifolium and
Cystoseira myrica were less preferred than those from ungrazed
algae in arti?cial diets.98 Nonpolar extracts of Lobophora variegata
from Brazil also suggested an induced chemical defense in response
to amphipod grazing.99
The brown alga Bifurcaria bifurcata was examined for seasonal
variation of extracts as inhibitors of two marine bacteria and
barnacle (Balanus amphitrite) settlement.100 Similar to other
algae57 a seasonal effect of the extracts against all three organisms
was observed with the most potent activities during April?June.
This increased activity corresponded to the seasonal maximum
concentration of the major diterpene eleganediol 30 found in the
crude extracts,101 but individual compounds were not tested for
their antifouling activity.100
Compared to studies with brown algae, and particularly on
phlorotannins, only a few recent studies have explored chemical
defenses in red and green macroalgae. One red alga (Hypnea
pannosa) from Kenya was examined for inducible defenses. In
tests with live algae, grazed H. pannosa was less preferred by the
amphipod than ungrazed controls, but this same result was not
observed with nonpolar extracts of grazed and control algae.98
Another study of inducible defenses of four red, four brown, and
one green algae from Brazil demonstrated that the live red alga
Pterocladiella capillacea was less preferred after it was grazed by
amphipods than non-grazed controls.99 Whether these two red
algae were defended by an induced chemical defense is not clear,
but changes in palatability were observed.98,99
Lack of information about heritability of algal secondary
metabolites is a gap in our understanding of the variability and
evolution of these compounds. The red alga Delisea pulchra pro-
duces halogenated furanones that vary in concentration spatially
and temporally. Wright et al.102 determined the heritability of the
four major furanones 31?34 and then determined the effects of
different concentrations on feeding by herbivores. Total furanone
concentration and the major furanone 33 showed signi?cant
genetic variation; however, all four furanones were genetically
correlated. All six common herbivores used in feeding assays,
except for the gastropod Phasianotrochus eximius, consumed D.
pulchra at lower rates than other macroalgae. The herbivores were
usually deterred by extracts and furanone 33 at concentrations that
spanned the range of concentrations found in the ?eld, but were
occasionally not deterred at the lower end of the concentration
range.102 The authors concluded that herbivores could provide an
adequate selective force to act upon this heritability.
The effects of varying levels of grazing by the sea urchin Helio-
cidaris erythrogramma on macroalgal community structure on a
temperate Australian reef was studied, and Wright et al.103 found
that the chemically defended Delisea pulchra had relatively high
survivorship when grazed by sea urchins. Grazing by high densities
of sea urchins caused the algal community to switch from a mix of
foliose algae before grazing, through an intermediate stage where
grazer-resistant algae such as D. pulchra coexisted with crustose al-
gae, to a community consisting almost entirely of crustose algae.103
Host use by the sea urchin Holopneustes purpurascens was
shown to be chemically mediated and changed from the red alga
Delisea pulchra to the brown alga Ecklonia radiata with ontogeny
of the sea urchin. Sea urchin larvae settled and metamorphosed
preferentially on D. pulchra because of its relatively high concen-
trations of histamine, which is a settlement cue for larvae of this
sea urchin.104,105 However, the major halogenated furanone 33 in
D. pulchra deterred feeding by H. purpurascens, and juvenile and
adult urchins grew better, were more fecund, and had higher
survival on E. radiata than on D. pulchra. Larger sea urchins
were found on E. radiata, and urchins moved from D. pulchra
to E. radiata habitats at night when predation risk was low from
diurnal predators such as ?sh. Settlement cues and chemical
defenses co-occurred in D. pulchra, and both played a role in host
use by the sea urchin.104
The Brazilian red alga Laurencia obtusa produces elatol 35 as
its major metabolite,106 a compound previously shown to deter
herbivores (reviewed by Paul et al.107). In this study, both crude
algal extracts and elatol at natural concentrations inhibited feeding
by two herbivores, the crab Pachygrapsus transversus and the
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sea urchin Lytechinus variegatus.106 In addition, a crude extract of
L. obtusa signi?cantly reduced fouling relative to controls after ?ve
weeks when incorporated into phytagelTM and placed in the ?eld.106
Crude extracts also inhibited settlement by mussel larvae (Perna
perna) in laboratory assays and overall fouling cover in ?eld assays
relative to controls or extracts of the brown alga Stypopodium
zonale.108 Elatol 35 was found in extracts of the surface of L.
obtusa dipped in hexane, but it was never tested independently for
its antifouling potential.
Several other studies examined antifouling activity in red algae.
The red alga Dilsea carnosa was observed to have very low
fouling cover in the ?eld.109 However, phytagelTM treated with
crude extract was readily fouled in the ?eld. Further ?eld trials
of live algae showed that D. carnosa is capable of sloughing
its outer tissue layer, a mechanical not chemical antifouling
strategy.109 Isethionic acid (2-hydroxyethane sulfonic acid) and
?oridoside (2-O-a-D-galactopyranosylglycerol) were isolated from
the invasive red alga Grateloupia turuturu collected in France.110
Isolated compounds were tested individually in the laboratory;
isethionic acid and ?oridoside reduced settlement of Balanus
amphitrite larvae at concentrations as low as 0.001 mg ml?1, and
together the compounds reduced settlement at 0.002 mg ml?1.
Isethionic acid was toxic to B. amphitrite nauplii while ?oridoside
was not, suggesting that ?oridoside acts as a chemical cue to reduce
barnacle settlement at concentrations that are non-toxic.
In a comparison of ?ve common red algae from the Swedish west
coast for their ability to inhibit bacterial growth and attachment,
the red alga Bonnemaisonia hamifera was found to be the most
active. Extracts of B. hamifera inhibited the growth of nine of the
eleven bacteria tested.111 Surface extracts of B. hamifera inhibited
the growth of three marine bacteria, indicating that the alga had
suf?cient levels of metabolites on its surface to reduce bacterial
growth. Bacterial abundances on ?eld collected algae were much
lower on B. hamifera than on another red alga Chondrus crispus.111
Primary and secondary metabolites were measured to determine
the cellular processes involved in the colonization of the red
alga Chondrus crispus by the green algal endophyte Acrochaete
operculata.112 Different life stages of C. crispus differed in their
susceptibility to A. operculata; tetrasporophytes were more sus-
ceptible than gametophytes, and susceptibility was mediated
by different types of carrageenans in the cell wall matrix of
C. crispus.112 Contact between C. crispus gametophytes and A.
operculata caused release of L-asparagine by A. operculata, which
C. crispus breaks down to release hydrogen peroxide, ammonium
ions, and a carbonyl compound (succinamic acid). Exposure to L-
asparagine decreased fouling on C. crispus by associated bacteria
and A. operculata spores.112 Additionally, H2O2 was more toxic to
A. operculata than to C. crispus.
Many recent studies of the chemical ecology of green algae
have focused on species of Caulerpa. There has been considerable
interest in natural products chemistry and chemical defenses in
these green algae because many species are highly invasive.113?115
Studies of the invasive Caulerpa taxifolia in the Mediterranean
examined the biosynthesis of the major metabolite caulerpenyne
36116 and its role in wound healing in the alga.117 Transformation of
caulerpenyne by an esterase to oxytoxin 2 37 occurs rapidly after
injury,118 and the resulting 1,4-dialdehyde is highly reactive and
cannot even be detected four minutes later.117 The decay kinetics
of oxytoxin 2 37 matched those of the formation of the external
wound plug of C. taxifolia.117 Adolph et al. conducted experi-
ments that suggested that proteins cross-linking with oxytoxins
and similar reactive aldehydes in C. taxifolia are essential for
wound-plug formation.117 This suggests an additional function for
caulerpenyne in addition to its antimicrobial and feeding deterrent
activities.107 Wound plug formation has been studied for another
green alga Dasycladus vermicularis,119 but the role of secondary
metabolites has not been demonstrated.
Extracts of Caulerpa prolifera collected from shallow habitats
in Greece were tested for antibacterial and antialgal activities.120
Extracts contained caulerpenyne esters and exhibited antimicro-
bial activity toward three of six marine isolates and antialgal
activity toward Phaedactylum tricornutum.120 Responses of native
herbivores to three species of Caulerpa that have recently appeared
in southeast Australia were studied by Davis et al.115 The snail
Turbo undulatus showed the lowest preference for C. ?liformis of
?ve live algae offered in choice and no-choice assays. Ethanol
extracts of C. ?liformis, C. taxifolia, and C. scalpelliformis were
tested for their effects on feeding by natural assemblages of
?sh, a sea urchin and molluscs. While few signi?cant results
were obtained, some extracts tended to reduce feeding relative
to controls. Unfortunately, in this study115 and the study of C.
prolifera from Greece,120 algae were frozen after collection, and
green algal metabolites including caulerpenyne 36 decompose
quickly under these handling conditions.121 Good methods of
extracting these compounds after shock freezing algae in liquid
nitrogen have been developed,122 and these should be carefully
followed by chemists and chemical ecologists working with species
of Caulerpa and other green algae.
Some green algal water soluble metabolites are potential
antifouling compounds; for example, extracts from the green alga
Ulva reticulata and an epibiont bacterium Vibrio sp. inhibited
attachment of the polychaete Hydroides elegans and the bryozoan
Bugula neritina.8 Each polar extract was deactivated with different
enzymes and was composed of different carbohydrate monomers,
suggesting that the Vibrio sp. inhibitory compound(s) were
different from U. reticulata compound(s).8 Allelopathic effects of
164 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
other species of bloom-forming Ulva have also been observed. Ulva
pertusa cultured together with the HAB-forming microalgae Het-
erosigma akasiwo and Alexandrium tamarense strongly inhibited
the growth of the microalgae.123 A similar study with U. pertusa and
U. linza co-cultured with the microalga Prorocentrum micans also
indicated the presence of allelopathic compounds released by Ulva.
Aqueous and methanol extracts of the Ulva spp. strongly inhibited
growth of P. micans.124 Aqueous extracts of Ulva fenestrata and
Ulvaria obscura inhibited zygote development of Fucus gardneri,
growth of epiphytic algae, and development of oyster larvae, also
indicating the allelopathic activities of these algae.125
Bio?lms are a key feature of marine systems that some algal
spores use as cues for recruitment into appropriate habitats.
Bio?lms are implicated in Enteromorpha settlement, and a recent
study shows that this green alga settles in response to a variety
of bacterial strains isolated from natural habitats.126 Quite a few
genetically distinct bacteria stimulated settlement, especially when
allowed to develop for 48 h, but not all species had a positive effect;
?ve out of 14 strains of Pseudoalteromonas inhibited spore settle-
ment. A possible explanation for this variable settlement response
is that Enteromorpha spores were found to settle in response to N-
acylhomoserine lactone (AHL), a bacterial growth promoter.127
AHL is a compound known for its role in quorum sensing in
bio?lm bacteria, and by creating mutant bacterial strains the
authors were able to show that settlement of Enteromorpha only
increased in response to the bacterial strains that could produce
AHL. Tests with synthetic AHLs showed that settlement activity
was lost when the lactone ring structure of AHL was opened.127
Our understanding of marine microbe bio?lms and diversity
has grown in recent years and is revealing complex chemical
interactions between microbes and many marine organisms. For
instance, microbial compounds not only in?uence settlement for
some marine algae but also mediate the differentiation of cells
into a thallus in the green alga Monostroma oxyspermum.128 In
the ?eld, M. oxyspermum has a leafy morphology similar to Ulva
spp. but when M. oxyspermum was raised in aseptic conditions
it grew as unorganized bunches of cells. Thallusin 38, isolated
from an epiphytic marine bacterium, induced differentiation of
the algal cells to form a thallus. Indeed when the M. oxyspermum
was deprived of thallusin the algal thallus began to disintegrate
and the alga reverted back to its unorganized state. Thallusin
38 also caused differentiation in other green algae such as Ulva
pertusa and Enteromorpha intestinalis.128
4 Seagrasses
There are few studies of chemical defenses of seagrasses against
herbivores compared to other benthic marine plants. In contrast,
plant?pathogen interactions have been addressed more often in
seagrasses than algae. This is due to major die-offs of near
shore seagrass beds caused by disease. Antimicrobial activities
observed in seagrass extracts are attributed to the presence of
phenolic compounds.129 A study of Thallasia testudinum demon-
strated that the seagrass produces an antibiotic ?avone glycoside,
luteolin 7-O-b-D-glucopyranosyl-2??-sulfate, which inhibits the
growth of Schizochytrium aggregatum at 1/10th of the natural
concentration.130 More recently, phenolic acids were reported to
accumulate above necrotic lesions on blades of T. testudinum
infected with Labyrinthula sp.131 However, the ?pseudo-induction?
of phenolics did not appear to increase resistance of the seagrass
to infection. The authors proposed that the increased biosynthesis
of phenolics is a response to the excess carbon available in
plant tissues because the infection site disrupts plant resource
allocation. In a broader study of the antimicrobial activities of the
methanol extracts from three cultured and ?eld collected estuarine
seagrasses, Potamogeton pectinatus, P. perfoliatus, and Ruppia
maritima, crude extracts were reported to inhibit the growth of all
12 strains of gram-positive and three of 12 gram-negative bacteria
included in the survey.132 Interestingly, antimicrobial activity was
found in the crude extracts from the natural populations collected
in the spring but absent in the extracts from plants collected in
the fall. This pattern correlated with new growth in the seagrass
shoots and higher nutrient levels in the surrounding waters.
5 Sponges
Sponges (phylum Porifera) continue to be a source of novel and
interesting natural products.133?135 Our knowledge of the ecological
roles of these compounds continues to grow as new techniques
are developed to understand the complex relationships between
sponges and their predators, competitors, microorganisms, and
associated invertebrates.136?138
Many Antarctic sponges are chemically defended against a
range of potential predators,138 and sponges in northern high lati-
tudes also have chemical defenses. In a series of feeding deterrence
assays of crude extracts from 17 benthic organisms from a sub-
Arctic fjord, only the sponge Haliclona viscosa and the cnidarian
Hormathia nodosa deterred amphipod feeding.139 Haliclona viscosa
also deterred feeding by the star?sh Asterias rubens, but deterrent
compounds were not isolated and characterized.
In tropical regions some sponges are chemically defended while
others are not. Walters and Pawlik140 arti?cially wounded some
Caribbean sponges, and found that those not chemically defended
healed at signi?cantly faster rates than the chemically defended
species. In addition, morphology was important since tubular
shaped individuals healed faster than vasiform shaped individuals
of Niphates digitalis. The authors proposed that Caribbean reef
sponges have adapted to overcome ?sh predation by investing in
the production of chemical defenses or by a rapid wound-healing
response after injury.
There are several examples in which one species of sponge
produces chemical defenses against multiple predators, competi-
tors, or microorganisms. Compounds from the Antarctic sponge
Isodictya erinacea deterred the sea star Perkinaster fuscus141 and
prevented the molting of the sponge boring amphipod Orchomene
plebs.142 Laboratory experiments demonstrated that amphipods
fed a continuous diet of erebsinone 39, isolated from I. erinacea,
exhibited high levels of premature mortality when they were
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unable to molt.142 A more recent study of the Brazilian sponge
Geodia corticostylifera showed that crude extracts are deterrent
to generalist ?shes, prevent fouling by the mussel Perna perna,
and act as a chemical cue for colonization by the ophioroid
Ophiactis savignyi.136 Unique laboratory experiments conducted
in ?ow-through tanks with sponge mimics constructed of sponge
skeleton and coated with phytagelTM alone or containing the
crude extract demonstrated that the ophioroid Ophiactis savignyi
chemically recognized and colonized the mimics containing the
sponge extracts.136
In past studies, spicules have rarely been shown to be an
important structural defense in sponges and are thought, instead,
to be important to the structural integrity of the animal. Two
recent studies of temperate and subtropical sponges show that
sponge spicules can deter ?sh and invertebrate predators when
offered alone or in combination with crude extracts.137,143 Arti?cial
foods containing spicules from the sponges Cliona celata and
Halichondria bowerbanki and a combination of crude extracts and
spicules from the sponges Microconia prolifera and Halichondria
bowerbanki collected from the Long Island Sound were unpalat-
able to the hermit crab Pagurus longicarpus.143 For M. prolifera, the
hermit crab was signi?cantly more deterred by the combination
of crude extracts and spicules than either offered alone. Their
results demonstrate that there is an interaction between the
chemical and structural defenses of M. prolifera that enhance the
feeding deterrence properties of the sponge against P. longicarpus.
Synergistic interactions between the chemical and structural
defenses against the wrasse Thalassoma bifasciatum were recently
reported for four of eight sponge species surveyed from the Florida
Keys and the Bahamas.137 Synergy was observed in arti?cial foods
with natural concentrations of crude extracts and spicules from
Cinachyrella alloclada, Clathria virgultosa, and Xestospongiamuta,
whereas the combination was only synergistic for Agelas clathrodes
when the concentration of crude extract was decreased by 3-fold
and the spicule concentration was increased by 8-fold of natural
concentrations. In addition, the spicules from three other sponges
effectively deterred feeding by T. bifasciatus. The results of these
studies suggest that spicules from some sponges are important
structural defenses against different predators.
There continue to be few studies of seasonal and temporal varia-
tion in the production of chemical defenses by sponges. Two recent
studies of the intraspeci?c variation of secondary metabolites that
did not directly address ecological questions could have important
ecological implications. In the ?rst study, concentrations of
the cytotoxic metabolites mycalamide A 40, pateamine 41, and
peluroside A 42 were shown to vary both spatially and temporally
in individuals of the New Zealand sponge Mycale hentschelli
from Pelorus Sound and Kapiti Island.144 Pelorus Sound, a
semi-estuarine sheltered area that receives freshwater runoff,
and Kabiti Island, a high-energy coastal environment, represent
two extremes in marine habitats that are proposed to account
for both morphological and chemical differences observed for
M. hentschelli. Concentrations of mycalamide A 40 were greater in
sponges found on the deeper reefs of Kapiti Island, whereas 41 was
only present in individuals from Pelorus Sound. Concentrations
of 42 varied signi?cantly among individuals at Pelorus Island,
but peluroside A 42 was rarely present in samples from Kapiti
Island. In addition, metabolite concentrations increased in the
spring as the water temperature increased, but declined before
peak temperatures in the summer.144 In a second study, Mart??
et al.145 demonstrate that the Microtox R? assay can be a useful
tool for quickly assessing intraspeci?c variability of avarol 43 and
palinurin 44 concentrations in Dysidea avara and Iricinia variabilis
populations, respectively.
The vast and complex nature of metabolites isolated from
sponges has raised the question of where the production of
secondary metabolites is taking place. Because of the similarity
in structures isolated from microorganisms and many sponges,
biosynthesis is often suggested to occur in a symbiont. We
previously reported several examples of sponge cell localization ex-
periments that suggested secondary metabolites are often localized
in bacterial or cyanobacterial cells.1 Ecological studies of defensive
compounds biosynthesized by sponge symbionts are rare. One
recent study of Dysidea granulosa in Guam by Becerro and Paul146
used transplant and shading experiments to test the prediction
that if the symbiotic cyanobacterium Oscillatoria spongeliae is
responsible for the production of polybrominated diphenyl ethers
45?47, changes in the concentrations of polybrominated diphenyl
ethers should be positively correlated with changes in chl a. While
45 and 47 and chl a concentrations were positively correlated
166 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
in natural populations, the results from the transplant showed a
decrease in chl a concentrations when sponges were transplanted
to a deeper depth while concentrations of 45?47 did not change.
Shading experiments showed that concentrations of 45?47 did
not change (but tended to decrease) in sponges under all types
of treatment plates (black, transparent and UV-?lter) while chl a
concentrations decreased only under the black treatment plate.
This study clearly demonstrates that unraveling the complex
relationships between symbionts and host sponges is a challenge
for chemical ecologists.
Several recent studies have shown that the biosynthetic genes
of some invertebrate secondary metabolites are localized in their
symbionts. For example, an uncultured Pseudomonas sp. bacterial
symbiont of Theonella swinhoei contained the PKS genes for the
biosynthesis of theopederin 48 and onnamide A 49.147 Flatt et al.148
used catalyzed reporter disposition ?uorescence in situ hybridiza-
tion (CARD-FISH) to localize the biosynthetic genes for the
production of polychlorinated peptides related to barbamide 50
in the cyanobacterial symbiont Oscillatoria spongeliae of Dysidea
(Lamellodysidea) herbacea. Ridley et al.149 examined chemical
variation in four dictyoceratid sponges containing chlorinated
amino derivatives or polybrominated diphenyl ethers and related
this chemical variation to genetic variation of O. spongeliae.
They found that only the strain of O. spongeliae in the sponge
containing chlorinated compounds possessed genes involved in the
biosynthesis of these compounds. The gene pathway was absent
from O. spongeliae in sponges that did not contain the compounds,
not a case of selective expression of the metabolites.149 In another
study by Vickery et al.,150 the bacterium Pseudomonas aeruginosa,
derived from the Antarctic sponge Isodictya setifera, produced two
phenazine alkaloids 51 and 52 when grown at ambient temperature
of 0 ?C. Although these studies provide direct evidence that sponge
symbionts contain the machinery for biosynthesizing sponge
metabolites, they provide little insight into the ecological role of
these compounds for the symbiont or the sponge.
One recent study of Suberites domuncula provided evidence that
this sponge is responsible for the production of 1-O-hexadecyl-
sn-glycero-3-phosphocholine 53 and 1-O-octadecyl-sn-glycero-3-
phosphocholine 54, active against the bacterial isolate SB1 isolated
from S. domuncula.151 Induction experiments exposing the sponge
to lipopolysaccharide, an endotoxin from the outer wall of gram-
negative bacteria, resulted in higher concentrations of 53 and
54 and increased expression of alkyl-dihydroxyacetonephosphate
synthase, a key enzyme in the biosynthetic pathway of this class
of compounds that was cloned from the sponge. In another
study of S. domuncula, Wiens et al.152 were able to localize the
major metabolite okadaic acid in the endopinacocytes, the cells
lining the canals of the aquiferous system of the sponge, and
in cells composing the choanocyte chambers using antibodies
speci?c to okadaic acid. Low concentrations of<100 nM okadaic
acid caused an increase in MAP kinase 38, potentially resulting
in stimulation of an antibacterial defense; whereas, at high
concentrations of >500 nM okadaic acid induced apoptosis.
With the growing recognition that some sponge metabolites are
produced by symbionts, there is an increased interest in the host-
speci?city of sponge-associated microbial communities within the
tissues and at the sponge surface. Taylor et al.153 proposed that
specialist bacteria are more common than generalists in microbial-
sponge associations for the temperate Australian sponges Cym-
bastela concentrica, Callyspongia sp. and Stylinos sp. Further
evidence for speci?c microbial-sponge associations comes from
a ?eld transplant experiment with colonies of Aplysina cavernicola
from the Mediterranean Sea in which the bacterial communities
and chemical pro?les of sponges transplanted to shallow reefs
remained largely unchanged after two months. The small variable
fraction of bacteria that did change during this experiment was
composed of bacteria common in seawater.154
There is growing evidence that interactions between sponges
and the bacterial communities with which they are associated
are chemically mediated. Crude extracts from sponges promoted
the growth of culturable strains of surface-associated bacteria
while exhibiting strong antibacterial activities against culturable
strains isolated from other species, hard substrates, and seawater.
In addition, surface-associated bacterial strains can inhibit the
growth of non-surface associated species. In a sponge surface
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colonization study, Lee and Qian155 showed that different bacterial
communities colonized the surface of surface-sterilized Mycale
adhaerens colonies and sterile polystyrene dishes after seven days.
Although the experimental design did not take into account the
physical differences between the sponges and polystyrene dish
surfaces, the resulting experiments clearly showed chemically me-
diated sponge?microbe and microbe?microbe interactions among
the isolated strains. In laboratory assays, 50% of the cultured
bacterial strains from the polystyrene dish were susceptible to
sponge extracts whereas none of the bacteria isolated from the
sponge surface was affected by the sponge extracts. In addition,
crude extracts from the cultured epibionts from the sponge surface
were inhibitory to the isolates from the polystyrene surface. Crude
extracts from the sponge Iricina fusca from the west coast of
India enhanced the growth of bacteria isolated from the surface
of sponges.156 Densities of culturable surface-associated bacteria
changed temporally, with the lowest densities in January. Strains
of Bacillus and Micrococcus were common during all sampling
months. Crude extracts from Bacillus and Micrococcus exhibited
antibacterial activity against fouling bacteria suggesting that I.
fusca promoted the growth of bacteria on its surface that may
assist the host in the control of epibiosis.156
A recent study examined the ability of marine strains of bacteria
to attach, swarm, and grow in the presence of sponge extracts.157
The results demonstrated that crude extracts from eight species
of sponges inhibited the attachment of bacterial isolates from
sponge surfaces, nearby strata, or adjacent seawater. Extracts
from four sponges, Ailochroia crassa, Agelas conifera, Amphimedon
compressa and Aplysina fulva, completely inhibited swarming for
six of the bacterial strains. Other sponge extracts enhanced or
had no effect on swarming, but attachment of 23 of the 24
bacterial isolates was inhibited by nearly all of the sponge crude
extracts. In contrast, few crude extracts inhibited the growth of the
bacterial isolates in standard agar disc assays. In this same study,
bioassay-guided fractionation of the sponge Ailochroia crassa
yielded ianthelline 55, which inhibited bacterial attachment, and
a bromotyrosine derivative 56 that inhibited swarming.
Sponge extracts can also inhibit settlement of other fouling
organisms. Dobretsov et al.158 showed that conditioned seawater
of Callyspongia pulvinata decreased the settlement and survival
of Hydroides elegans, and decreased the density of the diatom
Nitzschia paleacea, but had no effect on cultured bio?lm bacteria.
In the ?eld, live sponge decreased the total cover, diversity,
and species richness of the fouling community adjacent to the
sponge, which the authors attributed to water soluble sponge
compound(s). Three Hong Kong sponges, Haliclona cymae-
formis, Haliclona sp., and Callyspongia sp., had unique natural
bacterial communities.159 Crude extracts of Haliclona sp., and
Callyspongia sp. incorporated into phytagelTM and left in the
?eld for one week inhibited fouling and changed the bacterial
and diatom communities when compared to a solvent control.
The methanol extract from Haliclona sp. was the most effective
at decreasing the richness, diversity, and evenness of the fouling
diatom community. After three weeks all six of the sponge extracts
(methanol and dichloromethane extracts for each sponge) de-
creased the mean number of settlers on the gels in the ?eld.159
In laboratory experiments, compounds isolated from the Mediter-
ranean sponges Cacospongia scalaris, Dysidea sp., and acetates of
compounds from Ircinia spinosula, inhibited settlement by cyprids
of the barnacle Balanus amphitrite but were not toxic to them.160 A
recent review135 discussed the antifouling potential of derivatives
of natural sponge compounds including alkyl amines, isocyano
aromatic amines, and N-formyl aromatic amines. Synthetic ana-
logues of isocyano compounds inhibited the settlement of Balanus
amphitrite larvae, but were also toxic to microorganisms.161 This
research has industrial applications but does not consider the
ecological functions of these compounds for the sponges.
6 Cnidarians
Studies of the chemical ecology of Alcyonarians (Octocorallia),
especially the Alcyonacea (soft corals) and the Gorgonacea
(gorgonian corals) have addressed predator defense, antibacterial
defenses, and antifungal defenses against Aspergillus sydowii.
Recent studies of crude extracts and pure compounds from
gorgonian corals show that these serve multiple ecological roles.
For example, n-hexane extracts from Stereonephthya aff. curvata,
an exotic soft coral introduced to the Arraial do Cabo region
in Brazil about 10 years ago, deterred ?sh feeding and through
contact caused necrosis in the tissues of the Brazilian endemic
soft coral Phyllogorgia dilatata within three weeks. The authors
proposed that its chemical defenses promoted the spread of this
invasive species.162 The sesquiterpene ainigmaptilone A 57, isolated
from the Antarctic gorgonian coral Ainigmaptilon antarcticus, also
had multiple ecological roles; it deterred predation by the sea star
Odantaster validus and inhibited the growth of sympatric Antarctic
bacteria and diatoms.163
Aspergillosis, a disease caused by the fungal pathogen
Aspergillus sydowii, continues to infect Gorgonia ventalina pop-
ulations in the Caribbean. Gorgonia spp. are also threatened
with overgrowth by other invertebrates including the hydrozoan
Millepora alcicornis. Short-term induction experiments with G.
ventalina demonstrated that tissue necrosis results when healthy
G. ventalina is placed in contact with diseased G. ventalina
infected with aspergillosis or live M. alcicornis.164 In addition,
there was an increase in the number of purple sclerites at the
wound site. The antifungal activities of the non-polar crude
extracts against A. sydowii remained unchanged throughout the
experiment suggesting that natural products are not induced
defenses to these potential threats.164 In longer term experiments
168 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
with G. ventalina and G. ?abellum, mechanical damage caused by
scraping with a dive knife and cable ties attached to the sea fans did
not induce purpling, suggesting a recognition system that signals
the induction of purpling in response to infection and invading
biotic agents. In addition, most colonies of G. ventalina and G.
?abellum put in contact with diseased G. ventalina infected with
aspergillosis or live M. alcicornis recovered after seven months,
suggesting that the induction of the purpling response at the
infected regions is a successful defense facilitating the recovery
and survival of Gorgonia sea fans.164
Relatively few studies have addressed the potential of octocoral
symbionts as the source of bioactive secondary metabolites. Two
recent studies of the Caribbean gorgonians Pseudopterogorgia
bipinnata and P. elisabethae provide evidence that some diterpenes
may be produced by the cellular symbionts in the genus
Symbiodinium.165,166 Biosynthetic studies of the Caribbean
gorgonians P. elisabethae and P. bipinnata suggest that isolated
dino?agellate symbionts produce pseudopterosins A 58 and D 59
and kallolide A 60, respectively.165,166 Further, high concentrations
of pseudopterosins were positively correlated with a greater abun-
dance of the symbiont Symbiodinium sp. at the tips of P. elisabethae
colonies.165 In another recent study of P. elisabethae, Puyana
et al.167 investigated the intraspeci?c variation of pseudopterosins
in sea fans from the archipelago of San Andres and Provencia
in the southwestern Caribbean. Sea fans from Provencia were
chemically rich in pseudopterosins 61?69 and seco-pseudopterosin
70, whereas the sea fans from San Andres contained only low
concentrations of these compounds. The ecological implications
of these results have not been explored.
Symbionts may provide important chemical defenses for their
host, but the mechanisms of acquisition and maintenance of
symbionts are still poorly understood. Recent research on the
soft coral Sinularia lochmodes found that the lectin SLL-2 may
be used to selectively uptake speci?c symbionts.168 The lectin was
found on the surface of Symbiodinium cells released from this
soft coral. Three cultured strains of Symbiodinium exposed to
SSL-2 in the laboratory at less than natural concentrations had
reduced motility similar to that exhibited by natural symbiotic
zooxanthellae. Growth was not affected in two strains but was
inhibited in one strain. When non-symbiotic dino?agellates and
algae were tested, SLL-2 had no effect on the motility and growth
of the dino?agellate Alexandrium minutum but caused signi?cant
mortality for Gymnodinium catenatum and Prorocentrum micans.
The chlorophyte Tetraselmis sp. also lost cell motility and reduced
growth suggesting that this lectin not only modi?es the physiology
of symbiotic zooxanthellae but may also be a chemical mechanism
for selecting the appropriate symbiont.
The antimicrobial properties of the eggs of 11 species of scler-
actinian corals were evaluated by testing extracts for their ability
to deter surface attachment and inhibit growth of two laboratory
bacteria and 92 marine bacterial isolates.169 Antimicrobial activity
was found in eggs of only one species of coral, Montipora digitata,
which has been previously reported to produce montiporic acids170
and other cytotoxic diacetylenes.171
Chemical cues are often used by cnidarians for recruitment
into an appropriate habitat. Recent research on the larvae of
the stony corals Acropora tenuis and A. millepora showed that
these larvae selectively settled in response to crustose coralline
algae.172 The coral larvae had the highest settlement in response to
Titanoderma prototypum, which also caused the lowest coral post-
settlement mortality of the algae tested. Methanol extracts of T.
prototypum and Hydrolithon reinboldii both induced high levels of
metamorphosis at low concentrations.172
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7 Ascidians (tunicates)
The ascidians (Ascidiacea, Tunicata) are rich sources of bioactive
secondary metabolites133,134 that can deter invertebrate and ?sh
predators and inhibit the growth of microorganisms.173?175 Fresh
animal tissue and lipophilic crude extracts from the Antarctic
ascidian Distaplia cylindrica deterred predation by the sea star
Odantaster validus.173 Strong acids concentrated at the surface of
the tunic also deterred O. validus, suggesting that this tunicate
employs multiple defense strategies. Hydrophilic and the lipophilic
crude extracts of D. cylindrica caused mortality in a chain-forming
diatom demonstrating the potential of the crude extracts for
controlling surface fouling. The congeneric ascidian Distaplia
nathensis also has antifouling extracts; its water soluble extract
inhibited the growth of all 14 tested bacteria and attachment of
the mussel Perna indica.176 Extracts of three other ascidian species,
from seagrass habitats in the Caribbean, did not deter bacterial
attachment but did inhibit growth of some bacterial strains and
settlement of cyprid barnacle larvae (Balanus amphitrite).177 In the
?eld none of the ascidian extracts inhibited polychaete attachment;
however, methanol extracts of A. stellatum and B. planus inhibited
bryozoan attachment, and chloroform and methanol extracts of
these two ascidians inhibited settlement by barnacles.177
A broad survey of the chemical defenses of the adults and
larvae of six colonial ascidians from the Mediterranean showed
that defense strategies of tunicates can vary signi?cantly among
species.174 Overall, the tunic material from the adults was less
palatable than zooids (thorax and abdomen) when offered to
?sh and crustaceans, and unpalatability was found in all species
in at least one assay. Crude extracts from the tunic of two of
the six species tested signi?cantly deterred feeding by hermit
crabs. The authors suggested that predators may be deterred
by a combination of factors including chemical defenses and
low caloric content, digestibility, and pH. Overall, ascidians that
produced large larvae in low numbers had larvae that were better
chemically defended against ?sh or invertebrates than ascidians
that produced smaller and more numerous larvae.174 These results
suggest that chemical defenses are more prominent in the larvae
of invertebrates with low fecundity.
Few studies have assessed chemical variation in ascidi-
ans. Lo?pez-Legentil et al.178 surveyed the major alkaloids in
Mediterranean specimens of purple, blue, green, and brown
morphs of Cystodes dellechiajei. They identi?ed two distinct
chemotypes. The purple morph of C. dellechiajei contained the
pyridoacridines shermilamine B 71 and kuanoniamine D 72 in
the tunic and their deacetylated forms 73 and 74 in the zooids
while the blue and green morphs contained the C9-unsubstituted
pyrridoacridines, ascididemin 75 and 11-hydroxyascididemin 76
in the tunic and zooids. The brown morph only contained
minor concentrations of ascididemin. Further studies of the
mitochondrial DNA of the different color morphs of C. dellechiajei
showed little correlation between the chemotypes, morphotypes
(spicules), and genotypes179 with one exception. There was an
association between the color of the purple morph and the
pyridoacridines, which are purple under the acidic conditions
found in the tunic of Cystodytes.178 Crude extracts and ascididemin
75, the major metabolite from the blue morph of Cystodes from
the Mediterranean, deterred ?sh feeding in ?eld and laboratory
feeding assays, but were palatable to the sea urchin Diadema
savignyi. Tunic acidity and spicules from Cystodes spp. did not
deter predators.175 Indirect evidence from energy-dispersive X-
ray analysis indicated that pyridoacridine alkaloids present in
Cystodes dellechiajei were accumulated in granules of the pigment
cells in the purple morph. Turon et al.180 proposed that the
biosynthesis of the alkaloids occurs in the ascidian because sulfur
signals corresponding to shermilamine B or kuanoniamine D or
their deacetylated derivatives were not found in the bacterial cells
sparsely distributed throughout the tunic and the zooids.
Until recently, the origin of metabolites in Lissoclinum sp. has
been the subject of some debate. Schmidt et al.181 reported that
the biosynthetic genes for patellamides A 77 and C 78, isolated
from Lissoclinum patella, occur in the cyanobacterial symbiont
Prochloron didemni. The function of the biosynthetic genes was
170 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
con?rmed by heterologous expression of the whole pathway in
Escherichia coli. Long et al.182 also expressed genes for patellamide
biosynthesis in E. coli by ?shotgun? cloning. Their study utilized
a universal expression system in tandem with direct chemical
analysis to identify clones that produced patellamides. In another
study, Prochloron spp. symbionts of ?ve didemnin ascidians from
Okinawa, Japan have been implicated in the production of UV
absorbing mycosporine-like amino acids (MAAs) that accumulate
in the tunic of the transparent animals and protect them from
harmful UV exposure.183 Of the species surveyed, MAAs were
only detected in tunicates containing Prochloron symbionts.
Chemical alarm cues are known from many marine organisms
but have rarely been observed in ascidians. A recent study mea-
sured the electrophysiological responses associated with arrested
cilliary activity in the branchial chamber of the ascidian Clavelina
huntsmani.184 C. huntsmani responded to water soluble compounds
released from conspeci?cs by stopping water ?ow through its
branchial chamber, but did not respond to conspeci?c water after
it was boiled, water soluble cues from Corella willmeriana, or
cues from the mussel Mytilus californianus. The ascidian Corella
willmeriana did not respond to conspeci?c cues or those from C.
huntsmani. The author suggested that arrested water movement
through the ascidian is an alarm response to conspeci?c cues.
8 Bryozoans
The production of bryostatins by the bacterial symbiont En-
dobugula sertula of Bugula neritina is one of the ?rst examples in
which an invertebrate symbiont was demonstrated to biosynthe-
size secondary metabolites found in an invertebrate.185,186 Recent
studies of Bugula neritina by Lopanik et al.186,187 demonstrated
that three bryostatins protect the larvae of B. neritina from
North Carolina from predation by ?sh. Crude extracts prepared
from B. neritina larvae deterred feeding by pin?sh but crude
extracts from the adults were palatable. When the larvae were
raised without their symbionts they were also palatable. Bioassay-
guided fractionation of the active extracts resulted in the isolation
of three ichthyodeterrent compounds, bryostatins 1 79, 10 80
and the new bryostatin derivative, bryostatin 20 81.187 These
three compounds were present in larvae with symbionts but
absent in larvae without symbionts, and interestingly they were
absent in B. neritina larvae from Delaware.186 Using 16S rDNA
speci?c primers for the symbiont E. sertula, the authors found
that the variable presence of bryostatins in B. neritina larvae
correlated to the presence or absence of the bacterial symbiont.
This study illustrates the complex interactions between symbiont
and host, and the importance of secondary metabolites in defense
for marine larvae.
Bryostatins related to those isolated from Bugula neritina are
reported from B. simplex. A closely related bacterial symbiont
to Endobugula sertula was characterized from the pallial sinuses
of B. simplex larvae. The larval symbiont, Endobugula glebosa, is
consistently and uniquely associated with B. simplex exhibiting
an ecological and evolutionary relationship similar to that of
the invertebrate?symbiont relationship between B. neritina and
Endobugula sertula.188
The roles of chemical cues for settlement of bryozoan larvae
have recently been studied. Bacterial and diatom bio?lms were
used to assess settlement cues for B. neritina.189 Individual bacteria
species had no effect or a negative effect on the number of B.
neritina settlers, and settlement was negatively associated with
the density of the bacterium Pseudoalteromonas sp. The diatom
Amphora cofeaeformis induced higher rates of settlement than any
other individual diatom.
Chemical cues not only determine where bryozoans settle
but are also implicated in inducible structural defenses of the
bryozoan Membranipora membranacea.190 Waterborne cues from
fourteen species of nudibranchs were tested for their ability to
induce spines that inhibit nudibranch feeding. The nudibranch
conditioned seawater (water that held test species for six hours)
was incubated with M.membranacea for three days, and the growth
of spines was scored after ?ve days. Four nudibranch species that
were known to feed on M. membranacea and four nudibranchs
that do not feed on M. membranacea induced spine formation,
although the induction of spine formation was variable between
trials for all nudibranch species except for the M. membranacea
specialist Doridella steinbergae. Doridella steinbergae egg masses
and juveniles also induced spine growth. Interestingly there was
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no phylogenetic pattern for the nudibranch species that induced
spine formation.
9 Crustaceans
Chemical defenses in crustaceans are not well studied. The
isopod Santia sp. from Papua New Guinea is host to a complex
episymbiotic community that is dominated by bacteria and small
unicellular Synechococcus-type cyanobacteria.191 In the absence
of symbionts the isopod was palatable to reef ?shes, but crude
extracts prepared from the isopods with the episymbionts were un-
palatable, demonstrating that these microbial symbionts provided
a chemical defense for the isopod. Observation of newly emerged
juveniles climbing on the mother and inoculating themselves with
her episymbionts suggests that the chemically deterrent symbionts
are vertically transferred from the mother to the offspring.
While chemical defenses in crustaceans are rarely studied,
settlement and chemical cues are relatively well studied. A recent
review by Rittschof and Cohen192 discusses the structure?activity
relationship of peptides for chemical signaling in crustaceans.
Barnacles are often used as assay organisms to test antifouling
natural products in a variety of organisms,110,161,177 thus un-
derstanding their positive settlement cues has ecological and
industrial signi?cance. Settlement plates with a smooth surface
texture and the amount of time left in the ?eld increased settlement
of Balanus improvisus larvae. Recruitment was unaffected by crude
extract from conspeci?c adults.193 Surface texture was shown to be
important for barnacle cyprid behavior as they tended to swim
faster, more continuously and more randomly when offered a
textured surface compared to a smooth surface. The presence of
an adult extract also decreased the velocity and randomness of the
swimming cyprids. In ?ume experiments cyprids were more likely
to remain on smooth surfaces and leave rough surfaces.
The barnacle Balanus amphitrite preferentially settles in re-
sponse to marine bio?lms, and current research focuses on
teasing apart the multiple factors that in?uence the rates of
settlement. Qian et al.194 found distinct bio?lm communities
at high-, mid- and sub-tidal heights but the barnacle cyprids
preferred the bio?lm from a mid-intertidal height. This selection
was not correlated to bio?lm biomass or abundance but was likely
related to bacterial composition of the bio?lm. Hung et al.195
found that ultraviolet light did not affect bacterial density in
bio?lms but did decrease the percentage of respiring bacterial
cells as the dose of UV light increased. Even so, this did
not have an effect on the rates of settlement of B. amphitrite
cyprids. Individual diatom species were tested to determine if
they induced B. amphitrite metamorphosis.196 Bio?lm extracellular
polymeric substances (EPS), but not free EPS, induced the same
rates of larval settlement as the positive control of conspeci?c
adult extract. In ?ve axenic diatom cultures at three different
densities, the species and age of the diatom in?uenced barnacle
settlement. In two non-axenic diatom cultures only the bio?lm
age increased the metamorphosis rates of B. amphitrite.196 The
bacterium Pseudomonas aeruginosa, isolated from the shell surface
of B. amphitrite, also induced metamorphosis in B. amphitrite
cyprids, the rates of which varied under different salinity and
temperature treatments.197 When offered together the surface
bound components and the culture supernatant of P. aeruginosa
doubled the metamorphosis of barnacle cyprids, but the surface-
bound components alone inhibited metamorphosis. The media
on which P. aeruginosa was raised also had an in?uence on
cyprid metamorphosis with culture supernatants from a semi-solid
culture showing the highest rates of metamorphosis. Semi-solid
bacterial cultures showed higher protein content than that of the
other cultures, which were mostly composed of carbohydrates.
FTIR analysis showed that the semi-solid cultures contained
compounds with peaks characteristic of ketones, however these
were not tested for their in?uence on barnacle metamorphosis.
The eicosanoid hepoxilins and trioxlins were examined for their
roles in egg hatching and settlement of the barnacles Balanus
amphitrite and Elminius modestus.198 Trioxilin A3 82 did not induce
egg hatching in E. modestus but the unstable epoxide precursor
hepoxilin A3 83 did. None of the compounds tested induced
settlement in B. amphitrite cyprids.
Other barnacles, especially parasitic species, use chemical cues
to ?nd the appropriate host or habitat for settlement. The
parasitic rhizocephalan barnacle Loxothylacus texanus settles on
the external carapace of the blue crab Callinectes sapidus.199
Barnacle larvae settled less when proteins and carbohydrates were
removed, but settled more when lipids were removed from the
exoskeleton of Callinectes sapidus. These cues were associated with
the epicuticle layer of C. sapidus exoskeletons, not bio?lms on the
carapace. Larvae of another parasitic barnacle, Heterosaccus doll-
fusi, modi?ed their swimming behavior towards the host (direction
and velocity) in the presence of waterborne metabolite(s) from the
host crab Charybdis longicollis.200 Swimming behavior of nauplius
larvae of the commensal barnacle Trevathana dentata was also
modi?ed by water soluble cues from the host coral Cyphastrea
chalcidicum.201
Crabs also use chemical cues to ?nd appropriate habitats for
settlement. In the laboratory and the ?eld, megalopae of the
crab Callinectes sapidus preferred live eelgrass habitat over oysters
and mud.202 More ?rst stage juvenile crabs were recovered from
live oyster habitat (Crassostrea virginica) than eelgrass habitat;
however, in the ?eld both megalopae and the ?rst stage juveniles
settled in Zostera marina habitats. Another study found that
current speeds and waterborne cues were important for swimming
orientation and behavior of C. sapidus megalopae.203 At high
current speeds in ?ume studies, C. sapidus intermolt megalopae
were most often found at the top of the water column swimming
172 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
at faster speeds.203 This was not true for premolt megalopae
which had a constant proportion of megalopae oriented upstream
irrespective of current speed in offshore water, but increased their
swimming upstream in the presence of Zosteramarina conditioned
water. At lower current speeds, more premolt megalopae of C.
sapidus oriented themselves upstream in the presence of Zostera
marina and Spartina alterni?ora conditioned water when com-
pared to offshore water. The megalopae may perceive predators
by waterborne cues as the presence of crab Uca pugilator and
grass shrimp Palaemonetes pugio conditioned water added to the
Z. marina water resulted in less megalopae orienting upstream,
but water from the crab Panopeus herbstii had no effect. The study
showed that larvae could detect and respond both to positive and
negative environmental odors.203
Megalopae larvae of the crab Sesarma curacaoense rely on
conspeci?c water soluble chemical cues for fundamental physi-
ological and ecological processes including faster development,
less mortality and metamorphosis.204 Although quite variable,
interspeci?c cues from S. rectum also increased the rate of
development and the percentage of metamorphosis suggesting
that Sesarma spp. produce chemically similar waterborne cues.
Other grapsid crabs including Armasesmiersii and Chasmagnathus
granulata did not speed up development or increase the percentage
of metamorphosis in S. curacaoense larvae. In a set of ?eld
cage experiments megalopae of the ?ddler crab Uca minax were
suspended over a marsh and at increasing distances from the
marsh into a river site.205 After 12 days, molting response was
greatest in the marsh site with or without marsh sediment and
lowest at the river site, which was not different from the ?ltered
seawater control. A regression analysis showed that with increasing
distance from the marsh, molting rates decreased. Chemical
cues for molting of ?ddler crab megalopae originate in marshes
and decline in effectiveness a short distance away from marsh
habitats.205
Chemical cues aid crustaceans in ?nding the appropriate habitat
and in ?nding a mate. A water soluble female pheromone trail
changed the swimming direction and pattern of male copepods
(Centropages typicus).206 As the female trail aged (from 0?30 sec-
onds) fewer trails were found and the males increasingly swam in
the wrong direction. Male copepods (Tigriopus japonicus) engage
in pre-copulatory mate guarding to enhance their reproductive
success. Males use surface proteins similar to a2-macroglobulin to
recognize females; the actual proteins were partially sequenced
by mass spectrometry.207 Males of the Chinese mitten crab
Eriocheir sinensis did not use waterborne chemical cues to ?nd
females, but instead mate recognition occurred after physical
contact.208
10 Molluscs
Natural products in?uence the ecology of marine molluscs at
all life history stages. They function as chemical defenses and
in recruitment and fertilization success. The chemical defenses of
sacoglossan opisthobranchs have recently been reviewed by Mar??n
and Ros.211
A recent study of the sea hare Aplysia californica represents the
?rst neurophysiological study to demonstrate that chemical de-
fenses disrupt the sensory responses of a predator.209 A. californica
releases secretions when attacked by the spiny lobster Panulirus
interruptus that induced feeding and grooming behaviors, and
in some cases caused the lobsters to perform a defensive tail
?ip, allowing 60?67% of sea hares used in this study to escape
predation. Millimolar concentrations of amino acids in the ink-
opaline secretion induce ?phagomimicry? where the secreted
amino acids mimic the stimulatory properties of food to divert
predators.209 P. interruptus individuals were observed digging and
grabbing in the area where the ink-opaline was released into the
seawater, thus distracting the predator while the sea hare escaped.
Ink-opaline is a sticky substance that can also coat the spiny
lobster?s sensory and feeding appendages, which causes confusion
in sensory messages and interferes with the ability of lobsters to
attack their prey.
The feeding selectivity of marine consumers on benthic
cyanobacteria has been rarely studied but has the potential to
control large blooms. Feeding assays with the opisthobranchs Sty-
locheilus striatus and Bursatella leachii showed that they prefer the
cyanobacterium Lyngbya majuscula over diverse macroalgae.212
Even though S. striatus consumed more L. majuscula during
the ?rst eight days of the experiment, B. leachii had a higher
conversion ef?ciency (algal mass to body mass) and grew to
a larger size. To understand the fate of cyanobacterial toxins
in these opisthobranchs, Capper et al.213 described the different
mechanisms for toxin allocation and excretion in Stylocheilus
striatus, Bursatella leachii and Diniatys dentifer. S. striatus accu-
mulated more of both lyngbyatoxin A and debromoaplysiatoxin
in its digestive gland than in its head and foot. B. leachii only
accumulated lyngbyatoxin A in its digestive gland and trace
amounts in its head and foot. D. dentifer had trace amounts of
lyngbyatoxin A in its digestive gland and variable concentrations
of debromoaplysiatoxin in its head and foot. When the whole
animal was compared to its excretions, S. striatus accumulated
high concentrations of lyngbyatoxin A in its body (3.94 mg kg?1)
and excreted low concentrations in its ink (0.12 mg kg?1) and
its fecal matter (0.56 mg kg?1). B. leachii showed the opposite
trend with the lowest concentrations of lyngbyatoxin A in its body
(2.24 mg kg?1) and higher concentrations in its ink (5.41 mg kg?1)
and fecal matter (6.71 mg kg?1). This study illustrates that
consumers are not necessarily deterred by toxins and can have dif-
ferent strategies for accumulation and detoxi?cation of secondary
metabolites.
Recruitment of gastropod larvae is a complex process that is
often mediated by chemical cues. Larvae of the Australian blacklip
abalone Haliotis rubra settled in response to a crustose coralline
alga (Amphiroa anceps) and a green alga (Ulva australis), but not
the methanol extracts of these algae or to bio?lms associated with
the surfaces of the algae.214 In contrast, the nudibranch Phestilla
sibogae metamorphoses in response to a water soluble cue from
Porites spp. corals. Had?eld and Koehl215 found that P. sibogae
larvae stopped swimming and sank toward the bottom of a ?ume
tank when exposed to waterborne cues from Porites compressa
(obtained by soaking healthy P. compressa branches in seawater),
but resumed swimming when not exposed to the coral water. This
ability of larvae to sink in response to dissolved settlement cues
could enhance their transport to a suitable habitat for settlement
and metamorphosis. Behavioral responses to chemical cues from
prey or host species were also recently found for host speci?c
barnacles,199?201 and are important mechanisms for ?nding the right
habitats for recruitment.
This journal is ? The Royal Society of Chemistry 2006 Nat. Prod. Rep., 2006, 23, 153?180 | 173
Marine gastropods also used chemical cues to avoid predators.
The snail Tegula funebralis exhibited a ?ight response to water
soluble cues released during feeding by a crab predator.216 The
snails responded to ground up conspeci?cs and crabs actively
feeding on conspeci?cs but not to crabs feeding on other species
of snails or crabs not feeding. A study of the Antarctic limpet
Nacella concinna reported evasive behavior in response to physical
contact or crude extracts from a predatory sea star.217 N. concinna
displays evasion responses including extension of pallial tentacles,
raising its shell in a mushroom-like fashion, rotation, and ?ight
in response to the hydrophilic crude extracts from the sea star
Neosmilaster gergianus. No evidence that waterborne chemical
cues alerted the limpet to the presence of the predatory sea star
was found because evasive behaviors were only observed when
limpets made physical contact with N. gergianus.217
Inducible defenses in the form of a thicker shell have been
described for some molluscs. In a ?eld experiment where crab
populations were manipulated, multiple factors interacted to in-
?uence the shell thickness of Littorina subrotundata.210 In general,
snails collected adjacent to crab shelters (higher densities of the
predatory crab Hemigrapsus nudus) had greater shell mass, but
the results were only signi?cant for two of the six collection days.
To determine whether the presence of predators was the most
important factor, snails from the ?eld were brought into the
laboratory and raised in the presence of waterborne cues from
H. nudus feeding on L. subrotundata. Collection location and the
presence of waterborne cues from the feeding crab signi?cantly
increased L. subrotundata shell thickness.210 An induced defense
was also found in the green mussel Perna viridis in response to
crab and snail predators.218 Exposure to waterborne cues from
the portunid crab Thalamita danae and the muricid snail Thais
clavigera induced greater shell width, height and lip thickness of
Perna viridis when compared to the control. When Thais clavigera
individuals were offered a choice, they ate less of the mussels from
the snail treatment than from the control group. Thalamita danae
ate the same number of mussels from each treatment, but there
was an increase in the shell breaking time of Perna viridis from the
crab treatment compared to the control treatment.
The release of tryptophan plays a key role in gamete fertilization
for the red abalone Haliotis rufescens.219 In a well designed series
of experiments, sperm attraction to the egg was lost when the
treatment was ?ooded with exogenous tryptophan and with the
addition of the enzyme tryptophanase. Sperm swam faster towards
the egg at distances of 50 and 100 lm but not >150 lm from the
egg, probably related to a concentration gradient of tryptophan
released from the egg. This research shows that sperm chemotaxis
may be an important mechanism for fertilization success of mass
spawning benthic organisms.
11 Polychaetes
There are few reports investigating the chemical defenses of
marine worms. Chemical defenses of invertebrates from deep-
sea hydrothermal vents or cold-seep communities have not been
studied. In a survey of the palatability of deep-sea invertebrates
against shallow water consumers, the sharp setae from the
polychaete Archinome rosacea deterred feeding by the shore crab
Pachygrapsus crassipes.220 Crude extracts from parts of three out
of ten polychaetes and two bivalves deterred feeding by different
consumers including P. crassipes, the lesser blue crab Callinectes
similes, and the ?sh Fundulus heteroclitus and Leiostomus xanthu-
rus. The presence of chemoautotrophic bacteria may contribute to
the production of chemical defenses in deep-sea invertebrates. This
study demonstrates that organisms from extreme environments
may produce chemical defenses, but further studies need to be
conducted with predators from deep-sea hydrothermal vents or
cold-seep communities.
The recruitment of the polychaete Hydroides elegans is relatively
well studied because it is a common fouling organism. Under-
standing the positive cues for settlement has increased along with
our understanding of marine bio?lms. Individual diatoms isolated
from marine bio?lms induced high settlement rates of H. elegans
larvae, but their associated bacteria did not affect settlement.221
Inductive strains comprised seven different diatom genera. Two
individual diatom strains, Achnanthes sp. and Nitzschia constricta
were density dependent inducers of H. elegans settlement at the
same rates as natural marine bio?lms.222 Further work showed
that the >100 kDa fractions of the extracellular polymers from
Achnanthes sp. and Nitzschia constricta induced signi?cant larval
settlement.223 Another study of the speci?c H. elegans larval
metamorphosis cue examined a suite of amino acids.224 Nine
amino acids were toxic to the larvae and eight induced abnormal
metamorphosis. Three other amino acids (asparagine, aspartic
acid and glutamic acid) induced signi?cant rates of normal
metamorphosis but never at the same rate as the positive bio?lm
control.
Chemical cues as pheromones are well known in terrestrial
organisms and were recently implicated in reproductive isolation
for the polychaete Neanthes acuminata.225 Initial experiments
showed that exposure of male N. acuminata to females from
another population initiated higher levels of aggressive behavior.
Only when exposed to females from their own populations did the
males show little aggressive behavior. Further experimentation
showed that conditioned seawater from all populations caused
similar aggressive behavior as did the live worms, suggesting
that waterborne compound(s) were responsible for the observed
behavior.
12 Echinoderms
Echinoderms are reported to produce chemical defenses against
predators although recent studies are rare. The juveniles and
embryos from two brooding sea stars and an isopod deterred
feeding by the sea star Odontaster validus.226 Unpalatable organic
extracts from embryos of the sea star Lyasterias perrieri and
juveniles of the isopod Glyptonotus antarcticus provide evidence
that they are chemically defended.
Echinoderm natural products can have antifouling activity as
illustrated by two studies that used spores from the brown alga
Hincksia irregularis as a model system for testing antifouling
potential. The aqueous extracts of the sea stars Astropecten artic-
ulatus and Luidia clathrata and the brittle star Astrocyclus caecilia
had little effect on spore settlement, but all extracts inhibited
short and long term germination when tested at below natural
concentrations.227 Extracts from all three of these echinoderms
also changed the direction and speed of the swimming behavior of
H. irregularis spores.228 These studies showed that these extracts
can inhibit settlement of H. irregularis, but their ecological activity
174 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
in the ?eld against a suite of fouling organisms remains to be
con?rmed.
Few settlement cues for echinoderms have been isolated and
characterized. Previous research on the urchin Holopneustes
purpurascens had attributed settlement to an isethionic acid and
?oridoside complex.229 Through bioassay-guided fractionation
and the use of cation exchange chromatography the settlement cue
was isolated and identi?ed as histamine.105 Synthetic histamine
induced similar rates of urchin settlement as isolated natural
histamine. Histamine is found in especially high concentrations
(although quite variable) in the alga Delisea pulchra onto which
H. purpurascens recruits in the ?eld.
Chemical cues serve as alarm cues for some sea urchins.
In y-maze experiments, juvenile Strongylocentrotus franciscanus
urchins were attracted to adult conspeci?cs when the predator
Pycnopodia helianthoides was upstream of the adult.230 The only
signi?cant choice (other than the positive control of kelp) for
the juveniles was when the adult was downstream of the P.
helianthoides, suggesting that the adults produce a secondary
chemical cue in response to primary cues emitted by the predator,
since there was no signi?cant response when the adults were
upstream of the predator. This treatment also caused the juveniles
to orient upstream more often but did not change their crawling
speed. The authors consider this an alarm response, suggesting
that the juveniles were better protected when they were adjacent
to adults, a distribution pattern observed in the ?eld.
Water soluble feeding cues were implicated in another y-maze
experiment, which tested the chemotactic response of Pycnopodia
helianthoides to its prey the butter clam Saxidomus giganteus.231
Even though there was little reaction to whole live clams when
compared to a shell control, this sea star detected and reached
damaged clams faster than live clams in a y-maze. In ?eld
experiments with damaged S. giganteus suspended above the
benthos, P. helianthoides only approached its prey when it was
down current of a damaged clam. When P. helianthoides was
placed up current of the damaged S. giganteus, it did not approach
the prey; the response was similar to the control of empty clam
shells, which did not induce movement. In a ?eld manipulation
that directed sea stars through a corridor, 13 of 15 sea stars passed
over live clams to reach damaged clams. In a treatment of damaged
clams 14 of 15 sea stars reached the damaged clams, and in the live
clam treatment no sea stars reached the live clams.231 The study
showed that waterborne cues emitted by damaged prey changed
the foraging behavior of a key predator.
Water soluble cues may function in competitive interactions
by inducing a negative effect on the feeding rates of sea stars.232
In laboratory experiments the feeding rates of the sea stars
Leptasterias polaris and Asterias vulgaris increased when presented
with conspeci?cs and were reduced in the presence of the other
species, although feeding rates were also found to be temperature
dependent. The feeding rate of both sea stars was reduced in the
presence of water soluble cues from the other species feeding. In
the ?eld, combinations of sea stars were placed in 2 m diameter
circular plots with a 0.2 m2 bed of mussels. L. polaris reduced
its feeding and had a higher proportion of sea stars leave the
plot when introduced with A. vulgaris than when alone, however
it took seven days for this trend to develop. A. vulgaris did
not change its feeding behavior when introduced alone or with
L. polaris.
13 Other invertebrates
A recent study looked at the waterborne cues involved in settlement
of the actinotroch larva of Phoronis pallida.233 Adult P. pallida
are consistently found embedded in the burrow walls of the
mud shrimp Upogebia pugettensis. Seawater conditioned with U.
pugettensis, burrow walls, and Upogebia gut tissue modi?ed the
behavior of P. pallida larvae by increasing their swimming velocity
and benthic probing behavior. The U. pugettensis conditioned
seawater treated with arginase and lipase eliminated P. pallida
probing behavior by the larvae.
Chemical defenses were found in the Antarctic brachiopod
Liothyrella uva.234 Tissues and crude extracts from the exposed
pedicle of Antarctic brachiopod Liothyrella uva were unpalatable
to the sea star Odontaster validus and the ?sh Notothenia coriiceps.
In addition, crude extracts of the lophophore, intestine, and
stomach were effective growth inhibitors of several strains of
Antarctic bacteria suggesting that L. uva produces multiple
chemical defenses against predators and pathogens.
14 Vertebrates
14.1 Fish
Six species of coral dwelling gobies from the genus Gobiodon
were found to contain toxins in their excreted mucus.235 Each
goby species elicited a different response in a loss of equilibrium
bioassay with the planktivorous cardinal ?sh Apogon fragilis.
The wrasse Thalassoma lunare ate less of the food with skin
secretions of Gobiodon okinawae added. The rockcod Cephalopho-
lis cyanostigma also ate fewer pieces of food when whole G.
erythrospilus was concealed within the food. The bioassays
used ecologically relevant ?sh predators, but further chemical
characterization is necessary to determine what compounds are
deterrent and whether the different species of Gobiodon have
the same toxic compounds. Many marine natural products have
multiple ecological roles, and the skin toxins of Gobiodon spp.
may inhibit parasitism by gnathiid isopods.236 Even though just as
many parasites attached to three Gobiodon spp. as the control ?sh
Paragobiodon xanthosomus, the parasites preferentially settled on
the body of P. xanthosomus, but only on the ?ns of Gobiodon spp.
Since there are no toxin glands in the ?ns of Gobiodon spp. the
authors attributed the differential distribution of the parasites to
the skin toxin of Gobiodon spp., however, they did not test alternate
explanations for the observed patterns.
Chemical cues are used by ?sh to locate food. For example, a
recent study compared the responses of three ?sh species, Paci?c
halibut, walleye pollock and sable?sh, to squid extract (squid
mantle soaked in water).237 All three species exhibited increased
movement in response to squid extract. Sable?sh were the most
responsive to squid extract, with Paci?c halibut being the least
sensitive, although to some extent this is a re?ection of different
swimming behavior measured for each ?sh species. The size of the
?sh was found to be important as larger sable?sh were more active
than smaller sable?sh, however this could be a re?ection of the
different behavioral measures of swimming activity versus turning
rates.
Chemical cues function as ?sh alarm signals, and a recent
example is the coral goby Asterropteryx semipunctatus, which
This journal is ? The Royal Society of Chemistry 2006 Nat. Prod. Rep., 2006, 23, 153?180 | 175
modi?ed its swimming and feeding behavior when exposed to
conspeci?c skin extracts.238 In an interesting learning experiment,
?sh were exposed to the conspeci?c alarm cue along with a neutral
cue (extract from the damsel ?sh Acanthochromis polyacanthus),
and after three days the ?sh responded the same to the neutral
cue as to one exposure to the alarm cue, showing that the ?sh had
learned to associate an alarm signal with the previously neutral
cue. Studies of ?sh deterrence and chemical signaling remain
limited by a poor understanding of the natural product chemistry
responsible for the observed ?sh behavior.
Chemical cues affect many facets of ?sh ecology at different
life history stages including larval recruitment. A physiological
experiment using Pomacentrus nagasakiensis showed that larval
and post-settlement juvenile ?sh have a neurobiological response
to sound and olfactory conspeci?c cues.239 In a broad survey of 18
reef ?sh species, 13 species used conspeci?c cues to settle, 10 species
used visual cues, 10 species used chemical cues, and three species
used mechanical cues such as sounds or vibrations.240 In a more
in depth study, Lecchini et al.241 used the coral reef ?sh Chromis
viridis to determine which cues drive recruitment patterns. They
found that C. viridis responded to visual and acoustic/vibratory
cues from a distance less than 75 cm but responded to olfactory
cues from less than 375 cm. A major peak was separated by HPLC
from ethyl acetate extractions of C. viridis conditioned seawater
that caused 83% of the larvae to swim towards the compound(s)
in a compartment within an aquarium.
Adult ?sh also use chemical cues to home into their habitat.
In a series of experiments on the black rock?sh Sebastes inermis,
olfactory cues were more important for homing behavior than
visual cues.242 In experiments where the ?sh were blinded, a total
of six of seven blind and six of seven control ?sh returned to their
territories. The time taken to return was not different between the
blind and control ?sh. Only two out of six ?sh with their olfactory
pits blocked (with petroleum jelly) returned to their home site, and
the time required to return was longer.242 Contrary to this, pelagic
?sh did not appear to use olfactory cues to ?nd ?sh aggregation
devices (FADs) in Australia, and instead are thought to use sound
or vibrations to cue into FADs.243
14.2 Birds
Recent research has illustrated the importance of chemical cues
in marine birds. Feathers were collected from male and female
crested auklets, Aethia cristatella, and through chemical extraction
and GC-MS analysis were found to contain twenty volatile
compounds.244 The concentration of nine of these compounds was
variable between summer months (breeding season) and winter
months. When tested for attraction quality in a modi?ed y-maze,
the whole feather and a combination of cis-4-decenal and octanal
caused signi?cant attraction, while controls of amyl acetate and
mammalian musk did not cause any attraction. The volatile
compounds of the crested auklet may serve as a cue for sexual
selection, and they may also improve bird ?tness by serving an anti-
parasite role. Octanal and hexanal, which are known from insects
as potent invertebrate repellents, were also found in odors from
the crested auklet.245 Octanal and decanal from Aethia cristatella
feathers were found to repel tick ectoparasties.246 In a concentra-
tion gradient of crested auklet odorant in ethanol, duration of
tick attachment was signi?cantly reduced compared to an ethanol
control at 100% and 10%, but not at 1% of natural concentration.
Ticks exposed to octanal became moribund in less than 1 hour
and none recovered. Lice also became moribund within seconds of
exposure to octanal and (Z)-4-decenal.246 However when directly
tested against lice, auklet feathers did not reduce survival, and
in the ?eld, crested auklets had signi?cantly more lice than the
least auklet A. pusilla.247 The synthetic analogues of A. cristatella
compounds were extremely deterrent compared to the ethanol
control when tested against mosquitoes; however, the experiments
were not ecologically relevant as they measured the number of
mosquito landings on a human hand with the compounds on it.248
15 Conclusions
As the discipline of marine chemical ecology grows we are gaining
a better understanding of the diversity and range of ecological
functions of secondary metabolites. The fact that natural products
in?uence fundamental ecological processes such as predator?
prey interactions, trophic cascades, competition, prey capture,
reproduction and larval recruitment has become well established
in ecological theory. Recent advances in molecular identi?cation
of microbes are revealing their importance as symbionts as
well as pathogens and their in?uence on ecological processes.
Molecular techniques have revealed that some symbiotic microbes
are responsible for the production of secondary metabolites that
serve fundamental roles for their hosts.
The discipline of marine chemical ecology continues to be
limited by a poor understanding of the processes that control
chemical diversity and variation. Even though concentrations of
natural products can be highly variable, our understanding of the
physical and biological (especially genetic heritability) factors that
in?uence secondary metabolite production remains limited. The
phenotypic and genetic variability in the physiology of consumers
to overcome chemical defenses is another important aspect of
chemical ecology that remains poorly researched. While we were
able to expand the scope of this review to include more taxonomic
groups including microbes, worms, echinoderms, crustaceans, and
?sh, many of the secondary metabolites that function as chemical
cues for reproductive and behavioral processes for these groups
remain unidenti?ed. As researchers develop better techniques for
bioassay-guided fractionation and chemical characterization of
small amounts of compounds we expect more natural products
involved in chemical signaling to be characterized. Basic research
such as identifying and characterizing waterborne compounds is
advancing as analytical chemistry techniques improve.
Perhaps some of the most exciting and novel recent advances
in marine natural products are in isolating and describing biosyn-
thetic genes that are responsible for producing secondary metabo-
lites. These techniques can be applied to ecological questions
about localization of compound production within organisms and
environmental regulation of natural product biosynthesis. These
studies have the potential to answer fundamental evolutionary
and ecological questions such as which organisms in complex
symbioses are producing secondary metabolites and the effects
of genetic mutations on the structure and evolution of novel
compounds. Advances in other ?elds such as genomics, proteomics
and metabolomics249 also have potential applications in chemical
ecology.
176 | Nat. Prod. Rep., 2006, 23, 153?180 This journal is ? The Royal Society of Chemistry 2006
Interdisciplinary approaches are key to further advances in
marine chemical ecology. Continued collaborations between
microbiologists, molecular biologists, physiologists, biochemists,
cellular biologists, natural product chemists and ecologists will
allow the ?eld of chemical ecology to mature into a discipline that
is capable of describing many of the basic processes that explain
patterns of population, behavioral and community ecology.
16 Acknowledgements
We would like to thank all of the authors who provided us
with their reprints and papers that were in press. Special thanks
also to Commissioning Editor Marcel Jaspars, Greg Cronin and
Clifford Ross for their helpful comments that improved the clarity
of this manuscript. Recent research in our laboratory on algal
chemical defenses has been supported by the Smithsonian Marine
Science Network, and our work on settlement cues for marine
invertebrate larvae has been supported by NSF (OCE 9911867
and OCE 0317707). M. P. P. was supported by the Florida Center
of Excellence in Biomedical and Marine Biotechnology. This is
contribution #635 of the Smithsonian Marine Station at Fort
Pierce and P200603 from the Florida Center of Excellence in
Biomedical and Marine Biotechnology.
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