Marine Chemical Ecology - Chapter 15 pot

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure: UV-Absorbing Mycosporine-Like Amino Acids and Scytonemin Deneb Karentz CONTENTS I.. Chemical Defenses of Marine

Chemical Defenses of Marine Organisms Against Solar

Radiation Exposure:

UV-Absorbing Mycosporine-Like Amino Acids and Scytonemin

Deneb Karentz

CONTENTS

I Introduction 482

A Ultraviolet Radiation and the Solar Spectrum 482

B Ultraviolet Radiation in Marine Environments 483

C Biological Consequences of Ultraviolet Exposure 484

1 Absorption of UV by Organic Molecules 484

2 Photo-Oxidative Stress 484

D Biological Defenses Against Ultraviolet Radiation 484

1 Avoidance 484

2 Sunscreening 485

3 Antioxidants 486

II UV-Absorbing Compounds in Marine Organisms 486

A Mycosporine-Like Amino Acids (MAAs) 486

1 MAA Structure 486

2 MAA Synthesis 487

3 Phylogenetic Patterns of MAA Occurrence 491

4 Geographic Distribution of MAAs 493

5 MAAs in Freshwater Taxa 498

6 Concentration of MAAs in Cells and Tissues 499

7 Distribution of MAAs Relative to Radiation Exposure (Depth and Season) 500

8 Regulation of MAA Concentrations 501

9 Effectiveness of MAAs for UV Protection 505

10 Other Functions of MAAs in Marine Organisms 506

B Scytonemin 508

1 Scytonemin Structure and Localization 508

2 Scytonemin Distribution 508

3 Regulation of Scytonemin Concentration 508

4 Effectiveness of Scytonemin for Ultraviolet Protection 509

15

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482 Marine Chemical Ecology

III Evolutionary Aspects of Ultraviolet Protection 510Acknowledgments 511References 511

I INTRODUCTION

Ultraviolet radiation (UV, 100 to 400 nm) comprises the shortest wavelengths of the solar spectrumthat reach the Earth’s surface (~290 to 400 nm) Although UV spans a very small range within thesolar spectral band, it can elicit a wide variety of biological responses and it is impossible for mostorganisms to avoid UV exposure UVB wavelengths (280 to 320 nm) are injurious to cells (e.g.,cause direct molecular damage to DNA and proteins), while longer UVA wavelengths (320 to

400 nm) can be both harmful (e.g., initiate photo-oxidative damage) and beneficial (e.g., requiredfor vitamin D synthesis and DNA repair).1–3

It is generally accepted that incident UV wavelengths and intensities were much more ardous under ancient Earth atmospheres than they are today.4,5 As a result, nearly all organismshave some capability for protection against UV exposure and for repair of UV-induced damage;many of these biological defenses are common across very diverse taxa.2,6,7 Secondary metabolitesthat absorb UV radiation and provide protection from UV damage occur in most phylogeneticgroups.8 For example, melanins in bacteria, fungi, and animals and flavanoids in plants significantlyreduce the potential damage caused by direct exposure to UV.9–11 In aquatic organisms, mycospo-rine-like amino acids (MAAs) and scytonemin are assumed to serve a complementary or analogoussunscreen function.6,12–15

haz-A U LTRAVIOLET R ADIATION AND THE S OLAR S PECTRUM

Within the electromagnetic spectrum, UV is the wavelength band between X-rays (1 to 100 nm)and visible light (400 to 700 nm) UV wavelengths are further subdivided into four categories based

on physical properties and the biological consequences of exposure.3

• Vacuum UV (100 to 200 nm): Air and water absorb this portion of the UV spectrum;therefore, these wavelengths do not penetrate past the upper reaches of the Earth’satmosphere

• UVC (far UV, 200 to 280 nm): UVC wavelengths are the most damaging to organismsbecause they are most efficiently absorbed by nucleic acids and proteins Fortunately, assunlight is attenuated through the atmosphere, the entire UVC component is absorbedand these wavelengths do not penetrate past the stratosphere Although UVC was prob-ably a biologically important component of incident solar radiation during earlier geo-logic eras when life on Earth first originated and began to evolve (without benefit of anozone layer), UVC is not ecologically relevant in present day environments However,much of what is known about UV photobiology is the result of research investigatingthe response of cells to UVC exposure from artificial sources (e.g., germicidal lampsemitting 254 nm radiation)

• UVB (middle UV, 280 to 320 nm): UVB is extremely harmful to organisms and theprimary cause of erythema (sunburning of human skin) A large proportion of the UVBbelow 300 nm is absorbed by stratospheric ozone, but the small amount that does reachthe Earth’s surface is sufficient to cause significant damage and can be lethal The primaryconsequence of ozone depletion is an increase in the bandwidth and intensity of shorterwavelengths of incident UVB

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure 483

• UVA (near UV, 320 to 400 nm): These longest wavelengths of UV can be both harmfuland beneficial UVA is implicated in many photosensitive reactions and can compoundthe damage caused by UVB Despite the negative impact of UVA on cells, these wave-lengths trigger an array of fundamental responses in organisms through the action ofcryptochromes.16 UVA wavelengths are not significantly affected by stratospheric ozone

B U LTRAVIOLET R ADIATION IN M ARINE E NVIRONMENTS

After atmospheric attenuation of UV, marine organisms have an additional environmental UV filter

of the overlying water column Although intertidal species have the greatest risk of exposure andexperience the highest doses of UV, planktonic and subtidal benthic organisms are also subject toharmful levels of UV in surface waters and at shallow depths Pure water is transparent to UVwavelengths, but dissolved substances and particulate matter present in natural waters causesignificant absorption, reflection, and diffusion of UV within the water column This results invariable absorption of wavelengths from the incident UV spectrum and wide variation in attenu-ation coefficients between different water masses (Figure 15.1).17–20 Even in the clearest waters,UVB is usually attenuated within the upper 10 m of the water column, although UVB wavelengthshave been detected up to 60 m in the very transparent waters of the Southern Ocean duringspringtime ozone depletion.18,20–23 UVA wavelengths generally penetrate to depths of approximately

50 m or more

Characterization of the underwater UV regime is not extensive, although documentation of UVpenetration to ecologically significant depths has been known since at least the 1950s.24 Theimportance of daily and seasonal variations in UV or the role of vertical mixing in the temporalvariability of exposure have not yet been comprehensively studied or evaluated but are the focus

of current research efforts

FIGURE 15.1 A In-water attenuation of four indicated UV wavelengths in coastal Antarctic waters on 16 Oct 1996 measured with a PUV-500 spectroradiometer (Biospherical Instruments, San Diego, CA) B Spectral depth range of attenuation to 10% of incident UV levels in various water masses Number labels on lines refer to reference citations Modified from Booth, C.R and Morrow, J.E., Impacts of solar UVR on aquatic microorganisms: the penetration of UV into natural waters, Photochem Photobiol., 65:254–257, 1997, Figure 3 With permission from the Amercian Society for Photobiology, Augusta, GA.

20

17 17 19

18

UVA UVB

484 Marine Chemical Ecology

C B IOLOGICAL C ONSEQUENCES OF U LTRAVIOLET E XPOSURE

1 Absorption of UV by Organic Molecules

UV (especially UVB) is absorbed by two major groups of organic molecules — nucleic acids andproteins Absorption of UV photons causes these molecules to undergo conformational changes thatsubsequently interfere with or destroy their ability to participate in vital metabolic functions Theformation of UV photoproducts in DNA can significantly compromise the accuracy of transcriptionand replication; therefore, UV damage to DNA molecules by UV exposure can result in debilitating,mutagenic, and lethal effects.3 Damage to proteins is also problematic as these molecules function

as enzymes, hormones, and structural components of cells Generally, protein damage is not sidered as important as damage to DNA.25 Protein molecules are present in numerous copies andare readily degraded and resynthesized; however, prevention of UV-induced damage to defray theenergetic costs of repair and replacement of molecules is certainly advantageous

con-2 Photo-Oxidative Stress

In addition to the direct absorption as a biological hazard, UV can have additional indirect effects

on organisms.26,27 A number of UV photochemical reactions occur in solutions, both within cellsand in the external aquatic environment In the presence of UV, water itself is hydrolyzed, producinghydroxyl ions Related reactions involving dissolved substances and mediated by UV lead to theformation of peroxides, super oxide, and other radicals These reactive products are toxic by causingoxidative damage to biological molecules.28–31

D B IOLOGICAL D EFENSES AGAINST U LTRAVIOLET R ADIATION

In response to the ubiquitous presence of UV, most organisms have developed a variety of defenses

to tolerate exposure.2 These include adaptations to minimize UV exposure and to repair UV-induceddamage when protective measures are not adequate.2,6 Characteristics that are useful for evading

UV damage include avoidance, screening of UV, and antioxidant activity.6 The focus of this chapter

is on specific chemical defenses used by marine organisms to reduce the amount of UV radiationthat reaches vital molecular targets, but a very brief overview of other UV defense strategies ispresented below

1 Avoidance

Physically moving away from UV radiation is one of the most effective means of minimizingexposure This can be accomplished in a number of ways and does not necessarily require movinggreat distances However, it does require that an organism have the ability to detect the presence

of UV wavelengths (directly or indirectly) and that the organism is capable of movement UV canalter the behavior (motility and photo-orientation) of unicellular organisms and metazoans.32–34

Generally, organisms tend to avoid high light intensities, and many of the photoreceptors sible for light responses detect UVA and visible, not the more harmful, UVB radiation.16 Interrestrial plants, there are many examples of blue light receptors and numerous genes that areregulated by blue light exposure.35 In some cases (e.g., chalcone synthesis), the roles of variouswavelengths from the UV and visible spectra can be clearly distinguished on both a genetic andbiochemical basis

respon-While receptors for visible light are very common in marine species, less is known about thedetection of UV In aquatic invertebrates, UV photoreception is primarily in the UVA wavelengths,although variable UVB phototropic responses have been observed.36–38 Visual UV photosensitivity

is a specific characteristic in some animal species, including arthropods, reptiles, fish, birds, and

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure 485

mammals.39–41 It is presumed that the potential for ocular damage caused by UV is offset by thebenefits of UV vision (e.g., increased visual acuity for capture of prey).41,42

Obligate phototrophs have a dilemma with regard to solar exposure UVA and/or visiblewavelengths (also referred to as photosynthetically available radiation, PAR, 350 to 700 nm43) arerequired for photosynthesis, but exposure to UV can be harmful Benthic autotrophs (e.g., algalmacrophytes and seagrasses) cannot relocate once spores or seeds have germinated Phytoplanktonicspecies have more options for avoiding UV through passive transport by vertical mixing or activevertical migration behavior

Some biological processes are on a diurnal cycle and may be entrained to a circadian rhythm.When events are scheduled at night, this provides an opportunity for UV avoidance For example,dinoflagellates tend to undergo mitosis and cytokinesis during the dark, thereby avoiding UVexposure during a vulnerable period of the cell cycle A common temporal avoidance strategy ininvertebrates is spawning after sunset.44 In the ascidian Corella inflata, not only are embryosshielded within the adult body cavity during development, but release and settlement of competentlarvae are nocturnal events.45

Usually, intensities of UV and visible light co-vary so that an avoidance response to one includesavoiding the other However, under ozone depletion, incident UVB intensities increase whileUVA/visible light fields remain unchanged Therefore, if organisms are relying on visible or UVAwavelengths to provide accurate proportional cues for changes in UVB, ozone depletion can exert

an undue and unexpected selective pressure on populations and communities

2 Sunscreening

The outer covering of organisms can provide substantial protection against UV exposure At thecellular level, cell walls and membranes offer protection by blocking or attenuating incident UVbefore it reaches organelles and other intracellular components For example, it is estimated thatthe outer silicate wall of diatoms can absorb up to 30% of incident UVB radiation, affording asignificant primary UV defense for the cell.46 For metazoans, cuticles, carapaces, shells, scales,feathers, and fur all provide an effective optical barrier between incident UV and internal tissues.These external surfaces can absorb more than 95% of incident UVB, providing an effective radiationshield for internal cells and tissues.47,48 The environment can also provide passive shading from

UV In benthic cyanobacterial communities, the extracellular presence of ferric chloride in sedimentsfunctions as an adequate UV filter for cells.49

Across diverse taxonomic groups of marine organisms there are several classes of compoundsthat absorb UV and act as putative sunscreens These include scytonemin (Figure 15.9), an extra-cellular cyanobacterial sheath pigment, and the mycosporine-like amino acids (MAAs,Figures 15.3–15.6) that are usually located intracellularly in cyanobacteria, algae, invertebrates,and fish These compounds are the major focus of this chapter and will be discussed in detail below.Many marine species also possess the tyrosinase-mediated pathway to synthesize theUV-absorbing pigment melanin Melanin occurs in a wide range of taxa including bacteria, fungi,invertebrates, and chordates While much is known about the role of melanin in the UV protection

of mammalian skin, very little research has been conducted to examine the efficiency of melanin

as a UV-protective mechanism in aquatic taxa.9 It is known that melanin levels in juvenile merhead sharks, Sphyrna lewini, are directly correlated to solar UV exposure; in the freshwatercrustacean Daphnia pulex, melanin concentrations are genetically determined within populationsand are correlated to UV sensitivity.50,51 The few studies that have been undertaken suggest thatmelanin has an important role in UV protection in aquatic environments

ham-Several other UV-absorbing compounds have been implicated in the protection of aquaticorganisms from UV exposure These compounds do not seem to be as common as MAAs(Figures 15.3–15.6) or scytonemin (Figure 15.9), possibly because less effort has been made to9064_ch15/fm Page 485 Tuesday, April 24, 2001 5:26 AM

486 Marine Chemical Ecology

study them One such molecule is biopterin glucoside (BG), a UVA-absorbing compound isolatedfrom cyanobacteria and related to pteridine pigments.52 The synthesis of BG in Oscillatoria sp isinduced by exposure to UVA wavelengths, and the presence of BG has been shown to conferincreased UVA resistance to cells.53,54 However, in intertidal cyanobacterial mat communities, pterinconcentrations remain unchanged across seasons, suggesting a minor role in UV protection.55

Further studies suggest that pterins may have a regulatory role in UV protection In Chlorogloeopsis

sp., pterins appear to function as UVB receptors and may be involved with signaling the induction

of MAA synthesis.56 Further research on these compounds is required to better understand theircontribution to cell survival under solar stress

Other compounds such as phlorotannins, sporopollenin, coumarins, tridentatols, polyphenolics,and several as yet unidentified substances (e.g., P380) have also been implicated as UV protectantsthat can increase UV tolerance.57–63 With the rapidly accelerating rate of research in the area ofaquatic UV photobiology, it is highly likely that additional new types of UV-screening compoundswill continue to be discovered Many of these secondary metabolites probably have multiple protectivefunctions For example, tridentatols serve as allelopathic agents, antioxidants, and sunscreens.57,58,64

3 Antioxidants

Cells have substantial chemical defenses against the UV photoproducts produced in seawater andintracellular fluids Many organisms have antioxidants (e.g., carotenoids, ascorbate, tocopherols,anthocyanins, and tridentatols) that quench photo-oxidative reactions.64–67 Cells also have enzymes(e.g., catalase and superoxide dismutase) that can counteract the oxidative nature of peroxides andother radicals.26 Some compounds, such as the UV-absorbing pigment melanin, can act as bothoptical filter and antioxidant.68 The MAA mycosporine-glycine (Figure 15.3) functions in a similardual capacity.69 The role of UV-mediated reactions in seawater relative to biological effects is animportant current area of study

II UV-ABSORBING COMPOUNDS IN MARINE ORGANISMS

A M YCOSPORINE -L IKE A MINO A CIDS (MAA S )

In the late 1960s, studies of water extracts from marine cyanobacteria and cnidarians detectedunknown UV-absorbing compounds that were initially named for their maximum wavelengths ofabsorbance, e.g., substance-320 (S-320) exhibited maximum absorbance at 320 nm.70,71 Relativelyhigh concentrations of S-320 and related compounds were observed in a variety of marine organ-isms It was speculated that these compounds either have a solar protective function or are precursors

to common pigments During the subsequent decade, the widespread and taxonomically diversedistribution of S-320 and similar UV-absorbing compounds in marine organisms was confirmed.72–78

Additional investigations strengthened the notion that these compounds serve as sunscreens.73,79–81

With the subsequent elucidation of molecular structures (Figures 15.3–15.6), the S compounds wereidentified as mycosporine-like amino acids (MAAs).80,82–87

1 MAA Structure

MAAs found in aquatic organisms are closely related to fungal mycosporines that were first isolatedfrom sporulating mycelia.88–91 12 for a detailed comparison of MAA andmycosporine structure.) MAAs are colorless water-soluble compounds with absorption maxima(309 to 360 nm) within the UVB and UVA (Table 15.1).92 They are derivatives of aminocyclohex-enone or aminocyclohexenimine rings (Figure 15.2).93 Nineteen known MAA compounds resultfrom N-substitutions of different amino acid moieties to the cyclohexenone or cyclohexeniminechromophore There are only two aminocyclohexenone-derived MAAs from marine organisms:

(See Bandaranayake

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure 487

mycosporine-glycine and mycosporine-taurine (Figure 15.3).12 These are most similar in structure

to the fungal mycosporines and the only two known MAAs with maximum absorbance in the UVB

The remaining MAAs are iminomycosporines and absorb maximally at UVA wavelengths The

majority of iminomycosporines contain glycine (12 MAAs, including shinorine, porphyra-334,

palythine, asterina-330, and palythinol), and the other five iminomycosporines have serine or

threonine substitutions (Figures 15.4 and 15.5).94,95 Two MAAs isolated from reef-building corals

are sulfate esters (palythine:threonine-sulfate and palythine:serine-sulfate).96 It is most likely that

more types of MAAs and related compounds occur in marine organisms With increasing interest

in these molecules and further investigation, identification of additional forms is expected

Related UV-absorbing compounds found in marine organisms are gadusol

(1,4,5-trihydroxy-5-hydroxymethyl-2-methoxycyclohex-1-en-3-one) and deoxy-gadusol (Figure 15.6).97–99 Gadusol

is a colorless oil first observed in the eggs of fish and sea urchins.97–99 It is a derivative of cyclohexane

and is structurally similar to both the fungal mycosporines and MAAs An isomer of gadusol

(spinulosin quinol-hydrate) is synthesized in fungi and arises from an acetate precursor.97 Other

fungal mycosporines are synthesized via products of the shikimate pathway, and it is speculated

that the shikimate pathway is also a plausible synthetic route for gadusol.97

2 MAA Synthesis

Metabolic pathways for MAA synthesis, conversion, and degradation have not yet been elucidated

Based on structural affinity with gadusol and fungal mycosporines, MAAs are most likely

syn-thesized through the shikimate pathway.74,97 This is confirmed for the production of MAAs by the

zooxanthellae of the coral Stylophora pistillata and is probably true for other organisms as well.100

The shikimate pathway is present in a variety of taxa including bacteria, fungi, algae, and plants

The products of this pathway are involved in the synthesis of the aromatic amino acids tyrosine,

phenylalanine, and tryptophan There is no complementary or analogous pathway for the synthesis

of aromatic compounds in animals.101 Organisms that do not have the shikimate pathway have an

obligate requirement for ingestion of the aromatic amino acids phenylalanine and tryptophan

FIGURE 15.2 Aminocyclohexenone and aminocyclohexenimine ring structures.

FIGURE 15.3 Molecular structures and wavelengths of maximum absorbance ( λ max ) for two

aminomycospo-rines from marine organisms.

O

NHR OCH3

OH HO

NHR OCH3

OH HO

NH

aminocyclohexenone aminocyclohexenimine

OCH3

NH COOH HO

HO

OCH3

NH HO

HO

mycosporine-glycine λmax = 310 nm

mycosporine-taurine λmax = 309 nm

SO3H 9064_ch15/fm Page 487 Tuesday, April 24, 2001 5:26 AM

Marine Chemical Ecology

Chemical Def

Mytilus galloprovincialis74

Mytilus galloprovincialis74

Note: Abbreviations used in subsequent tables are shown (Abb.) along with chemical formulae, molecular weights (mw), wavelengths of maximum absorbance (λmax,

molecular weight.)

© 2001 by CRC Press LLC

490 Marine Chemical Ecology

(phenylalanine can be converted into tyrosine by animal metabolism) The dietary requirement

for aromatic amino acids probably precludes de novo synthesis of gadusol or MAAs in

inverte-brates and chordates.101 It has been proposed that ingested MAAs could serve as precursors forconversion to gadusol and can be interconverted into different MAAs by animal metabolism orenteric bacteria.14,101–103

If MAAs are synthesized via the shikimate pathway, then in the marine environment they would

be produced by bacteria and primary producers (algae) and transferred by ingestion/assimilation

to consumer organisms It has been demonstrated that diet can regulate MAA content in invertebratesand fish.104–106 The first direct evidence of this was obtained from controlled feeding experiments

with the temperate sea urchin Strongylocentrotus droebachiensis.104 Furthermore, the assimilation

of MAAs from food sources can be very efficient In medaka fish, the MAA concentrations inocular tissues increase by nearly 800% over a 5-month period by providing fish with a MAA-enriched diet.106

MAA acquisition by ingestion/assimilation and not de novo synthesis in consumer organisms

is further supported by observed gradients of MAAs along the digestive tract of sea urchins In

Strongylocentrotus droebachiensis, the posterior portion of the gut has over three times the

concentration of MAAs as the anterior portion of the digestive tract, indicating sequential tion of ingested material.104 Similar observations have been made in holothuroid species.102

absorp-FIGURE 15.4 Molecular structures and wavelengths of maximum absorbance ( λ max ) for 12 ing iminomycosporines from marine organisms.

glycine-contain-N OCH3NH COOH HO

HO HO

N OCH3NH COOH HO

HO

COOH

H3C COOH

N OCH3NH COOH HO

HO COOH

N OCH3NH COOH HO

HO

COOH HOOC

N OCH3NH COOH HO

HO

H3C

N OCH3NH COOH HO

HO

HO

CH3

N OCH3NH COOH HO

HO HO

N

H3C

OCH3NH COOH HO

HO COOH

NH OCH3NH COOH HO

HO

N

H3C

OCH3NH COOH HO

HO

HO

glutamic acid:glycine λmax = 330 nm

mycosporine-shinorine λmax = 334 nm

porphyra-334 λmax = 334 nm

mycosporine-2 glycine λmax = 331 nm

mycosprine-glycine:valine λmax = 335 nm

asterina-330 λmax = 330 nm

usujirene λmax = 357 nm

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure 491

Degradation by invertebrate digestive enzymes cannot be excluded as an alternative explanationfor the observed intestinal gradients, although this is unlikely since MAAs are not susceptible tochemical digestion by mammalian digestive enzymes (virtually all of the shinorine eaten by miceappears in their feces).106

3 Phylogenetic Patterns of MAA Occurrence

Nineteen MAA compounds have been reported from 382 species of marine organisms collectedfrom tropical, temperate and polar latitudes (Tables 15.1, 15.2, and 15.3) Approximately 75% ofthe data available on MAA presence/absence and quantification in individual taxa come from onlyone or several specimens or cultures While a complete evaluation of intraspecific variation,geographical distribution or phylogenetic trends is not possible, a number of generalizations aboutMAA occurrence are evident

FIGURE 15.5 Molecular structures and wavelengths of maximum absorbance ( λ max ) for five nonglycine containing iminomycosporines from marine organisms.

FIGURE 15.6 Molecular structures of gadusol and deoxy-gadusol with wavelengths of maximum bance ( λ max ).

absor- methylamine:serine λmax = 325 nm

methylamine:threonine λmax = 330 nm

mycosporine-palythine-serine λmax = 320 nm

palythine-serine suflate λmax = 321 nm palythine-threonine sulfateλmax = 321 nm

NH OCH3HO

HO3SO

H3C NH COOH OH

NH OCH3HO

HO NH

COOH OH

NH OCH3HO

HO3SO NH

COOH OH

N OCH3HO

HO NH

COOH OH

H3C

N OCH3HO

HO NH

COOH OH

HO OH

deoxy-gadusol

λmax = 268 nm (pH<2)

λmax = 294 nm (pH>7)

492 Marine Chemical Ecology

The majority of marine cyanobacteria, algae, invertebrates, ascidians, and fish probably containMAAs, as 87% of marine taxa examined have detectable levels (Table 15.2) Palythine (seeFigure 15.4) is the most common MAA found in marine organisms (61% of all species analyzed),followed by shinorine (57%), mycosporine-glycine (52%), porphyra-334 (48%), asterina-330(35%), palythinol (29%), and palythene (23%) (Figure 15.7) Chordata (68 species examined, nobirds or mammals included), Cnidaria (59 species), and Rhodophyta (49 species) have been moreextensively studied than other groups One hundred percent of cnidarians, 96% of chordates, and82% of rhodophytes analyzed contain MAAs (Table 15.2) MAAs have not been observed in theChaetognatha, Ctenophora, or Protozoa, but these phyla are, thus far, only represented by one ortwo specimens from one geographic location (Antarctica).107 In all other phyla and divisions, 57

to 100% of species examined contain MAAs

Most species have a complement of at least several MAA compounds (Figure 15.8A) eight percent of all species examined contain at least three MAAs; 36% of algal species, 74% ofinvertebrate taxa, and 62% of chordates have a suite of three or more MAAs The maximum reported

Fifty-number of MAAs in a single individual is ten in the coral Stylophora pistillata.100 Two species

have been reported with eight MAAs in individual specimens: the Antarctic krill, Euphausia

superba, and the Antarctic amphipod, Pontogeneia sp.107 Since wavelengths of maximum bance of MAAs span a 55-nm range across the UVB and UVA, the presence of multiple MAAs

absor-is assumed to provide a broader band and more effective optical filter than the presence of oneMAA alone (Figure 15.8B).108,109

In comparing algal divisions (total of 143 species examined, including cyanobacteria), tebrate phyla (171 species), and the chordates (68 species), there are distinct differences in theoccurrence of MAAs (Table 15.2) Palythine, shinorine, porphyra-334, and mycosporine-glycineare the four most abundant MAAs within each group, but their rankings are not consistent Shinorine

inver-is the most common MAA in algae (found in 59% of species), palythine inver-is the most frequent MAA

in invertebrates (occurs in 80% of species), and asterina-330 is the predominant MAA in chordates(present in 81% of species) (Figure 15.7)

There are sufficient data on MAA distribution from three algal divisions (Chlorophyta, greenalgae; Phaeophyta, brown algae; and Rhodophyta, red algae) to allow for a phylogenetic compar-ison of MAA content The Rhodophyta have the highest levels of MAAs, particularly in shallowbenthic environments.107,110,111 Nearly the same proportion of rhodophyte species posses MAAs(82%) as found in the Chlorophyta (81%), but concentrations of MAAs in red algae are an order

of magnitude larger (<1300 nmol mg–1 protein) than maximum concentrations found in phytes (<240 nmol mg–1 protein) Only one examined chlorophyte species, Halimeda opuntia, has

chloro-a high concentrchloro-ation of MAAs (885 nmol MAAs mg–1 protein).112 Many chlorophtes have other

types of UV-absorbing compounds (e.g., sporopollenin and coumarins).60,113,114 In the macrophyte Dasycladus vermicularis, colored substances are excreted during UV stress and release is correlated

with light intensity.63 The exudate does not contain known MAAs, and the colored substances aremost likely coumarins

A smaller percentage of the Phaeophyta (59% of taxa) contain MAAs and concentrations inthalli are relatively low (<300 nmol mg–1 protein) The Phaeophyta also excrete colored compoundswith UV-absorbing properties.62 Some of these exudates are polyphenolic substances that are usually

associated with alleopathy However, in Ascophyllum nodosum, thallus concentrations are regulated

by UV exposure and small herbivores are not deterred, rather they feed preferentially on irradiatedalgae.59 Since the Chlorophyta and Phaeophyta successfully inhabit intertidal and shallow subtidalareas, they have apparently evolved very efficient protective and repair mechanisms for dealingwith UV exposure, but MAAs are probably not the key to their fitness in high light environments

A general survey of UV-absorbing characteristics in 152 species (206 strains) of microalgaeshows that all taxa absorb within the UVA (between 320 and 340 nm, with most maxima near

337 nm), but only 11% absorb in the UVB.115 While there is a large range of variation among

strains, the highest ratios of UV attenuation to chlorophyll a concentration (UV:chl) are in

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure 493

bloom-forming flagellate taxa In this study, MAAs were analyzed in only five species, and avariety of MAA combinations (0–4 of five identified MAAs) occur From these results, theauthors conclude that UV:chl values greater than 1 are indicative of the presence of MAAs inunicellular algae

There are varied patterns in MAA occurrence within phylogenetically related invertebrates andfish In a wide geographic study (including temperate and tropical regions), similar complements

of UV-absorbing compounds occur in the eye lenses of fish at the family level Concentration isnot a taxonomic feature, but is related to the radiation regime of the habitat.116 On a smallergeographic scale, little taxonomic diversity is evident in ocular tissues of 52 species (19 families)

of fishes and sharks from the Great Barrier Reef.117 These taxa generally contain the same fourMAAs: palythine, asterina-330, palythinol, and palythene Sea cucumbers (12 species examined)from the Great Barrier Reef have three MAAs in common with local fishes (no palythene), and anadditional four MAAs (shinorine, porphyra-334, mycosporine-2 glycine, and mycosporine-glycine)are consistently present in various tissues.102 Corals (23 species) from the South Pacific (FrenchPolynesia) typically contain palythine, shinorine, porphyra-334, mycosporine-glycine, and palythi-nol.118 Four species of the temperate sea anemone Anthopleura have been found to have the same

complement of MAAs (shinorine, porphyra-334, mycosporine-2 glycine, and mycosporine-taurine),although proportions of each compound vary among taxa.119–121 Mycosporine-taurine has only been

reported from these four species of Anthopleura and may be unique to this genus A more detailed

assessment of phylogenetic patterns of MAA occurrence will require more extensive data sets

4 Geographic Distribution of MAAs

Several surveys have been undertaken to evaluate possible habitat or behavioral patterns in MAAdistribution and content, but only a few general trends in MAA occurrence are evident.107,110,112,117

Analyses of 382 species reveal that 95% of tropical species, 80% of temperate species and 82% ofpolar species have detectable levels of MAAs (Table 15.3) Palythine, shinorine, porphyra-334,mycosporine-glycine, asterina-330, palythinol, palythene, and mycosporine-2-glycine have beenfound at all latitudes Other MAAs are less frequently reported and have more limited distributions;however, this may be a function of insufficient data and not a true representation of MAA occurrence.Tropical marine organisms are not only more likely to have MAAs, but also to have the highestMAA concentrations.110 Among 25 of the highest reported MAA values, all but one sample (the

temperate red macrophyte, Cystoclonium purpureum112) are from tropical areas (Table 15.4) Whilehigh values for MAA concentrations are in keeping with a photoprotective function given thattropical environments experience the highest radiation exposures, not all tropical species have highMAA content.103,117,122

Forty-eight invertebrate species examined from the Antarctic Peninsula most often have variouscombinations of mycosporine-glycine, shinorine, porphyra-334, palythine and mycosporine-glycine:valine.107 Thirty-eight species of invertebrates from farther south (McMurdo Sound) have

a similar (but less concentrated) suite of MAAs with palythine the most common and, at times,the only MAA present.123 While many of the species are found at both locations, there are distinctdifferences between the benthic habitats of the Antarctic Peninsula and McMurdo Sound In theSound, benthic organisms are restricted to depths deeper than 20 m because of more intense seasonalice scour, and this results in lower UV exposure for these organisms Coastal ice cover also causeslower PAR intensities, resulting in lower macroalgal biomass, so most of the local invertebratesare not herbivores Since MAAs must be ingested by animals, this would further contribute to lowerMAA contents Antarctic diatoms (26 species examined) tend to have only shinorine and porphyra-

334 with less frequent occurrence of mycosporine-glycine.107,124–127 Antarctic benthic macroalgae(eight species studied) have a larger complement of MAAs (mycosporine-glycine, shinorine, por-phyra-334, palythine, asterina-330, palythinol, and palythene), and these occur in much higherconcentrations than observed in the local microalgae.107

Marine Chemical Ecology

Note: Total number of taxa examined for each group (#T), number (#) and percent (%) of species containing a specific MAA (see Table 15.1 for key to MAA acronyms), and number and percentage of species that

contain any MAAs ( Σ) are indicated Where multiple samples of a species are available and show variable presence of specific MAAs, observations were pooled to represent the maximum total number of MAAs

that can occur in a given taxon.

© 2001 by CRC Press LLC

Marine Chemical Ecology

Note: Total number of species examined (#T), number (#) and percent (%) of species containing a specific MAA (see Table 15.1 for key to MAA acronyms), and number and percent of

© 2001 by CRC Press LLC

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure 497

An MAA with a possible restricted geographic occurrence is mycosporine-glycine:valine ThisMAA was first described from a survey of Antarctic species and was initially observed only inconsumer organisms (invertebrates and fish), not any of the sampled primary producers.107 Subse-

quent studies in the Antarctic have identified this MAA in the diatom Fragilariopsis cylindrus, the haptophyte Phaeocystis sp., and mixed assemblages of Southern Ocean phytoplankton.124,126,128

There have been a few tentative identifications of mycosporine-glycine:valine in organisms from

FIGURE 15.7 Phylogenetic distribution of MAAs by percent occurrence in individual species within algal (this category includes photosynthetic prokaryotic taxa indicated in Table 15.2 ), invertebrate, and chordate groups (see Table 15.2 for reference citations and groups included in these categories; ∑ = percent values for all taxa and all MAAs combined).

FIGURE 15.8 A Number of MAAs occurring in individual species within algal, invertebrate, and chordate

groups (see Table 15.2 for reference citations and groups included in these categories) B Broad spectrum UV screening afforded by the presence of multiple MAA compounds (1 = palythine, 2 = asterina-330, 3 = palythinol,

4 = palythene) From Dunlap, W C., Williams, D M., Chalker, B., and Banaszak, A., Biochemical

photoadap-tation in vision: UV-absorbing pigments in fish eye tissues, Comparative Biochemistry and Physiology B,

Elsevier Science, 93B(3), 1989, 604, Figure 4, reprinted with permission from Elsevier Science, Oxford, UK.

number of MAAs species-1

A

0 1 2 3 4 5 6 7 8 9 100

0.20.4

498 Marine Chemical Ecology

outside the Antarctic; these include the temperate dinoflagellate Lingulodinium (Gonyaulax)

poly-edra and the tropical corals Porites compressa and Pocillopora spp.112,129 Further investigations will

be necessary to establish if this MAA may have a more widespread geographic distribution thanoriginally assumed

5 MAAs in Freshwater Taxa

MAAs have not been studied as extensively in freshwater as in marine species, but these compoundshave been reported in freshwater cyanobacteria, microalgae, invertebrates, and chordates(fish).106,116,130–133 There may be several MAAs unique to freshwater organisms Aqueous extracts

from the cyanobacterium Nostoc commune contain a mixture of UV-absorbing compounds with

two distinct chromophores that have maximum absorbance at 312 and 335 nm.134,135 These pounds are comprised of the usual mycosporine cyclohexenone ring structure; however, it is

com-TABLE 15.4

Twenty-Five Maxima Reported for MAA Concentrations in Marine Species

Species Phylum/Division Common Name [MAA] Notes Value

4 Pearsonothuria graeffe102 Echinodermata Sea cucumber 2100 Epidermis Single

5 Holothuria nobilis102 Echinodermata Sea cucumber 1600 Respiratory tree Single

11 Actinopyga echinites102 Echinodermata Sea cucumber 1000 Epidermis Single

13 Bohadschia argus102 Echinodermata Sea cucumber 850 Cuverian tubules Single

Note: Species name, phylum or division, common name, and concentration of total MAAs ([MAA], in nmol mg–1 protein) are indicated “Notes” describe whether samples are from a whole organism or specific tissues, and “value” indicates if data are from a single measurement, a maximum value from several samples (max), or a maximum mean value (mean) reported See text Section II.A.6 for an explanation of how rankings were determined The comparison of values from different research laboratories may be somewhat problematic as there are currently no commercial standards available for MAAs and calibration of instruments is achieved by a variety of means.

a Not from a natural collection, value from experimental radiation exposure.

b The only temperate species in this table, all others are from tropical latitudes.

Chemical Defenses of Marine Organisms Against Solar Radiation Exposure 499

conjugated to both amino acids and oligosaccharides MAAs not found in marine species also occur

in the terrestrial chlorophyte Prasiola crispa, but these MAAs have not yet been characterized.136

Since limited data are available, the actual extent of MAA habitat specificity (if any) relative togeographic region or marine/freshwater environments is not known

6 Concentration of MAAs in Cells and Tissues

Absolute concentrations of MAAs have been reported in a variety of units, most commonly as µgMAAs g–1 dry weight or nmol MAAs mg–1 protein Unfortunately, reliable comparisons of con-centrations standardized by dry weight to those expressed on a mg–1 protein basis are not possible.Assuming a mean MAA molecular weight of 280 and making a broad estimation of the meanprotein content of organisms at 50% of dry weight, more than 350 published concentrations oftotal MAA content from individual tissues and whole organisms were standardized to µg MAAs g–1

dry weight Based on these calculations more than 50 of the highest values were from measurementsoriginally made in nmol mg–1 protein The samples with the 25 highest concentrations of MAAsare listed in Table 15.4

The highest reported MAA concentration is 8852 nmol mg–1 protein in the tropical coral

Diphora strigosa.112 However, this equates to a remarkably high concentration of 2.5 mgMAAs mg–1 protein and bears further investigation Within the 25 highest reported values for MAAs,all but one is from a tropical organism, and 21 of the 25 values are from invertebrates The remainingfour species are primary producers (two Rhodophyta, one Chlorophyta, and one Cyanophytaspecies) The observation of highest concentrations in consumer organisms further supports thenotion that MAAs are bioaccumulated, as this fits the pattern expected for biological magnificationthrough sequential trophic levels However, the assimilation of MAAs is apparently restricted toinvertebrates, lower chordates, and fish (including sharks) Based on a study of mice, mammals donot have the metabolic pathways necessary to assimilate MAAs from their diet.106 (There have been

no studies of birds.)

The concentration of MAAs among individual organisms or strains of the same species can beextremely variable, even if individuals are collected at the same location and depth.118,137,138 More-over, MAA concentrations can vary across a considerable range within the tissues of a singleindividual In rhodophytes, meristematic apices are not fully pigmented (they are green rather thanred) and can have as much as eight times the MAA concentration of mature red-pigmented portions

of the thallus.111,139–142 A similar pattern of MAA distribution occurs in the tropical coral Pocillopora

damicornis, where branch tips can also have an eight-fold higher concentration of MAAs than

branch portions closer to the central part of the colony.143 (The MAA gradient observed along coralbranches is evident for palythine and palythinol, but not obvious for mycosporine-glycine.)

In more complex metazoans, MAA concentrations can vary by orders of magnitude between

various tissues For example, within one individual of the Antarctic limpet Nacella concinna, the

ovary has 5778 µg MAAs g–1 dry weight, the digestive tract has 525 µg MAAs g–1 dry weight, andthe remaining body tissues (minus shell) have 69 µg MAAs g–1 dry weight.144 A number ofinvertebrate species show a similar pattern of MAA allocation with maximum amounts of MAAsconcentrated in the female gonads.104,107,137,144–146 In cod, gadusol is primarily concentrated in matureovaries, with barely detectable levels in liver and muscle tissue and no evidence in testes.98 Little

is known or understood about how the selective partitioning of MAAs into specific cells and tissues

is achieved

Testes and sperm have very low or nondetectable levels of MAAs, even though sperm areexpected to be more vulnerable to UV damage because of their small size The high MAA content

in ovaries translates into high MAA content for eggs that is passed on to the developing embryo

In the temperate sea urchin Strongylocentrotus droebachiensis, eggs have over twice the MAA

concentration as ovary tissue.145 In tropical corals, the suite of MAAs in eggs is different fromadult tissue, and total MAA concentrations in eggs can be nearly seven times higher than in the

500 Marine Chemical Ecology

adult coral.147 In the Antarctic limpet, Nacella concinna, eggs and ovary tissue have the same MAA

complement in equally high concentrations.148

There is a significant ecological advantage to packaging MAAs into eggs, and consequentlyinto developing embryos, since these early developmental stages are often exposed to higher UVdoses than benthic adults, particularly in species with planktonic development In the tunicate

Ascidia ceratodes, MAAs are distinctly partitioned between the egg and the surrounding cells that

form the follicle and test.149 Eggs contain predominantly mycosporine-glycine and palythine, whilefollicle and test cells contain shinorine and palythine Removal of the external cell layers results

in a 60% delay in cell division under experimental UV exposures Several other studies havedemonstrated that successful development under UV exposure is directly correlated to MAAconcentrations in embryos and larvae (see also Section II.A.9).105,145,150,151

Some adult invertebrates in tropical areas have relatively high MAA concentrations in externalsurfaces Holothuroids preferentially accumulate MAAs in the epidermis, giant clams have highestconcentrations of MAAs in the outermost layers of siphonal mantle tissue (more than four timesthe concentrations in subsurface mantle layers), sea hares have high MAA levels in skin, andascidian tunics have higher MAA concentrations in the surface cells than in basal dermal lay-

than the less irradiated vertical faces.155 These topical distribution patterns reinforce the premisethat MAAs have a photoprotective function

Discrepancies between the types of MAAs found in algae (or other food sources such assediments for deposit feeders) and animals from the same region have been observed in a number

of studies.104–107,112,123,130,154,156 Even when the MAAs in primary producers and consumers are thesame, the relative concentrations often are not At present, the commonly accepted explanation forsuch differences is that intestinal microflora or animal enzymes are facilitating interconversionsamong various MAAs.14,101–103,156

There are distinct patterns of MAA distribution between algae and hosts in marine symbioticrelationships, with higher MAA concentrations in the host tissue than in the resident algal cells

Endosymbionts of the anemone Anthopleura elegantissima contain approximately 12% of the

MAAs present in the host tissue.120 (Note: isolated zooxanthellae in this study may have beencontaminated with MAAs from host tissue, as a previous study detected no MAAs in isolated or

cultured endosymbionts from Anthopleura.119,157) In the coral Acropora microphthalma, animal

tissues contain more than 90% of the MAA complement within the host–algal association.158 Inthis species, the MAA content of endosymbiotic algae is constant with depth, while MAA concen-trations in host tissues exhibit a vertical gradient with highest concentrations in shallow specimens

Cells of Prochloron sp that are endosymbiotic with the ascidian Lissoclinum patella have only

half the MAA concentration of the host.152 Zooxanthellae (Symbiodinium sp.) of the giant clam

Tridacna crocea contain no detectable MAAs, while the host tissue has appreciable amounts of

four MAAs.153

It is presumed that MAAs are synthesized by zooxanthellae and released into the host tissueenvironment where they are available for uptake and/or assimilation into host cells The endosym-bionts then rely on the host for UV protection rather than their own possession of screeningcompounds.152,153,158 It is of interest that endosymbionts are not required for MAAs to be present

in animal tissues MAAs occur in both symbiotic and aposymbiotic species of clams and insymbiotic and aposymbiotic anemones of the same species.120,121,153

7 Distribution of MAAs Relative to Radiation Exposure (Depth and Season)

In a number of species, the highest concentrations of MAAs occur in individuals inhabitingshallow waters where solar exposures are most intense or they occur during summer whenexposure to solar radiation is maximal relative to day length and sun angle.108,111,137,141,150,158–162