Ultraviolet Radiation and Coral Reefs' Hawai'i Institute of Marine Biology
HA@AU-W 94-005 C3
ULTRAVIOLET RADIATION
AND CORAL REEFS
EDITED BY:
D. GULKQ AND P. L. JOKIEL
HAWAI'I INSTITUTE OF MARINE BIOLOGY
UNIVERSITY OF HAWAI'I
UNIVERSITY OF HAWAI'I HAWAI'I INSTITUTE OF MARINE BIOLOGY
HONOLULU, HAWAI'I TECHNICAL REPORT NQ. 41 DEC. 1995
SEA GRANT PUBLICATION UNIHI-SEAGRANT-CR-95-03
TABLE OF CONTENTS
Liat of Workshop 4 Summer Program Participants.
An Introduction to the study of UV on coral reefs Paul Jokiel
Introduction: Plenary Addreai: Ultraviolet light and the origin of life David Mauzerall.
Dh no, not another workshop: A summary of previous UV workshops Michael P. Lesser 13
General overview of Inetrumentatlon, experimental methods, and attenuation of UV radiation into natural waters Michael P. Lesser .. ... ,......
Introduction to materials and methods commonly used by participants in the 1994 HIIIB Summer Program on 'UV Radiation and Coral Reefs' Dave Gulko, Michael P. Lesser, and Michael Ondrusek....,...... ,. ., . 19
An introduction to the UV environment of Kana'ohe Bay, Hawal'i Dave Gulko. ,25
Attenuation ln Kane'ohe Bay aa interpreted from PUV profilee Karen W. Patterson.
Plankton: The influence of solar UV-8 radiation on copepoda in the lagoon at Coconut island, Hawai'i kiroaki Saito and Satoru Taguchl...
A biological weighting function for phytopiankton growth inhibition Peter J. M. Peterson, Ray C. Smith, Karen W. Patterson,and Paul L. Jokiel...... 51
Physiological and biochemical effect of UV radiation on the marine phytoplanirton Nannochlorepaie ap. and Dunegefle ap. Barbara Butow and Tamar Fisher 61
Gorais: Horizontal and vertical distribution of MAAs In Hawaiian coraia: a depth profile and a survey llsa Kuffner, Michael E, Ondrusek, and Michael Lesser
UV-absorbing compounds in the coral PoNIlopora damfcorrrir. effects of light, water flow and ultraviolet radiation Paul L. Jokiel, Michael P. Lesser, and Michael E. Ondrusek. . 87
Reaponee of a Pacific stony coral to short-term exposure of ultraviolet and visible light Sarah K. Lawis...... ,. .... , ... ...... ,...,, 89
Bleaching and lipida in the Pacific coral Monfipora verrucoea Andrea G. Grottoli-Everett ....107 Uneven bleaching within a coral coiony ln responseto differing levels of solar radiation Andrea G, Grottoli-Evaritt and Ilsa Kuffner. 115
The metabolic response of Fung a ecutarla to elevated temperatures under various UV radiation regimes SophiaV. Hohlbauch. 121 Preliminaryreport on the occurrenoeof mycoaporlne-likeamino acids in the eggs of the Hawaiianscleractinian corals Morrtlpora verrucoaa and Fungus scutarfa DavidA. Kruppand JacquelineBlanck 129
Effects of ultraviolet radiation on fertllizatlon in the Hawaiian cor3I Furrfife soutarfa Dave Gulko...... 135
The effect of UV on settlement of the planula larvae of pocillopora damieornls Andrew Baker. ... 149
Zooxsnthellae: Ultravioletradiation: helpful or harmful to zooxanthellaeculture growths Scott R. Santos 165
Seaweeds: Surveyof mycosporlne-likeamino acids ln rnacrophyteaof Kane'oheBay Prnastazia T. Banaszak and Michael P. Lesser ... ,...... 1 71 Kffecta of ultraviolet radiation and nitrogen enrichment on growth In the coral reef chlorophytes DIctjroaphaeriecavernosa and D~oeptraerfa versluyaii. Scott Lamed ...... 181 phototoxlcity: phototoxlclty ln a coral reef flat community Rite Peacheyand Don Crosby...... 193
UV IL VIske: UV vision by marine animals: mainly questions Nadav Shashar . 201 polarizationvision aa a mechanismfor detection of transparentobjects Nadav.'lMarhar, Loana Arldesi, and Thomas W. Cronin .. .207
INalgne for subraeralble imaging polarirneters NadaVShrurhar, Thcmaa W. Crcnin, CeeOrgeJOhnSOn, and LmvrenoeB. Wciff...... 213
Ultraviolet imagery GeorgeLossy, Craig w. Ha~ehls1, WilliamN. McFarhnd, Ellis R. Lowe, Tom W. Cronin, and Dhne Flore. 219 Conclusions: Work!hop roundtable dlscuaelon on -Directions in Marine UV Reeearch"...,...223
Appendices: Appendix I: Couree syllabus for the 18S4 Edwin W. Pauley Summer Program ln Marine Biology - "VV Radiation on Coral Reefs"...... 229
Appendix II: Ultraviolet Radiation ln Tropical Coastal Ecoeyetema
Workshop Schedule 231
Appendix III: UV Measurement Instrumentatlon Comparison Chart...... 233
Subject index. 237 Participants:
1994 Edwift W. Peuiey Summer Program 'Ultraviolet Radiation and Coral Reefs' Hawai'i institute of Marine Biology
Workshop on Meseuremeftt of Ultraviolet Radiation Irt Tropical Coastal Ecosysterns Held at the East-WestCenter, Universityof Hawai'i, August 3rd - 5th, 1994.
A~, Loana Ms.!' Butow,Barbara Ms.! ' Department of Oceanography Lake Kinneret Limnological Lab. University of Hawai'i at Manoa POB 345 Honolulu, Hl 98744 Blat ISRAEL
Amrami,Dov Mr.! ' Chadwick-Furman, Nanette Dr.! Department of Life Sciences Interunivemity of Eilat Bar-lian University 52900 M. Steinitr Marine Laboritory Ramet~ P.O. Box 489 ISRAEL Elat ISRAEL
Baker, Andrew Mr.! ' Cronin, ~mes Dr,! Marine Biology and Fisheries DepL of Biological Sciences University of Miami Univ. of Maryland BatbmoreCtny. 4800 RickenbackerCswy 5401 Wiikens Avenue Miami, FL 33149 Baltimore, MD 21228-5398
Banaszak,Ania Ms.! ' Crosby,Donald Dr.!" Department of Biological Sci. Dept. of Env. Toxicology U.C.S.B U.C, Davis Santa Barbara, CA 93106 Davis, CA 9561M$88
Bidwell, Roy N. Dr.! Dubinsky, Zvy Dr.! Dep't. of Biological Sciences Dept. of Ufe Sciences Los Positas College Bar-lian University 52900 LIvermore, CA 94550 Ramat-Gan ISRAEL
BIanck, Jaqueline Ms.! Fiore, Diane Ms.! Hawai'i Instituteof Manna Biology Optics For Research P. O. Box 1348 P.O. Box 82 Kana'ohe, Hl 96744 Cakfwell, NJ 07006 Rsher,Tamer Ms.! Kinzie,Bob Dr.!" Dept. of Ufe Sciences HawalffInstitute of MarineBiology Bar-lian University 52900 Uniwasiiy of Hawal'I Ramat~ P.O. Box 1346 ISRAEL Kana'ohe, HI 96744
Gitelson,Anatoly Dr.! Krupp,David Dr.! " J.Baustein institute for Desert Research Hawal1I~ of MarineBiology Ben GurionUniversity of the Negev Universityof Hawai'i Sade Boker Campus 84993 P.O. Box 1346 ISRAEL Kana'ohe, Hl 96744
Grottol4Everett,Andrea Ms.! ' Kuffner,Ilsa B. Ms,! ' Defertment of Biology Hawai'iInstitute of MarineBiolog University of Houston Ureversityof Hawai'i 8282 CambridgeN904 P.O. Box 1348 House, TX 77054 Kana'ohe, Hl 96744
Gutko,David Mr.!' Lamed,Scott Mr.! ' Hawai'IInsltuie of MarineBiology Dept. of Zoology Unlvenity of Hawait Un~ of Hawaii P.O. Box 1346 Honolulu, HI.96822 Kana'ohe, Hl 96744
Hawqehyn,Cnig Dr.! Lesser,Michael Dr.!" Universityof Victoria Dept. of Zoology P.O. Box 1700 Spalding Building Vlciorta, BC VSW 2Y2 Universityof New Hampshire GIANT Durham, NH 03824
Hohlbauch,Sophia Ms.! ' Lewis, Sarah Ms.! Universityof Calff,Santa Barbar instituteof Ecology 130Anoqul Rd. Universityof Georgia Santa Barbara, CA 93108 . P.O. Box 2829 Athens, GA 30602-2202
Iluz,David Mr.!' Loew, Ellis Dr.! Departmentof Uts Sciervxe Dept. of Physiology Zxcv Bar-ffanUrlrersity 5290 Coffege of Vet. Medicine Ran ~ Cornell University ISRAEL ~ Naw York 14653
Jokiel,Paul Dr.! Losey,George Dr.! Hawai'iinstffute of MarineBiohgy Havvw1 Inshtuie of Manna Bnlogy Universityof Hawai1 University of Hawai'i P.O. Box 1346 P.O. Box 1346 Kana'ohe, HI 96744 Kane'ohe, Hl 96744 Mauzersll,David Dr.! Santos,Scott Mr.! ' The Rockefeller University Dept.of Zoology 1230 York Avenue Box 293 Universityof Hawai'i New York, New York 10021 2538 The Mall Honoluiu, Hl 96822
Morrow,John H. Dr.! Shashar.Nadav Mr.! BiospherfcalInstruments, Inc. Dept.of BiologicalSciences 5340 RileySt. BaltimoreCounty Campus San Diego, CA 92110-2621 Baltimore, Maryland 21228-5398
McFarland,William Dr.! Stambler, Noga Dr.! Director,Philip K. WrigleyMarine Science Alfred-Wegener-Institute Center at Catalina Inst. for Polar & Marine Research P.O. Box 398 Postfach 120161, Avalon, Calif. 90704 Columbusstrase, D-285D Bremerhaven, GERMANY
Ondiusek,Michael Mr.!' Suttie,Curtis Dr.! Dept.of Oceanography Universityof Texasat Austin MarineScience Bldg. 607 Marine Science Insbtu Universityof Hawsi'iat Manoa P.O. Box 1267 Honolulu, Hawai'i 96822 Port ArantuLs,TX 78373-1267
Patterson,Karen Ms,! Taguchi, Satoru Dr.! Dept.of Geography Chief,Biologicaf Ocn. Univsrsityof California Nat. Fish. Research InsL Santa Bartiara, CA 93106 Katsura4oi 116, Kushiro,E-Mail: Representing Ray Smith's Iab! Hokkaido 085 JAPAN
Peachey,Rita Ms.!' Yakobi, Yosef Dr.! Dept.of Zoology The YigalAIIon Kinneret Universityof Hawai'i LimnologicalLatxiratory 2538 The Mali Lake KinneretLimnological Lab. Honolulu, Hl 96822 POB 345, Tiberias 14102 ISRAEL
Reeks-Kudla,Marjorie Dr.! Dept.of Zoology Also registered as a student in the 1994 Universityof Maryland PauleySummer Program on Ultraviolet CofiedgePark, MD 20742 Radiationand Coral Reefs; held at the Hawai'iInstitute of MarineBiology June- August,1994. Saito,Hiroaki Mr.! Hokkaido Nat. Fish. Research Served as an instructor for the 1994 Katsura-koi 116, Kushiro PauleySummer Program on Ultraviolet Hohkaich 085 Radiationand Coral Reefs; held at the JAPAN Hawai'iinstituteof Marine Biology June- August, 1994. Ultrevlorereedierien enrr Carel Reefe. 1885, D, Gulknd P, L. Jokielleds,i. HIMS Tech. Reprrrt 441. UHIHI-See Grent48-lr543.
Introduction
Paul L. Jokiel HeerM'IIneiikrie ar Marineekrlogy P. O. Be 1348 Kene'ohe,HI 86744
It has beenmany years since solar ultraviolet radiation was dearly identmedas an important ecologicalfactor on coralreefs Jokiel,1980!, so it seemedtlrrely to organizea majorrnulti- disciplinary project designed to evaluate the state of the art, conduct research, train new researchers in the field and evaluate techniques and methods of measurement in common use todayon coral reefs. Majarfunding for the researchand educationalfunctian was grantedby the EdwinW. PauleyFoundation for an advancedresearch and training program at the Hawai'i Inetituteaf Marine Bialagy HIMB! entitled UltraViakktRadiation and COralReefS" that waS held fromJune 15 to August2, 1994. ln addition,the Universityof HawaiiSea GrantProgram funded an internationalworkshop entNed Measurementaf UltravioletRadiation in TropicalNearshare Environments"that was heldat the East-WestCenter Universityof Hawai'i~ Honolulu, Hawai'i! fromAugust 3-5, 1994. The US-Israel Binational Science Foundation supplied additional funding for participantsfrom israeland supported the participationby Or.Dave Mauzerall, our keynote speaker for the workshop. This volumecontains much of the informationdeveloped during the 1994program. Additionalresearch papers are stillin preparationby someof the participantsand will be forthcomingin variousjournals. A veryimportant intangible outcome was the sharingof ideas, eetablIShmentOf reeearahlinks between VariauSgraupS and the fOrmulatiOnOf neWreekearah directions.One exampleof this was the designof an underwatervideo system capable of detecting visual patterns in the UV portion of the spectrum. Many fish and invertebrates have the ability to visually detect UV, so they must somehow use UV to obtain informationabout their environment. This totally new instrument will allow us to "see" as these fish and invertebrates "see in the UV-rangefor the first time. The constructionof the instrumentdesigned during the t 994program was recentlyfunded by the US NationalScience Foundation. Although the instrumentwiII be locatedat HIMB,it will be usedby an internationalgroup of scientistsincluding the workshop participants that contributed ta its design. The 1994program addressed topics from the molecularto the ecosystemlevel. Onecan simplyexamine the following"Table af Contents"to gainappreciation for the wide rangeof UV topicscovered by the participants.By the endaf the program,there was a generalconsensus on the following major points: ~ UV is an importantenvironmental factor in shallowtropical ecoeystems, inffuencing living systems at all levels of organizationfrom molecularta community. The importanceof UV shouid not be surprising, given the role of this factor in the origins and evolution of life as described in Dr. Mauzerall's plenaryaddress. ~ Althoughthe all-pervasiveinfiuence of UVcan be shownby experimentaltreatments of "UV present"vs. "UV absent", it is anothermatter to conductexperiments that evaluatethe possible importanceof futureincreases in UV resultingfrom anthrapogenicozone thinning. Major obstaclesto progressin this areaindude: 1.!lackaf dataon spectralirradiance reaching the earth'ssurface at low htitudes,2.! lackof a reliablepredkWre model that can providedata on futureinareasee in Sf:IeatralirradianCe, and 3.! taskaf biOlagiaaldata an passibleimpartanae of suchincreases. An irnrnense expenditure of researchresources will be requiredto gainthe needed information in these areas due to the technical difficulties encountered in measurementof UV,difficulties in simulatingexperlmentaI regimes of increasedUV and complexitiesin evaluatingeffects of a slightUV increaseon an ~rstem. ~ Thechief obstacle toassessrrent ofincreased UVon shallow water tropical reef communities liesin their demonstrated complexity, susceptibility andadaptability toUV. Using the terms suscepHbHity"and'adaptabiiity appears tobe a paradox.Onone hand, reef organisms are livingatthe highest ktvels ofUV found inthe oceans, andshow remarkable ability toadapt to ertiwrelyhigh levels of UV.Can these organisms readily adapt to levelsof UVthat are even higherthan presently encountered atthe surface ofthe ocean? This is probable, buthas yet to bedemonstrated. TheUV4kxking compounds inreef corals serve as a goodexample ofa mechanismaHowing corals to adapt to a widerange of UV environments atlittle metabolic cost. Onthe other hand, UV has been shown to influence most aspsicts ofcoral metabolism and impactsaHshayn In the Ne cycles cfvarfous organisms. Inaddition, interaction ofUV with other phIfelcalfactors e.g. temperature-UV synergism's! orprocesses suchas UV-phototoxicity can exacerbatetheintensity ofother environmental stretises acting on reefs, and in thesecases UV picxfucesmore damage than anticipated, Every argument asto the sensitivity ofreefs to UV dsnistgeseemingly canbe met with a counterargument thatadaptation oracclimation willreturn the reef to equilibrium. ~ Parlcfpanfsinthe 1gg4 program came tothe conclusion thatUV reses0eh mustalways be queeHon LfTERATURE CITED Jokiei,P.L. 980!. Soktr ultraviolet radiation andcoral reef epifauna. Science 207: I069107l . NraelefetRacism aadCecal Reefe. 1555. PINaayikhase - D.Nfwaerall D. Guked P.L Jokiel ede.!. HI%Is Tech. Reporl 541. UHlHI-See Genl48-9549. Ultraviolet Light end the Origin of Life David Mauzerall Rockefeller University,New York, NY 10021 Sincemost of thetatka at thissymposium will be Onthe naet'NraapeW OfUV light,I WiShtc putin a goodword for this active radiation. I will describehow it mayhave provided redox reactions for the chemicalorigin of life on the primitiveearth. I will beginby describingthe surprisinglyvoracious appetiteof livingcells for energy, We willreach the nottoo surprising conclusion that evolution REQUIREDthe useof solarenergy, and that is in facthow it occurred.The argumentfor the photochemicalorigin of lifeis lesssecure, but either lt didso or the earlycells were vastly more simplein metabolismand reproductivemechanisms than those we knowabout today. LifeiS a highlyorganized system whiCh requires a cOntinualflux of flee energytO remain in the INingstate and a muchgreater fiux of bothenergy and matterfor repmducbon,It is convfmientto expressthis fiux in unitscf powerper area,since the biosphereis a ratherthin layer on the earth,and theseunits are just thoseof photonfluxes. TableI showsthat only solar energy can supplythe energyflux required by modemorganisms. The secondlargest energy flux is thatof lightening, whichfalls to supplythe energyrequired by slowgrowing yeast by two to threeorders of magnitude. Brodagave a striidngillustration ofthis energy utilization by pointing out that a hardworking azotobacterium,fixin nitrogenin the aerobicatmosphere and duplicatingrapidly, consumes 10 bmesas muchenergy aS the Sunemits on a parweight baaiS, EVen a quieeoenthuman Sinka 105 timesthe solaroutput Uvingcells are Iittfeblack hofes for energy. TNsenergy requirement can be calculated on a globalbasis. Given a biomassof 6x10'7g carbon,the area of the earth, Sx101S Crn andthe energy requirement Of10 W gC!-1One reaCheS thesame energy flux as above, 10 W cm+.This value is quite independent ofdetails. Given a biomassturnover time of 2 - 20 yearsand the energy needed to fix a moteof 0, 500kJ mole!' one againobtains 10 to 1& W cm+.Based on the isotopic ratios of C, andalso cf S, invarious mcks, Schidlowskiet GJ. 983! arguethat the biomasshas beenconstant for some S.5 Gyr. One is forced to the conclusionthat solar energy was requiredfor evoiution. W%5therthe originOf life requiredSOlar energy is an openqusaNon. The earlycell may have beensimpler, but it was probablyalso less efficient, if thatword can be appliedto the modernpower- guzzlingceil. Ifso, much energy would be neededto assemblethe selfAuplicating organism. The alternativeis an extremelysimpis, metabolically and duplicatimly, organism of whichwe haveno knowledge. We shallreduce the problemto a yetmore simpie level: the origin of the semi-reducedorganic moleculesthat are the componentsof all livingmatter, The reasonthat this is a problemis thefailure of Urey'smodel of the primitiveatmosphere. He believedin a coldaccretion of the earth,aliowing timefor the metallic iron to reducenitrogen to ammonia and carbon dklide to methane.Miller's gnat experimentshowed that electric discharge in suchan atmospherealong with water vapor hd Io the copiousformation of aminoacids and other anthropologically desirable rnolecules. Unfortunately geophysicistsnow argue that the earth accreted rapidly enough to loseiron metal to the core very early. The primitiveatmcephere was composed of nitrogenand carbon dioxide. The fact that volcanicemissions are largelyCO2 with only traces of CH4 supportsthis argument More unfortunately,but predictably,the quantitativeexperiments of Millerand coworkers have shown that theyield of aminoacids decreases dnunatically as the rafioaf H toC inthe presumedatmosphere decreases,becoming undeteCtable with CO2 alOne.Thue we havea dilemma. Whatis neededis a sourceof reducing equivalents to make the carbon biochemically useful. It is Interestingthat of thefive stable formal valence slates of carbon: +4! +2! o! -2! t! CO, HCO2HHCHO CHGOHCH4 TabbI. EnegyFtumi RequhmarN and &e45m. ENERGY FI.UXES Wcm ~ ACTIVE BACTERIA -10 ~ YEAST -10-5 SOLAR, VISIBLE X +10 UGHTNING +10 - 10+ ENERGY REQUIREMENTS Wg1 AZOTOBACTERIA -10 QUIET HUMAN -'IO~ SUN +10 BROOA,1975! LIVINGCELLS ARE BLACK HOLES FOR ENERGY ENERGY AND EVOLUTION BIOMASS 6X10 79C EARTH'S AREA 5X10'6~~ so10-' gCcm2 IF10+ W gC1 104g Ccm+ BIOMASS CONSTANT BYCARBON, SULFUR ISOTOPES, 3.5Gyr SCHISM.OWSKIE7AL, 1983! SOLARENERGY REQUIRED FOR EVOLUTION biologyoperates atthe zeroth orformaii~ide level. This is also by far the most reactive ofthe simplechemical forms ofcarbon. The selt-zero valence level of carbon; graphite, diamond or amotgAouscarbon, is chemicallyinert. Enterphotochemistry. A most common form of photochemicai reaction isthat of electron ~. Theseelementary re~ reduceand oxicfize the reactants and will even drive reactions thsimod!msmicallyuphill,sloilng some ofthe energy ofthe absorbed photon. The epitome of thesersaclians is photosynthesisItself. Ultravioletphotochemistry isparticularly relevant since even the simpkiet ofmolecuies absorb su%&rrlyshort wavelength UV.More to the point UV was available onthe primitive earth because ofths absence ofoxygen and thus of ozone. Table IIshows theavailable energy onthe primitive earlh.There srs one lo two orders ofmatfnitude more energy ineven the extreme UV <200nm! thanIn the other rin-photonic eneigy sources. The energy ofsuch photons, 8 eV, is sufncient to breakchemicel bonds and oxidize orreduce any knovm chemical compound, Infact the abundance theatmosphere isa very active fiekf and the elaborate ixsnputer models ofthese complex systems havebeen ex1erv~ tomodels ofprimitive atmospheres. Thepho~eis of CO2 and H~ wiNform somemay rain out. pinto et el 980! haveeslimated that some 10" meiesHCHO per yearcould be formed even al the Present level of COz. Unear extraf:iolation tothe estimated level of TableII, A~ Energyon the Primae Earth. Source Energy mW cm 2! Energyyield nntol J 1! Far UV<200nm 0.004 1xya UV 200 - 300 nrn 0.4 2xp Near UV 300- 400 nm 9 3x fr Visible 400 - 800 nm ex ft Electric discharge 0.0006 10 Radioactivity 0.0001 ? Volcanoes 0.00001 ? Shock waves, meteors, etc. 0.00001 100 arsstsr.Date from Mf lier and Orgei 974!. a ftis the qusttsm yield of the phOteteeOIIOn: ntetesof fwoc4aSBnetsfns ofphotons ~. Theenergy yiefd is wavsfsngth deperr~ E = hr= hort.!for photoohsmiosf ~ in the primitiveatmosphere,1 atm, wouldyield a fixationrate about a tenth that of modern phOtOSynthesis,101S molesC peryear. Howeverthis eatirnate seema to ignOrethe photolysis of HCHOitself which would be extremelyefficient ghren Its absorptktn down to 300 nm wherethe photonflux is 103greater than that required for the photolysis of HEOand CO2. Thus the quoted yieldsshould be decreasedby thisfactor. A criticmay argue that more complex rnoiecules will absorb at longerwavelengths, encounter a largerflux of photonsand so be destroyed,While this is true forsome molecules, many others such as polyenesand heterocydicaromatic are resistantto radiation.The photoresislanceof such molecules can be attributedto two different causes. For loose, fhppy molacules, the excitation energyis dissipatedas heat in picosecondtimes. For motterigid, aromatic molecules the excited stateslive longenough to emit lowerenergy radiation, fluorescence. These are alsothe photochemicafiyactive molecules. Thus these photostabile molecuies would enjoy a formof selectionand accumulate. Since they absorb stronglyIn the UV they would screen the less stablemoleculesfrom this radiation. The photoactivemolecules would contribute to the desired chemistry. In Table III we showthe compoundsmaking up the presentand a possibleprimiNve atmosphere andocean together with their light absorpfions. The atmosphericcomponents are limitedto simple molefxfles andto absorptionin thevery far UV. We shallconcentrate on the componentsin the ocean. We sae thatseveral components of interestto biologysuch as sulphateand phosphateions are protectedby the strongabsorption by waterbeyond 185 nm. In fact,the UV absorptionby water attsnuatesrapidly 0 meters!afi wavelengths shorter than 300 nfn see below!, Howeverthe rapid mbdngof the topwater layer of about100 m guaranteesexposure of the ionsto theseintermediate wavelengths.The nitrateand ferrous ions will be particulaftyvulnerabkt to photolysisand are discussedbelow. Figure1 showsthe photonflux availabkr to the primitiveocean, The cumulative molesof photonsor Einsteinsper cm per year are plotledversus the maximumwavelength of cumulation.This is a usefulway to approximatethe absorbedphoton flux by a givenmolecule. To be exact one should convolute the photon flux with the molecular absorptionas a function of wavelength.However since afi mofecules continue to absorbat shorterwavelengths, give or take a Ntfie,and sincethe photonflux is a rapidlydecreasing function with decreasing wavelength, the simpleproduct of thecufnulated photon flux and the absorptionof the moleculeat thatwavelength wiNgive a good approximationto the absorbed flux. The rateof nitrateion formation ffcm the fluxof oxidesof nitrogenproduced in the primitive atmctspherehas been gu!es5matsdto be 10 moiesper year. The nitratein theocean will be photofysedby the UV light. For a sourcelimited photosystem, and lrtdudingattenuafion by the water column, the steady state concentrationof the photolyta is: wheres is the source rate, a isthe attenuation ofwater P cm' at350 nm!, g isthe quantum yield of thephotolysis 0.1!, s isthe extinction ofnitrate ion 20 M' cm'! andfo is the cumulativesurface photonffux at this wavelength Fig. 1! times the cross section of theearth 0's cmz!. Thesteady state ccecentrationof nitrateIon is calculatedto be 10 M, Assuming the same source rates, the calculatedconcentra5ons of nitrate and sulfite iona are similarand are givenin the table. The value quotedforferrous ionis that estimated withoutpotolysis, Usingthe ferrous ionflux estimated from theHamrnersley ~ ion formation also 10 molesper year, see below!,its steady state wouldbe 2x1& M. Althoughthe steady state values are small, they represent an enormousflux of pholochemicallyformed reactive intermediates, in fact the total of the ion source rate. Thereare two importantaspecls of photochemistryin solutionthat we mustconsider. One is the cageaffect and the other is the encounteror diffusionlimited rate of reaction. Reacbonsof simplemolecules in the gas phaseoften require a thirdbody to carryoff the excess energyof reacbonllbenated by sayforming a newchemical bond. The cageeffect is causedby the surplusof thirdbodies in the condensedphase. The energycan dissipateon the femtosecondtime scalebut the molecular products require almost nanoseconds to changeplaces with solventmolecules, i.e.tc escapethe solventcage. Thussome fraction will be iostto recombination.The effectof this factoris containedin the quantumyield of the specified reaction. FQI a first order or unimolecular reaction that is all there is to it. But for a second order or birnolecular reaction,there is the rateof theirmeefing. The fastestrate possible for reactionoccurs when the eactantsreact at theirfirst encounter. For spherically symmetric molecules reacting at the sumof their hydrodIimamicradii, the secondorder rate constant depends only on the temperatureand the viscosity ofthe solvent, ~T/3000h or2x10 M s ' forwater at 25 C Fig.2!, Sincean excited state lives at most for a fsw nanosetxmds,efficient reaction requires &.1 M reactant, Since only unreactive Na+CI fitsthis criterion, other paths are required.There are threecommon possibilities: 1! aggregationor comI:Ames,inwhich the proximity of the reactantaon excitationallows rapid to femtosecond!reaction; 2! solvent,in which the electronis ejectedand trapped in the solvent;and 3! quantummechanical spin, in whichthe spin of the electronsunpair say from a singlet state, no unpaired electrons, to the triplet statewith two unpaired electrons. The return to the original state can be slowed by a millionfold. The resuIngmillisecond Netirne allows quantitative reaclion with substances at the micrornotarlevel. Examplesare given in Fg, 2. It is knownthat the quantumyield of solvatedelectrons from iodide and bromideanions are raapecfivaly, 0.2 54 nm!and 0.5 29 nm! at pH 7 Adamsonand Reischauer, 1975!. The commonproduct of reeWons of type 2 See Fig. 2! is the solvated electron, a very powerfulreductant. The remainingradical from the photoreactive anion is a powerful oxidant. These neednot simply recombine since they are formedat -10 M. Anyorganic molecule at a higher concentrafionwould preferentially react with these highlyactive species. Thus a widerange of redox reactionsare possiblewith these speciale. Thephotochemisby of the modernocean is completelydetermined by the highconcentration of oxygen,3x10 M. Reacbonwith the solvated electrons or tripletstates occurs in k2 x concentration of Oz! 100nsand pnxkces the strongoxidant superoxide ion or the reactivesingle stateof oxygen.These oxidize the reduceddissolved organic matter producing the prevalent'yellow stuff". Thastudy of this enormously active photosyatem has onlybegun recently.Unfortunately none of this workis directly rel~ertt to the anaerobicphotochemistry of the primitiveocean. In additionthe oxygenalso forms the ozone layer which depletes, or usedto deplete,those wavelengths shorter than300 nrn,Thus the modernoceanic photochemistry is attenuatedand the syntheticpossibilities areminbrxzad. However, in theprimitive ocean and atmosphere, these reactions would produce an abundanceof rnoleculesuseful for the beginningof biogenesis. Onshas now arrived at theuse of all this photoexcitation: to oxidize and reduce the biochemical proklenItors,making them more reactive and storing energy in the products. Examples ofthis scenatioare unfortunately rather rare @@diesofoceanic photochemistry under strictly anaerobic o~lons aN verylimited. Since the fernws ion was prevalent in the primitive ocean and some work hasbeen done on itaI:halochemistry, we will discuss its reactions. Thsphoto~eal formatkeof hydrogenfrom famous ion as a functionofpH is peculiar,as showni«ig 3. Thenda decreases from pH 1 topH 4, thenrebounds at pM6 andmore TableIII. COrnpoerttonof1he preeere end d a ~ PrimitiveAtmct9rhere end Ocean Atmosphericpressure P atm! Atmospheric Wavelength e component rmt! atm1 cm 1! 02 0.2 <1p-10 150 300 H2 5X107 103 110 H20 Variable Variable 170 150 N2 0.8 140 NO 10 9 NO2! 10 9 225 3 NH3 10-8 0-10 230 200 CO2 3X10+ 1 150 't5 CO 1P-7 -10 2 150 2 CH4 10-6 <1p-10 130 600 SO2 1 p-10 10 1o 290 10 H2S 1 p-10 10-10 200 150 Oceanic concentration !M} Oceanic Wavelength component Ancient rmt! Na+ 0.5 0.5 Mg + 0,05 0.05 0,5 0.5 74! Cl 2 X 10 198 er 10 3 103 1 X10 228 10-6 1P-6 1 X 10 -1 190 HCO3 2 X 10 5X10 0.03 75! SO4 10 2 3X10 -0 227 SO3 p-10! -10 1P-6 10-6 80! H2PO4 10 10-8 200 NO3 104 300 10 210 NO2 4X10 109 5X10 355 20 -0 320 F 3+ 107 colloid! 5X10 F 2+ -0 10-4 350 3 pH 5-6 Sane.' Datafrom Holland 984!, WeLer 977!, enclKeetlng er el. 999!. e TheCOnCenlreban OfCOmpenente in perentheeie are determined inthie paper tO be phctcohremioel y imfted. The maXima Of the iengeet wavelentrthabeorptixr bends and the ebezpbanindex a Ors Ofthe rXmgenenteere lated. Thewevekrngthe tn ptrenfheeee ere ~ by, eo thetaiona are ~. Rff.f ~alrrulrarrdEnds' rnoferrofeofar phoarrre! ~ up & a peel rrerrafrrngfhfrfooad on a fogarffhrrrioeo ~aferffffral fhe rru&ce and Ik f -end 'fOmehr defrfhs of fheflnffffte orararr. ffre ouloff Bt -180 rIn + a 8lrongowner ~ ~ aOaeNen e baaedon dafa of Wfffeow reel ~ 956!, Fig,2. Phxodmmistryin eolurion. solutions. Replacing the quartz cell with one of pyrex gives the explanation:the reaction in acid requires short wavelength UV, while that beyond pH 6 can make do with wavelengths >300 nm. The reactionis linear in light intensityand the minimumquantum yield, i.e. assumingall light is absorbed, is 0.3 between300 and 370 nm. The reactionin acid solutionfrom exciting the monmerichydrated ferrousion produceshydrogen atoms, which combine to givehydrogen. In contrast,the ferrous hydroxidereaction seems to be concertedoccurring from an aggregateto formhydrogen directly Fig. 4! by reductionof the intermediateby a neighboringferrous ion. The amount available is appreciable. From the Hamersley basin, well studied by Trendall 972!, one calculatesthe formationof3 x10" molesof Hp yr . Totalemission from volcanoes isestimated tobe about the samebut the Harnersleybasin occupies only 10 of the earth'ssurface; many such basins may have beenpresent at onetime, The hydrogenwoukl be usefulto reduceCO2. The chemicalorigin of IIfe needs all the help it can get. The oxidationof the bandediron formations was originallybelieved to be causedby the photosyntheticallyformed oxygen. They would have acted as a vastsink for the gas beforeit could accumulate in the atmosphere. However the productwould then be hematite, as it is in the red banks of age c2Gyr. The photochemicalreaction is stronglyinhibited by the presence of the feme ion,and thus cannot go to completion.The bandediron formations have muchmagnetite, the ferrous-femeoxide, as expectedfram this mechanism. At a particularpH, 8.5, ferroushydroxide can formhydrogen thermally and this reaction also is onlypartiall complete, Fg.3.~~ ~ rrt hydrogenframferrrrus erras a kssr5orr rHpH p faUzenS sfrrf-. f ~. Unfortunatelythere is no directreduction of bicarbonateion by ferroushydroxide either photochemicallyor thermally.The reductionis reportedto occurin acidsolution where tree COz alkalineis the reactantand short wavelength UV, 240 nm,is required.There are also reportsthat ferroushydroxide can reduceNs to ammoniain thedark at pH 8.5. These reactionsare criticalto the chemicalorigin of life and deserve further study. Thenitrite anion also absorbs weakly at 350nm, dissociating to NO' andOH' onadding a proton!.The quantumyield is 0.1, an exampleof the cage effect, The hydroxylradical can oxidizeall organicmolecules includinghydrocarbons. Reactionwith aramatics results in phenols which are readilyfurther hydroxylatsd at theortho or para positionsto formo- or p- hydroquinoneswhich are thecombined eleclron-proton carriers in biologicalsystems. The previousestimate of 10 rnoles peryear of NO fromatmospheric photochemistry would thus supplya roughlyequivalent amount of oxidant to the ocean. Fig,4. Direct~ ef hydrggenvie the ferrous ftytfraxitle teecben. Nate the driving force in this~ Fe -0-o Fe -0, hH = t5 ktstlrnol f Meuzeralietei. t993! CONCLUSION Photoreactiortsonthe primitive earth would lead to a vanetyof reactivemoiecuies that would txirttritxiteto the chemcal origin of life. Whether they would be decisive remains to be seen. A tentai}veconclusion isthat the vast amounts of compounds sufficient to fill theoceans as proposed bythe original chemca} origin of lifescenanos the "soup'! are unlikely to beavailable without the rapid,early development of photosynthesis. The next step fnxn the inorganicphotochemistry described here would be that based on organicmdecules, first absotting in the UV, then progressing into the visible, culminating in the magnificenporphyrins which opened the flood of ailphotons accessible in water to thepower of . A cnjcia}step was the development of photocydes wherein the excited pigment reacts set~ntlallywith boih donor and accepter thus regenerating the pigment for another cycle of activity iig.5!. Bythe addlthn of antennapigment molecules the systemcould make do witha tenthof the tttaX}muirtavalabie SOlar phctOn flux. An even more cruCial Step Waa the closing of thefeedback loap eothai molecules generated by the photoreactions can make themselves form more pigment and othernecessary components ofthe system. Finally inevitably?! a se}f-duplicating system capable ol aroseand the rest is history.To paraphraseLewis Thomas: There may have been elementsof tuckin the emergenceof photosyttthe4csystems, but once thesethings were on the scenetheir evolution was ordained. There was simplyno otherway tc go. TabtlIV. Fhaand Rseeek d Rahdng Equhaticehihthe Hernsndey Basin. AGE. 2.5 X 10 YEARS AREA: 150,000KM2 TIMEOF FORMATION: 106YEARS DEPTH OF SEA: 200 M DEPOSITION RATE. 0.5 MMOLE FE/CM /YR 1 X1012 MOLE FE/YR TOTALFE: 2 X10 6 MOLEFE 2/3 FE3' THUS AVERAGE CONCENTRAT!ON: 0.5 MMOLE/20L/YR = OF FE 2.5 X 105M PROFESSOR TRENDALL ESTIMATES 0.5 X 10 M SINCE ONLY 1 P%%dSEDIMENTS PRESENTTOTALATMOSPHERICCO2: 6x10 MoLEsC PRESENTTOTAL OCEANIC HCO3 !: 3 X10 MOLESC PRESENTTOTAL eiolIIASS: 5 x10'6 MmEsC THUS HAMERSLEY BASIN COULD REDUCE1/6 OF TOTAL OCEANIC CARBONATE ANDTENS OF TIMESTHE CO2 IN THE ATMOSPHERE OR THE PRESENT BIOMASS. PRESENT PHOTOSYNTHETIC PRODUCTION: 3MMOLE C/CM /YR e ! THUS HAMERSLEY PRODUC1lON COULD BE 3 X 10 OF PRESENT. Rg.5. Ev6utkmCI Phcbaernthesia. Aofskrrsercloemenfs.Much cf thispaper wes taken from D, ~, Oceansunlight and the origin of ae', in THE ENC'1rCLOPEDIAOF EARTHSYSTEM SCIENCE, VOL 3, pp445 - 453,Academic Press, 1992; and from D. Meuzerel,z. Borowskaand i. Zielinsid,'pikxo end thermal ~ of Ienoushrdrowde", Origins of Life,23:105- 114, 1993. LITERATURE CITED Adamson,A. W. &. Fleischauer,P. D 975!. "Conceptsof InorganicPhotochemistry.' Wiley, New York, Broda,E. 975!. "TheEvolution of the BIoenergeticProcess . PergarnfnonPress, Oxford. Calvert,J. G. & Pitts,J. N. Jr. 966!. 'Photochemistry.'Wiley, New York. Fox,R. F. 988!. 'Energyand the Evolutionof Life.'W. H. Freemanand Co., New York. Hoitand,H. D, 984!. 'The ChemicalEvolution of the Atmosphereand Oceans.' Princeton Univ, Press, Princeton, New Jersey. Kasting,J. F., Zagnle,K. J., Pinot,J. P., & Young,A. T. 989!, Sulfur,ultraviolet radiation, and the earlyevolution of life. Originsof Lifeand Evolutionof the Slosiphere19: 95 - 108. Mauzersll,D. 978!. Electron-transferphotoreactions of porphyrins.In: 'The Porphyrins' D. Dolphin,ed.!, Vol. 5, Part C, pp 29 - 52. AcademicPress, New York. Miller,S. L. 8. Orgel,L. E 974!. 'The origins of Life on the Earth.' Prentice-Hall, EnglewoodCliffs, New Jersey. Pinto,J. P.~ Gladstone,G. R. fL Yung, Y. L. 980!, Photochemicalproduction of formaldehydein earth's prirnIve atmosphere. Bcience 21 0: 183 - 185. SchidkenN,M. ~ Hayes,J. M,4 Kaptan,l. R. 983!. Isotopicinferences of ancientbiochemistries: carbon,sulfur, hydrogen and nitrogen. Irr. 'Earth's Earliest Biosphere, its Originand Evolution" J. W. Schopf,ed,!. PrincetonUniv. Press, Princeton,NJ. Schopf,J. W.,ed. 983!, 'Earth'sEarliest Biosphere, Its Origin and Evolution," PrincetonUniv. Press,Princeton, New Jersey, Trendall,A. F. 972!. Revolutionin earth history. J. QecI. Soc. AustarIIe19: 287 - 311. Waker,J, C. G. 977!. 'Evolutionof theAtra~here." Macmillan,New YoH<. Withrow,R. B, 8 Withrow,A. P. 956!. Generation,control and measurementof visible and near- v@bieradisntenergy. Irr. RadiationBiolo@r" A. Holktnder,ed.! McGraw-kill, New York, 3: 196. 12 uletnrloistttsttlattoo and Coral itsstl. 1QQS, D,Gulita b P. L.Jokiel 48.!. HIMB Tech. Report f41. uhilHI-3esGrant4R45-ta. Oh no, not another workshop: A summary of previous UV workshops Michael P. Lesser Dept.of Zoology8 Centerfor Marine Biology, Univsnttty ot NewHampshire, Durham, NH 03824 Thetoltowin9 was the open>ng presentation given to the delegates at the Measutementof UltravioletRadiation in Tropicat CoastalEoosystems Workshop held August 3 - 5, 1994at theEast-West Center, University of Hawari. Whywas it decidedto havea UVphotobiology workshop concerned specificaily with tropical ecosystems?Recently the focus of UV researchhas been on polar regions and principally concernedwith the Antarcticozone hole. A 60-70%total ozonedepletion occurs during the australspring. A 5-10%depletion has now been described over the Arctic as well,although that systemis a lotmore dynamic, and globaltrends are likely to be abouta 2-3%total decrease in ozoneheavily weighted at thepoles. These small incremental decreases will lead to stillhigher UV at the Equator.SpectficaHy, while equatorial regions show highly variable decadal decreases in ozone, areas within 20' N show a 12 to 14% decadal decrease in ozone. INSTR UMENTATtON What kindsof problemsdo we havewith instrumentation,both air measurementsand underwatermeasurements? Do we needfult spectral? Do we settleon wide-bandradiometers? What kindsof precisionare we lookingfor in our instruments? Do we createa seriesof primarycenters where we have very sophisticatedinstruments as in the network in and around Antartica!, and then to a secondary series of places where we are measuringboth air and underwater, and measuring perhaps with less sophistication? These measurementscould then be complementedwith radiativetransfer models of the atmosphere andwater. How do we interwatibratethese instruments7This bringsup a wholesuite of problems. BIOLOGICAL EFFECTS What are the biologicaleffects of UV radiation?Most of us here are biologistsor photobiologistsof one sort or another,so we arevery interested in that particular aspect of this workshopand I willcomment again on tropicalsystems as a uniqueiaboratory. UV radiationis, and has been an importantecological factor, Mostof the workin the poles has been stimulatedby acuteeffects, acute exposures to organismsthat over evolutionarytime have not been exposedto enhancedfluxes of UV radiation.Tropical systems have high biologicaldiversity that has been exposedto highirradiances af UV radiationfor a verylong evolutionarytime. We nowhave a veryunique opportunity to study these systems. The irradiancesof UV thatoccur in tropicalecosystems are muchhigher than anythingthat occurs in the Antarcticduring the ozonehole, So we havea whole suiteof organismsthat have evolved variousadaptations to protectthemselves from the detrimentaleffects of UV radiation.What sortsof mechanismshave they evolvedto protect,repair, and/or avoid ultraviolet radiation? OTHER WORKSHOPS What recommendationshave come from other workshops? The results of nine previous workshopshave alreadybeen summarized Weiter, 1993!. It'sa verynice documentthat Sue gaveus during another workshop Lake Lacawac, PA! when we first startedto lookat freshwater systemsIf youthink there is very littiework done on UV photobiologyin the tropicsor the Antarctic,you should took at the literatureon lakes. it is an openfield and onlya few investigatorsunderstand what is goingon in lakesystems in relationto UV photobiology,There maybe someinformation about UV radiationeffects on biogeochemicalcycling, but biological effectson freshwater organisms iscompletely anlargely unknown. please see the major recommendationsthat Sue compiledfrom those workshops. Weshould monitor UV and visible radiaticn using wavelength-s pacific equipment and establishprimary and secondary sites for the monitoring to get long-term data sets. The operathreword here kr 'monitor.' The government agencies are also saying we mustmonitor UV andvisible radiaffon with highly sophisticated instruments but,for whatever reasons, they are not forthomlngwith funds to do that. So one of the tasks here, and the task assigned toany workshop especially the ones from Antarctica! isto provide a convincing document for tropical systemsthat can be usedas evidence that we havea needin tropicalecosystems for this kind of work. Theother major recommendations include obtaining robust action spectra to assess wavelength-specNceffects, especially on phytoplankton, the primary producers in theoceans. Theaction spectra are very important formany reasons, especially for their predictive capabilities.Wecan also use action spectra for cross-comparisons oflaboratory experiments. If wehave action spectra we can compare different lamps and different filter combinations that we usein our laboratory experiments because all of usare using different systems. Usingthe Antarctic asa biologicallaboratory foracute effects of experiments af UV radiation, therewas a recommendationtotry to standardize UVsources, filters, and all otheroptical equipmentthat is being used in ktboratory-based studies and even extending over into field work as welt A majorconcern especially interms of global elemental cycling, was to look at thestudy of UVradkttkxi and biogerxhemical cycles. A lotof important UV photochemistry isgoing on in the ooeansthat may affect the global carbon cycle, for instance. We need to identify celluiar targets ofUV radiation and study the responsiveness, repair mechanisms, seasonal adjustments, acclimatizationresponse, and evolutionary responses. And the last one that almost all ofthe workshopstalked about was the study of the role of UV absorbing compounds inthe system you areworking with. In the case of marine systems we have rnycosporine-like amino acids MAAs!, buttenestrial s rrsteiirs have f levineand other kinds of compounds that presumably provide some kindof protection. But the evidence here is primarilycorrelative rather than cause and effect. So there ls a need to address that, Lastly,and again, one of the major points of those workshops was to study ecosystem effects.Recent work by ecologist Max Bothwell see editorial by Culotte, 1994! points out this needvery well, in termsof his studiesin freshwater systems. This was a casewhere he was lookingatshort-term exposures toperiphyton instreams in Canadaand found a UVresponse of decreasedbiomass and decreased photosynthesis overshort periods oftime, Serendipitously, theexperiment was feft nrnning for a longerperiod of time and what he ended up finding, and publishinginScience, was that there was a primaryeffect of UV on a veryimportant herbivore in thesystem. And that this effect was more profound than the short-term effects to the algae themselves.ItesserrtiaIIy killed the important herbivore, the 'keystone species", inthat system, sndshorrred thai in long-term exposure toUV the algal biomass inthose systems actually increasedrather than deceased. Sothese are very complicated syslams. There is a needto take a lookat the ecosystem- leveland it is very difficult todesign experiments because you have to monitor them for a very kingtime. You have to havethe right instrumentatlon, Weneed to provide a good case to funding agencies, tofund photobiology centers like they aretrying to doat Universityof Hawal'i.To fund monitoring networks, not just in theCaribbean basin,but in the Pacific as well Wehave lo convince our own government agencies that the pacifcis an important theater of study as well as the Caribbean. The Caribbean just happens to be in theirpolitical back door. LITERATURE CITED Culiota,E. 994!. UV-Beffects: Bad for insect larvae means good for algae. Science 26S: 30. Welier,C. S, 993!. UV-Beffects on aquaticorganisms and ecosystems: A summaryof recommendationsfrom a selectionof UV workshopreports. Unpublished report prepared by C. S, Weiler Oep't, of Biology,Whitman College, Walla Walla,WA 99362! for use by workshopparticipants, "The impact of UV-8 radiationon pelagicfreshwater ecosystems", Sept 13 - 18, 1993, LakeLacawac, Pennsylvania, 19 pages. UltravlotetRadiation and Cora! Recta, i!f05. D. Golkott P. L. Jokie! eda.!.H! MB Tech. Report 041. UNIHI4ea Grant-CR-95-i'. General overview of instrumentation, experimental methods, and attenuation of UV radiation in natural waters Michael P. Lesser Dept. orZOOtOQy 8 Center fOr hlarine atOtotfy, Uhhteraity of New Hampehire,Durham, NH 03824 INTRODUCTION I wouldlike to startoff the sessionwith a brief,and general, overview of the fieldinstrumentation that is availableand then lookat the differencesin spectroradiometersversus radiometers. Finally, I wouldlike to presentsome data thatwas collectedjust outsideof Kane'oheBay that showsthe variabilityin attenuationin variousnatural water types even here withinthe tropics, INSTRUMENTS & HARDWARE Someof the things we wantto talk aboutin regardsto instrumentationis air versusunderwater measurements, and the kinds of instrumentsthat are available for those particular kinds of measurements.What shouidwe be investingin, interms of money,should be the very high resolutioninstruments for air measurements. We should then be able to model the underwater light fieldand groundtruth that with underwater instruments that may not have quitethe resolution,but wouldbe goodfor monitoringand couldbe intercalibratedto a goodair measurementsystem and used with appropriate modeling. The typesof instrumentsthat we are talkingabout fall intotwo largeclass groups: radiometers and spectroradiometers,Radiometers are currentlyrepresented primarily by the InternationalUght broadband model and the BiosphericalInstruments narrow bandrnodel, Spectroradiometers, such as UCor's,have a scanningmonochrometer built into it. There are advantagesto bothof these typesof systems.Radiometers in general, likethe InternationalLight System, consist of measuringUV radiationwithin a broadwaveband sayfor instance,a UV-B instrumentthat could measurefrom 290 to 320 nm!and integratingover allof thosewavelengths in orderto give a single valuefor that particularwave band, The BiosphericalInstrument model has fourchannels on it plus a PARchannel that integratesover the 400 to 700 nm range, The advantageof someof these instrumentsis thatthey are portableand are small. The BiospherfcalInstrument model gives continuousreadings with depth and is very easy to use witha boat, Even thoughit doesn'tgive youfull spectral information, it doesgive you information at 305, 320, 340, 380 nm and PAR. The UCor spectroradiometeris a singlemonochrometer that givesyou full spectral information at the lowest resolutionof 1 nm, It works with a monochrometerand a filter wheel assembly that Sacks outall of the portionsof the spectrumother than theones beingmeasured at that time. There was somequestions about stray light affecting such unitsbut recentcomparisons between the LiCor and BiosphericalInstruments models show good agreement with each other,and reasonable agreementwith radiativetransfer models. Sometimesyou haveto be subjecbveabout the data, especiallyat the iowerend of the spectrum,as youstart getting into water that is attenuated. Occasionaliyyou get downto the 300 to 305 nm rangeof the LiCorinstrument and you start gettingnegative and positivenumbers and you knowthat the data belowa certainpoint is not appropriateso youjust have to subjectivelyseiect a cutoff where you see progressivelyincreasing positivenumbers and say thatthis is gooddata, thisis baddata. This occursfor spectracollected at progressivelydeeper depths where the shorterwaveiengths start to be completelyattenuated. We talked about biologicaland chemical dosimetersthis morning and I put them up here with instrumentationbecause of somaof the factorsthat we talkedabout this morning. Ease of depioyment,cost effectiveness,and intercalibrationneeds to be workedout withsome of the instrumentsthat we are usingin the field, I talkedagain this rnoming about the Smithsonianwhich is in the middle of developing a high resolution 12 channel instrument for measuring ambient levels of solar radiation, UV-6 in particular. Recently I was at a remote sensing meeting where a group of peopie were interested in utilizing remote sensing to do monitoringwork on coral reefs. All of the 15 peapieimelved with remote sensing satellites were telling us what we couldand couldn'I do, what waereal and nat real. Dr. Chalice Mazel fram MIT brought an instrumentalOng with himthat I found fascinatingfarsome of the work that we maybe interestedin as coralreef biologists. It was a hand heldspectroradiometer with a hardcircuited monochromeier, noi a mechanicalrnonachrometer. It wesheld in twosmall cases, one was a batlerypack and onewas all of the instrumentation.It was ableto do several things. It wasa fiberapticprobe that was hand held and just bya seriesof dffferentclicks you coukf get tull spectrum information from 275 to 850 nrn. I don'tknow what the resolutionaf that scan was. With the fiberoptic probe you could get this instrument in hardto reach places.lt alsohad the capability to measurefluorescence as well just by clicking on to another channel.Within seconds yau couldget bothspectra for thoseof us who are interestedin how prImaryproductivity and how fluorescence relates. It was builtfor a totalcost af $10,000 to $15,000.There was a recentinstrumentation 'shoot~ at LakeLacawac in Septemberof 1993 whereseveral people, including the manufacturers,brought representativesof the instrumentsI justtalked abaut. Therewere BiosphedcalInstruments models, LiCor Li-1800 underwater of thatintercompartsan worlakop was impressive Kirk et al. 1994!. It talksabout the different Instrumentsand their particular characteristics and includesa lot of spectralmeasurements, You wouldbe suprised,despite the pitfallsassociated with any one of these instruments,how close theseinstruments compared with each other under mast situations especially the Biosphericai Instrumentsand LiCor models!. Againthe consensus, interms of instrumentation,is to havea networkof primarymonitoring siteswith high resolutian full spectral capability spectral radiometers very similarto the system esbtbfishedbyNSF and Office of PolarPrograms OPP!, Thosetypes af instrumentsare vep expensive.Maybe something like the Smithsonian12 channelcould suffic as a highresolution machinefor air measurementif it fallswithin specife guidelinesthat have been addressedin anotherworkshop on UV-BRadiatian. One couldtake instrumentslike the broad bandradiometer Intercatfbratingand usethose instruments as secondarysites for terrestrialand underwater measutements.We needto considerthis since we can't afford and are notgoing to get the money!to buildenough high resolution systems to get the kindof coveragethat we want. We wilt haveto usesome kind of combinationcf the three typesof systemsmentioned above. EXPERIMENTAL METHODS In tenneof UV phatobiolagy,it is no longeradequate to just describethe lamp systemthat yauare using. The bestway to da it is to get a spectrumof that sourceand publishit alongwith yourexperiments, lt givesa chanceto compareour data withyour data. Againaction spectra play a rolein thisas well. Thepressure is goingto be put on youto providethat information more and more in the future. We hadsome goad discussion about subtraction and additionexperiments and what is approfxiatato assessthe effach of UV radiationon marineorganisms in particular.Most of usdo sut'~ifan experimentswhere we eliminateUV fromthe spectralregime and lookat a relaxation andameliarsbm af thestress response that we assumeis alreadythere. In termsof assessing whatthe effects of ozonedepletion, where we have enhancedfluxes of UV-B, it mightbe more appropnate and has beendone in terrestrialsystems! to develophigh resolutionsystems where we cancontrol incrementally, the Incseasesin UV-B irradianceimpinging on the systemthat we are Interestedin instsadol removingUV to measureif thereis somemycese!. It maybe more appropriateto develop a systemin whichwe canimpose higher irradiances of UV,even in the tropics,for experimental work. I thinkyou needan actionspectre to obtainbiologically effective doses for yoursystem. Thereis going to be evenmore pressure for people to developthere own action spectra or ta describetheir system that they are usingwith someone else's action spectra for biological dosesfor DNA, eqrtfxsna, photosynthesis, and then we willbe betterable to compare thoseresults from laboratory to laboratoryor fromffeld situation ta fieldsituation. CONCLUSION Whatwe need to considerin termsof tropicalecosystems is the following.What kindof monitoringdo we wantIo dof Whatkind of instrumentationshould we considerifwe areto establish sometype of monitoring network7 It doesn'tmatter if it isin the Caribbeanor Pacifictheater, people needto considerif it is costeffective, what will work in differentnations, what willhappen to the data, whowill calibrate it, whowill maintainit, andwhat is feasiblein termsof gettingthis kindof data on a long term basis. EXEMPLARY RESULTS Thisis a profilein Kane'oheBay Figure1! withinthe Sampan Channel, dght in the middia of the bay,within the barrier reef, Atthe surface on this profile,in termsof UV-Birradiances, we areseeing 2.89 W/m' at the surfaceand at 10 meterswe are seeingno UV-B. Sornepiacebetween 7 and 10 meterswe have tremendousattenuation. We do not see anythingbelow 328 nm at 10 meterin Kane'oheBay. Kane'oheBay is noi pristinetropical waters. It has tremendousimput of sediments, dissolvedorganics, and highchlorophyll levels, and is betterdescribed as a tropicalestuary. 1 000E-01 1.000E-02 1,0008413 1,000EO4 1.000E05300350 400 450 500 550 600 650 700 Wavelengtb nm! R9Lsa1. proSeof sfwtotfS inttdisnea ISCG - 700 nm! ai a seriesof depths in the Sampan Channsi, inside Kans'oha say, 0'ahlj, Hawai'i, If we goto a patchreef system north of the previous profile, but still withinKane'ohe Bay Figure 2!, againat 10 meterand below 10 meterswe seeno UV-B penetration. At this depth we stillsee a considerableamount of livecoral growth, Subsurface irradiance is stillat 2.8 - 2,9W m~of UV-B, There is somecontinuity in the attenuationof UV-Band someof this has been verifiedusing a BiosphericalInstruments PUV-500 see K. Pattersonthis volume! and foundthat attenuationat 308 is verycomparable Io what we are seeingusing the spectroradiometer,especially the dropbetween 5 and 10 meters. At 7.5 meters no more UV-B penetrates into these waters, Watersjust outsidethe bay Figure3! are a muchdifferent system. Here is foundmuch dearer tropical-likewater case 1 waters!,and in thissystem we are stillseeing UV-B penetration downto 18,5 meters and actuallystin seeing UV-B irradiances of way less than 0.1 W m at 21 meters!.Within a veryshort geographical scale we are seeingdramatic differences in the attenuationof UV-B within and outside the bay. I would like to reiterate the need for not only Iong termcoverage in termsof UV measurementsbut good spatial coverage in termsof whereyou are 17 doingyour experiments and what is thequestion you are asking. i thinkit is veryimportant to gst iongterm dale setson dNerentspatiai scales. 1.000&01 1.60fl3t00 1 000E01 1.000602 1,000M3 IN.1.MX&05300 350400450 500 550 600 650 700 Wavelcstgth am! RQUsS2 preae Of apeetra ~ fto- 700rmr! at a serieSof depthSOff Ot the Stiver Rect, inside Kana'Ohe Bay, 0'alW, Hawall, 1.000&01 1,0008+00 1 000841 1 000FA3 1.000EO4300350 400 450 500 550 600 650 700 Wavtlonyh sttn! Rgtae3, ProaeOf Speatnrf ~ 00- 700 rvn!ef a eerieeOf depthe Ofr Of MOtrtt frfanu ISfend, dlrerXfy OMtatde Of Kene'ohs Bay.Vahtr, HEN, TURE CiTED Kirk,J.T.O. eral. f994! MeasurementaafUVN radiatiOnin twOfreShwater lakes: An instrument intefotmparisort.E~sse der Umnolo~. 43: 7t - 99. 18 tfHrevfotatRadiation end Coral Reefs. 1905, D. Gvlko3 P, L. Jokiet etkr.!.HIMB Tech. Report «41. UNINI-Saa Grartt~-95-tt3. Introduction to materials and methods commonly used by participants in the 1994 H. I. M. 8. Summer Program on UV Radiation and Coral Reefs Dave Gulko', Michael P. Lesser, Michael Ondrusek f Hawal'iinstitute ofMarine Biology Kana'ohe Hawal'i 96744-f346 Dept.of ~, SpatdingBuilding, U~ of New HampeNre,Durham, NH C3S34 3 Dept.of Daranography, Mrefne Science Bldg, otfcy, Unnreraity cfHawat, Honolulu, Hf96822 ABSTRACT:The measurement of the enacts of ultraviolet UV! radiation on tropicalmarine organisms during lhe 1 994Havrai1 Instttu@ofMarine Bictoy/s summer program on UV~ and CoralReefs invdwd ~ uing a varietyof similarequipment and materials to measureincident uv, spacingUv, penetrationof UVthrough the trader column end effectsOf UV on theOrgariam liVing in neteeherewatere. A ComrrrehereeredeSCrfptton iegtVen Cent~ theeClufprnent usedto getherUV informarion, along with trtfttrmatfcn COncening tfte plastics used lo screenUV and the method used to erttrecrtmycrcepcrinrHike amino acids MAAs! from the organisms~. INTRODUCTION The 1994 Edwin W, Pauley Summer Program in Marine Biology held at the Hawai'i Institute of Marine Biologyfocused on the effects of ultraviolet radiation on coral reefs and associated habitats. Participantsin this program used a wide variety of methods to conduct their research. Often the same materialsand equipmentwere used to gather data about incident or spectral ultraviolet UV! radiation,penetration of UV in the water column or to manipulateUV radiation coming into contact with test organisms. What follows is a basic descriptionof the common equipmentand materials used in this research. UV RADIATION MEASUREMENT: Data measuringUV radiationwas collected concerningthree primary areas - Incident solar radiation - Spectralsolar radiation - Underwaterpenetration of radiation Incident solar radiation was measured in three different fashions: 1!. An Eppley UltravioletRadiometer Eppley Laboratoryinc Newport, R.l.! was used to measureradiation in the UV-A and UV-B range 80 - 320nm and 320 - 400nm respectively!, This rnachine used a hermetically-sealed selenium barrier-level photoelectric cell whose spectral response was limited to the wavelength interval 295 - 385 nm by an encapsulated narrow bandpass filter. Measurement values were in mifliwath per square centimeter mW crn !. This machine had been recently re-calibrated May, 1994!. 2!. Both the direct component and the diffuse component of sunlight that is, irradiance from the sun plus sky!, termed Giobal Solar Radiation, was measured through use of a LiCor LI-200SA Pyranometer LiCor, Lincoln, Nebraska!.Measurement values were in Calcm-3 min-t. Thismachine had been recently re-calibrated May, 1994!, 3!. Photosynthetically Aclive Radiation PAR!, the energy between 400-700nrn that piants use for photosynthesis, was measured using a LICor LI- 190SA Quantum Sensor LiCor, Lincoln, Nebraska!. The sensor measured the PhotosyntheticPhoton Flux Density PPFD!: the number of photons between 400 and 700 nm! that came in contact per unit time with a unit area. This machine had also been recently re-calibrated May, 1994!. Measurement values were in Itmol photons sec rn All of the above measurements were made in air, on a 24 hr basis, from the Point lab Weather Station located at the Hawai'i Institute of Marine Biology, Coconut Island Moku o Lo'e!, Kane'ohe Bay, Hawai'i. 19 Underwaterand Specter Measurements: Themajority of measufemsnts were recorded using a LiCorLI-1800UW scanning spectroradiometer UCor, Uncoln, Nebraska!. This unit measured spectral data 00-700 nm!. The cosine-correctedcollector and sensorswere programmed to scan from300- 700 nmin 2 nmintervals. The instrument was deployed using SCUBA or froma boat at predetermineddepths. All measurementsof ambient solar irradiance were made at approximatelyt 2 PM. Cafewas taken to minimizemeasurements taken duringovercast conditions.For each depth threes scans were taken and the meanreported in order to minimizeeffects of fluctuatingradiation. Measurement values were in urits of mW m nrn '. Integratedvalues of unweighted UV radiation XWOO nm! and UV-8 radiation 00- 320 nm!for eachdepth were also provided. PLASTICS: Plasticsused were generally of twotypes: thin films and thick sheets. Transmission propertkLswere as shownin figs 1-5 Pleasenote that figs, 1-5 are in 'YoTransmission, not Absofbance;all scanswere conducted ~n 290 and 800nm usinga HewlettPackard 8452ADiode Array Spectfophotorneter!, Transmission cutoffs for each plastic were deflnsdas thewavelength where 5tyL of the maximumvalue was transmitted,plastics wereused to filter solar radiation into the following three categories: Nff UVTrantyafenL UV-B+ UV-A+ PAR!: ~ PlexiglaseG UVT Acrylic sheet produced by Rohm K Haas Philadelphia,PA! 6.0 mmthickness ~ Aciaf.33c Ruoropolymer filmproduced by Allied Signal Plastics Pottsville, PA! 127 microns thickness gauge! suefor1,lhlS WSvslsogfh fssSSc. ~ OrPissfglsse G coroAeylc sheet tuVT Shssti; verbcal lee~ fho RyyfrSnSrosMsoo CLSoff 20 Rgure 2. with scan of Cttot433c 5 mil Rouropoiymerfilm UVT film!; ver5calline dence the 50% transmissioncutoff forthis plasac. UVA UV-A+ PAR!: ~ Mylafe Type D Fluoropolyrnerfilm producedby DuPont 127 rnicrons thickrtar55 mil! F~re 3 Wavelengthscan or ytaarrrr Type D 5rril Rouropohrmer ttlm UV-A atm!; vertical line ttanotss the 5tytt trarnsmtasron cutoff for this ptasttc, Rgure4, Wav¹eogtham of MyfareirType D 5 mffRouropcffvnsr Ilrrr UV-AIlfro! and plerdgfas8 G Acrylicsheet UVT rrhssgSrgefhar, verfcrrf line ~ the5 y%tnrnsmierrfcn cuarff far ihaee plaetioe, UVO fUV Opaque: PAR only! v PlexigLasS6 UF-3 Acrylic sheet produced by Polycag TechnologyCorporat on Qsmford, CO! 6,0 mm thickness ~ 100%Clear Acrylic Safety Glazing sheet produced by K-S-H,Inc. 2.5 rnm thic r5tess Rgrss5. Wavefrrngfhscan cf plerdgfaeftr G UF-35mrrr Acrylc sheet UYO sheet!; varbcrd line denolm the RrY transrrv'rr¹on roerfffor this ~ 22 FigonrS. Wavelengthscanof 100' Qeer Acrylic Safety Gazing 2.5rnm Sheet UVO thin sheetj; vertteS linedsnolee the5tp%%d transmissioncutoff for this ptestic. NEUTRAL DENSITY FILTERS: Black,plastic mesh of variousthickness were used to ~if Lfobfdecrease the amount of lightin orderto approximateambient tight levels associated with the depths that the testorganisms were gathered from, Scans with the LiCor Spectroradiofneter showed thatthese materials are truly neutral density at PARand UVwavelengths. MYCOSPORINE-LIKE AMINO ACIDS MAAS!: Theextraction and analysis of MAAswere performed according to theprocedures in Dunlapand Shick et al. 1992and Stochja et al.,1994, Samples were extracted in 5 cm HPLC grade100% methanol, Individual MAAs were separated byreverse-phase isocratic HPLC on a BrownleeRP-8 column protected with an RP-8guard, in an aqueousmobiie phase including0.1% acetic acid and 45'A methanol. Detection of peakswas by UVabeofbance at 313and 340 nrn. Identities of peakswere confirmed by co-chromatography withstandards of mycosporine-glycine,shinorine, porphyra-334, palythine, asterina-330, palythinol, and palythene.Peaks were integrated and quantification of individual MAAS was accomplished usingthe quantitative standards listed and by on-line diode array spectroscopy. All MAAs werenormalized using the soluble protein from an aliquot of theextracted sample. Protein wasanalyzed using the procedureof Lowry 951.! LITERATURE CITED Dykens,J. A Schick,J. M.,Benoit, C,, Buetlner, G. R. 8 Winston,G. W. 992!. Oxygenradical produCtiOnin the sea anemOne Anrhopleura elegantissima and its symbioticalgae. J. Exp Biol. 168: 219 - 241. Lowry,O. H., Rosebrough, N.J., Farr,A. I . 8 Randall,P. J. 951!. Proteinmeasurement with the folin phenol reagent J. Biol. Chem. 193: 265 - 275. Stochaj,W. R., Dunlap, W, C. 8.Schick, J. M. 994!. Twonew UV-absorbing fnycosporine-like amino acids from the sea anemone Anthopleura eiegartfissimaand the effects of Zooxantheilaeand speCtralirradianCe On Chemicalcompositicn and content. Mar, Biol. 118: 149- 156. 23 UtlraektletRadiation and Coral Rests. 1NS. D. Gulkeif P. l.. Jckiel sds.!.HIMS Tech. Aepert e41. IIIIIHI-Sea Grant-CR-95-03. The ultraviolet radiation environment of Kane'ohe Bay, 0'ahu Dave Guiko Hawai'i Instituteof Marine Biology P. D. 8ox 1346 Kana'Che,Hawts'I 96744 ABSTRACT:Kans'che Bay is a uniquecairneratar coral reef environment ~ off thewkedward sideof the iektnd of 0'ahu. Datafrem the Hawai'i ~ Cf MarineBiology's point Weather StaaOn were used tc kekat both amual and daily trends in UVsurface inadianca. Factors such as doud cover, air-bcurne paNkdea, and albedo funckm in modlhrfngthe arncurri of irradiancereaChing the surface of the bay, ~ variat'm inUV aurfaCe i~ peakedInttste and July and directly~ minimums~ tO the duringHawai'iwinter l~months of MarinaDeceenber Biologythnwgh ahmuatas ~!. quiddy ~mand may ofbe uvimpeokaÃtO tfvoqyh the alagewater tsderacobra by both ~ and oceanicinputs. INTRODUCTION Kane'oheBay, on the windward side of theisland of 0'ahu,consists of a large 1.5 krrP!bay, 12.7km long and 4.3 km wide Fig.1!. Thelandform surrounding the bay is characterizedbythe steeply-slopedwindward side of theKoolau mountain range, The cliff-like mountain faces, termed"the Paii", were initially formed by a massivelandslide in whicha targeportion of the kflend of 0'ahu slippedinto the sea a millionyears ago Mooreef ai., 1989!,and was subsequently moNiedby erosion into a seriesof amphitheater-headedvalleys University of Hawai'iDepartment of Geography,1983!. The Paii itself provides an abrupt orographic surface for the prevailing northeasterlytradewinds and is an importantfeature in cloudformation near the bay. The Kane'oheBay watershed is madeup of sevendiscrete drainage sub-basins encompassing an areaequivalent to 97km' Hunter& Evans,1993!. The ocean-facingside of the bay is bordered bya "barrierreef" with an extensivebackreef, while the interior of thebay contains roughly 80 patchreefs of assortedsizes. The internal coastline has a seriesof fringing reefs, rnangroves and ancientfish ponds spread along its length.Two channels provide access into and out of the bay forship traffic; the ship channel was dredged by the US Navy in 1940to a depthof 12m, whilethe undredgedSampan Channel is only2 -3 rndeep. Marine water circulation in thebay involves rnovernentof water across the back reef, and then out through either of the two channels; this resultsin the southernportion of the bay havingthe mostresttfctlve water circulaffon with the openocean. Such a situationmakes the southern portion of thebay most vulnerable to the effects of runoff from coastal areas. However the entire bay is extremelyestuarine due to restricted exchange with the open ocean. Priorto the 1950s,the coastaland inlandareas fronting Kane'ohe Bay were utilized primarily foragriculture, raising a varietyof cropssuch as taro, rice, sugarcane, and pineapple. Many open areaswere used as pasturesfor horses,cattle end goats Hunter& Evans,1993!, Distinctive south-north urbanization of the coastline and valleys surrounding Kane'ohe started in the 1960s andcontinues today. Accompanyingthis has comea varietyof impactson the bay suchas increased sedimentation, freshwater runoff, increased nutrient input, etc. Banner, 1974; Smith et el., 1981!. Sewagedischarge directly into the bayfrom Kane'oheTown wss divertedto a deep ocean ouffall in the late 1970s, Sewagefrom a treatmentplant serving the central coastal portion of thebay was divertsd in 1986 Hunter & Evans,1993!, Many coastal areas from the mid- to north bayare still on septicsystems & cesspools P. Jokiel,pers, comm.!, and maybe contributingto the bay via groundwater. Annual rainfall in Kane'ohe Bay averages 140- 240 cm/yr with a daily stream discharge estimatedto be around 214,000 m Jokiel et al., 1993; Hunter & Evans, 1993!. Occasional heavy storms result in freshwaterfloods which inundate the bay. Such catastrophicflooding is rare, occurringonce every 20 - 50years Jokielef al, 1993!. Still,when floods such as the December 1987flood occur,they often resultin massivedie-offs of benthicinvertebrates sponges, corais, zoanthids,etc.! in Kane'oheBay Jokielef al., 1993;Banner, 1968!. Largescale phytoplankton blooms often take place shorffy after such flooding events, and may be partially in response to 25 highnutrient njn-off from non-point source outlets such as streams and storm culverts, along with decompositionof deadbenthic organisms Jokiel et al., 1993!. Muchof thelagoonal 1oor is rnsdaup of softmud. Long-term studies on turbiditysuggest thatwaters of the bay,especially the southernportion, are bestdescribed as turbidwhen comparedtothose directly off-shore Smith ef al 1981! and is consistentwith the effects usually seenwith nsstrfcted circulation and urbanization influences Hunter & Evans,1993!. Even so, Kans'oheBay is oneof the richest coral reef areas in Hawaii Jokielet a!., 1994! and is thecenter for much of the research that is done on coaU and coral reefs. Biologistsattempting to understandthe importance of UVon the biota of Kane'oheBay and tropicallatitudes fn general!must have information on spectrum, intensity and modulation of UV input.This paper summarizes avaflaNe data on seasonal and daily variation in surfaceincident UV anddescribes attenuation of UVin thewater column of Kana'oheBay. is' pygmy>, Qgp gf ~~ asr, Q'aha ~ ~ af dissrerrtrs8f tgx%. 26 UV PENETRATION THROUGH THE TROPICAL ATMOSPHERE SolarUV irradiancereaching the surface of thesea is greater in thetropics than in either temperateorsub-polar regions Calkins & Thordardottir,1980!. Yoshihara 8 Ekem 977! calculatedthat the amount of total solar radiation reaching the surfacein Hawai'i was 25'/o greater thanthat reaching most areas af the US mainland. Overall,the solar zenith angle changes with both latitude and time seasonaland daily!; this angleaffects the path length that spectral irradiation takes through the atmosphere prior to striking anobject and its distribution onthe surface, Tropical areas have smaller such angles at solar noon!than temperate areas Madronich, 1993!, and this contributes tothe greater amount of UV presentat the tropicalsea surface. Radiationpassing through the tropical atmosphere isaffected by three major factors: atmosphericgasses, atmospheric particles, and cloud formations Fig. 2!. Atmosphericgasses differentlallyabsorb various wavelengths of spectral irradiance. The gasses of primary importance inthis regard are ozone, oxygen, and carbon dioxide. Ozone is theprimary absorber ofUV-B in theupper atmosphere. More UV passesthrough the tropical atmosphere because the ozone columnis thinner over the tropics than over temperate latitudes Madronich, 1993!. Particlesin theair bothabsorb and reflect radiation, causing a diffusionof theincoming irradiance. The air overmost tropical Pacific areas is characterized byvery low concentrations of particles often less than100! per cubic centimeter cc!, as compared to thatfound over temperate cities such as Los Angelesor NewYork upto 100,000particles per cc! Schaefer& Day,1981! where large densitiesof urban-derived particles result in the formation of smog.Hawai'i can have a higher particiecount than most tropical areas due to a combinationof occasional wind conditions and continuousrecent volcanic activity producing a natural version of smogcalled "vog". This vog oftencauses a noticeablehaze throughout the southernmost Hawaiian islands and alters the spectralirradiance reaching the surface, Particlesof air-bornematerials can alsofunction as cloudcondensation nuclei which result in theformation of clouds Schaefer & Day,1981!. Cloud formation can occur at various attitudes anddensities and are ciassNed into various groups dependent upon their shape and altitude. Cloudsoften function to decreasethe amountof UV reachingthe surfacesbeneath them, Most ofthe soiar irradiance absorption results from particles other than water in themake-up of the cloud Madronich, 1993!, whiie most of the scattering of transmitted radiation is dueto thewater dropletswithin the doud. The resulting diffuse radiation that reaches the surface is usuallyless than 20% of the overall spectral irradiance Fig. 2!. A final considerationis albedo, the amount of reflected irradiance relative to the amount incidentupon a surface.Such reflections, be they from terrestrial, aerial clouds!or even aquatic sources,can increasethe amountof UV radiationboth directly and indirectly.Directly, the radiationis increased by increasing the amount of illumination ofthe target object; indirectly, the radiationcan be increasedby re-illuminatingthe atmosphere, which then scatters the reflectance back down towards the surface. Under such conditions,distinctions must be made between localizedreflections which primarily lurninate only the targetarea, and regionalones which influencethe sky surroundingthe targetarea Madronich,1993!, Manytropical areas are characterizedby oceanicsurfaces which serve as strong reflecting bodies; additionally, cloud coverover islands also may serve to reflectmeasurable amounts of irradiancetowards both the atmosphereand towards the surface other than underneath the cloud!, Urban development can resultin highlyconcentrated reflective surfaces suchas concrete,glass, etc.! that can serveto increase the localized albedo within an area. 27 F. ~ effaoison radiation~ ihroU9n aie rorsraai~here Modiraidfrom Aiien Q~trrrtve alQQ,I8tlLi~,vol. 1 «ssd by Rorkins Mason,~ press, NewYork, 1960, p. 487 FACTORSAFFECTING SPECTRAL IRRADIANCE REACHING THE SURFACE OF KANE'OHEBAY, O'AHU TheHawai'i Institute of MarineBiology has had a continuousNational Oceanic and AtmosphericAssociation NOAA! tide gauge and weather stat~on in operationsince the 1950s. Solardata started tobe collected in1970, and sires 1985 an Eppley 295 - 385nm UV radiometer anda UCor400- 700 nm quantameter havebeen used to collect data which is averagedhouriy andstored ina computerdatabase. This database isaccessed monthly and printouts provided for useby resident andvisiting researchers, Specie information concerning technical features of theseinstruments iscontained within Gulko etal. thisvolume!. Totalradiant exposure, representing dailyintegration ofUV values, isshown for a o«yea' period t/94- 1?J94! inFig. 3. Note that the UV sensor was recalibrated dunng 'this time penod andthe data had tobe adjusted forthis recalibration. Thearrangement ofthe upnght sensor precludesdetection ofUV reflected fromsurface sources such as the wate rsof the bay itse 5.0 E k 4.0 s 3.0 2.0 I 0 0.0 FigureS. Daily mtudrnurnSurface UV mw cm'! St Hlhle, Kane'OheBay, Oehu: t/t/g4 - t 2/31B4. Data repreeentemean vs!ues taken during hour of highestUV intenSity. Obviously,seasonal shifts in the amount of UV present at the surfacewill havea directimpact on the amount of UV that penetrates the water column and the biologically effective dose rates that marine organismsare exposedto at any givendepth. Many of the experimentsdescribed in latter papers in this volume took place between June and August, 1994, in order to take advantage of the high UV levels. Short-termdaily variation Fig, 3! results primaniyfrom cloudcover and occasionallythe relationship between wind condition and the amount of vog present. During periods when the northeast tradewinds aren't active termed "Kona winds"!, strong hazy conditions are present over Kane'oheBay pers. obs,!. Otherpublished reportsfrom outside Hawai'i show that at sea level, for the samesolar angle 0'!, total direct UV irradianceunder a very hazy sky km visibility! is roughly one-third of that measured under a clear atmosphere Kneizys et al., 1988; cited by Mobley, 1994!. Hourly variations in UV levels are shown in Fig, 4 for a randomly chosen week in August 1994. Levels of soiar ultraviolet radiation characteristically rise steadily in the morning hours, waver and peak during rnid-day, and then decline less steadily in the aflemoon, The difference in the fluidity of the afternoondecline may be due in a large part to the common daily formation of clouds over the island of 0'ahu. 29 0 Date Fig.4. Meanhourly surface uv ImWcm'I inKans'ohe say, 0'ahu: 8/144- sPrg4. Data points ~t theaverage value for eachhcur taken over a Oneweek periOd. Nurreeta near each peak repreaent the bme Of day fOr that peak value; numbere nearIhe babe Oi Ihe graph epreeera the limeSOf rtay fOrthe ifrat anrl lect meaSuredValueS. lt is notunusual for daily solar values to fluctuate25 4 ormore of themean monthly value Yoshihara& Ekem, 1977!, based on such factors as wind,clouds, etc. Althoughweather patternsvary, in general,relative humidityon 0'ahu is faiffy constant at around 80%, The prevailingnortheast tradewinds result in a daily patternof orographiclifting of the moist sea air with associatedcloud formation over the mountainsfronting the windwardside of 0'ahu in the late fitoiningand early afternoon. This occursin additionto seasonalvariations in rainfall dueto Pacific Oceaninfluences which result in a characteristicwet seasonfrom October to May,and a dry seasofffrom June through September Smithef a/., 1981!, These daily cloud formationstend to formprimarily over the mountains and windward coastal areas and may have the effectof Produciffgan additionalatmospheric reflecting body to enhanceUV reachingthe surfacewaters ofKana'ohe Say Fig,5!, Thismay help to explainthe earlyafternoon as opposed to noon! Peaksin UVmeasured on individual days of a givenweek Fig.4!. FACTORSAFFECTING THE PENETRATION OF SPECTRAL IRRADIANCE THROUGH THE ytfATERSOF KANE'OHE BAY, O'AHU UVPenetrates to considerable depths in clearoceanic waters Jerlov, 1950!. Coastally,the dePthof Penetration varies with latitude Smith& Baker,197S; Calkins & Thordardottir,1982; Fleischrnann,1989! and season Levy, 1974!. In termsof water properties, UV penetration is dePendenttc a largedegree on turbidity, Turbiditycan be influencedby biogenous i,e, dissolvedorganic material DOM!, plankton, gelbstoff, etc.! andterngenous material Smith& Ba«r i 97S!.Kana'ohe Bay has beencharacterized as havingmeasurable amounts of terrestrial- 30 Figrrre5, Differencesin UV irradiancebetween mid morningand earlyafternoon rn Kana'oha Bay,0'ahu. derivedsediment, marine particulate calcium carbonate,organic detritus and plankton. Organic carbon is found at levels four times higher than those directly offshore Smith et al., 1981!. penetration of spectral irradiance through the water column was recorded using a LiCor LI- 1 800UWScanning Spectroradiometer see Gulko et a!., this volume!. Spectraldata 00-700 nm! was collectedin 2 nrn intervals. The instrumentwas deployeddirectly off of the Point Laboratory adlacentto the weatherstation at predetermineddepths above water, directly subsurface, 2 m, 3,5 m, 5 rn,7 m, and 10 m!. Ambient solar irradiancemeasurements were taken continuously around noon at times that minimized overcast conditions. Three scans were taken at each depth and the mean reported in order to minimize effects of fluctuating radiation Fig. 6!. l 1,00x io !.00x lO E Air l,00 x l0 Snhswfaee g l.00 x lO 3.5 rn l.00x 103 7 m R. l0 m l.00 x l 0 i 00x105300350 400 450 500 550 600 650 700 Wavelength nrn! Fi9ure6. Perrefrafronof ~ i ~ rn Kans'oheBay; measured around noon on a doudless tuiyday, f 994. UV-8penetration in Kane'ohe Bay is relatively limited when contrastedwith clear oceanic watersimmediately adjacent to it Lesser,this volume!. Yet,the characteristicsof spectral irradiancepenetration into the water column within Kana'ohe Bay is comparableto, and in-line with,measurements made for othercoastal waters Kullenberg,1982; Smith 8 Baker, 1979!, A moredetailed examination of UV attenuation in various sections of Kane'ohe Bay is available in Patterson this voiurne!. Tffediffuse attenuation coefficient for UV k! is definedas a functionof the wavelengthof radialion that has the lowest attenuation between 490 nm and 390 nm. This optical property acts to relatesubsurface irradiance with irradiance at depth Smith & Baker, 1979! and decreases exponentiallywith depth. As UV intensitydecreases logarithrnically as a functionof depth,this relationshipcan !Mused to calcLilatea UVextinction coefficient K!. Lamed thisvolume! calculatedsuch a coefficientdirectly adjacent to HfMB within Kane'ohe Bay, to be equivalentto- o 53 m' Thissuggests that UV attenuates relatively quickly with depth in Kane'oheBay, Smith eraI, 981! calculatedextinction COefficients for viSible light uSing a SeCChidisc and a quantum 32 radiometer;their values for 1979 ranged from -0.25 m' for thenorthern portion of thebay down to -0.41 m' for portions of the south part of the bay, Justas solar irradiance transmission through air is affectedby particles suspended in the atmosphere,solar irradiance transmission through water is affected by the size, type and distributionofparticles suspended inthe medium. The most obvious component ofsea water, thesalts themselves, actually increase scattering uniformly by about 30'to over transmission throughpure water, and may be involved inincreased absorbance of UV wavelengths Mobley, 1994!.At the most basic level, Kane'ohe Bay which,due to the surrounding watersheds, has varyinginputs of freshwater! would be expected tohave variations inthe penetration ofspectral irradiancedependant upon the amount, mixing, and interaction offreshwater inputs. Such an effectwould often be overshadowed pardon the pun!by the amountor typesof sedimentsor nutrients introduced by such inputs. Asstated earNer, a numberof studies Jokiel et al.,1993; Hunter 8 Evans,1993; Smith et al., 1981!have shown multiple inputs into the bay which might be expected to affectspectral transmissionthrough the water column, Among these, sedimentation and the effects of nutrients standout. Dissolvedorganic matter such as gelbstoff usually originates with decaying plant material often of terrestrial origin! and absorbs very strongly inthe blue and ultraviolet wavelengths Mobley, 1994!. Small, suspended particles would be expected tobe non-specific in theirreflectance of spectral irradiance overall, in additionto theamount of irradiancethat they absorbed.Kane'ohe Bay's waters also support high standing crops of bothphytoplankton and zooplankton Smith et al.,1981!; concentrations of plankters would obviously increase attenuationofUV through the water column. Overall, wind speed is thought to playa criticalrole in determiningtheamount of resuspended material inthe bay's waters Smith et al., 1981!. As wind speedincreases, wave action increases, causing dramatic changes inthe surface penetration of solarirradiance. High wind speed results in formation of subsurfacebubbles which tend to scatter irradianceinall directions this is why an air bubble produced by a diverappears white underwater! andresults in an increasein backscatteringof downward spectral irradiance Mobley, 1994!. Such wind-associatedvertical mixing would not only effect the depth of spectralirradiance penetration andits intensity,but would also contribute to thetypes of organisms primarily planktonic! that wereexposed to biologicallyeffective doses. The residence time under which organisms are exposedto higherlevels of UVnear the surface depends strongly upon the amount of vertical mixing Kullenberg, 1982!, Wind speed also has an effect on wind-generatedsurface waves withinthe bay, Suchwaves contribute to surfacescattering of spectralirradiance. Thepenetration ofUV-B and to a lesserextent UV-A! has a directeffect on the biologically effectivedose rate that marineorganisms in Kane'oheBay are exposed to and mayserve an importantrole in structuring the distribution ofvarious benthic components within the various habitats found within the Bay. ACKfVOWLEDGEAfEIVTS.MuchOf thiS wOrk waS funded through the Pauley FOrrndaecn andthe HaiNai'i lnatriute ofManna Biology,university ofHawari. Special appreciation goesto Dr. p. L, Jokiel for all of his assistance andmentorship. andDr M.Lesser for his assistance and use of his Li~r undenNater~diometer Thanksto L. Waterei and F Tefor assistancein coaxingthe HIMB Weatherstation to give up its secrets. LITE RATUR E CITED Banner,A. H. 968!. A freshwater "kIII" on the coral reefs of Hawaii.Haw. Inst. Mar. Bio. Tech. Rep. 37: 1 - 29. Banner,A. H. 974!. Kane'oheBay, Hawaii: Urban pollution and a coralreef ecosystem, Proc. 2nd Int,CoralReef Symp., Brisbane, Australia, 1974. 2: 685- 702. 33 Calkins,J, & Thordardottir,T. 982!. Penetrationof solar UV-B into waters off Iceland. In: 'The Roleof SolarUltraviolet Radiation in MarineEcosystems'. NATO Conf. Ser. 4, Mar.Sci. Vol. 7. Plenum Publ. Pgs. 309- 319. Calkins,J. & Thordardottir,T. 980!. The ecologicalsignificance of solarUV radiationon aquatic organisms. Nature 283. 563 - 566. Fleischmann,E. M. 989!. The measurementand penetration of ultravioletradiation into tropical marinewater. LImnol Oceanogr. 34 8!: 1623 - 1629. Florkin,F. & Mason,T. eds.! 960!, ComparativeBiochemistry, vol. 1. AcademicPress, New York, 1960, p. 467. Hunter,C. L. & Evans,C. W. 993!, Reefsin Kane'oheBay, Hawai'i: Two centunes of western influenceand two decades of data. Proc. Conf.on GlobalAspects of CoralReefs, University of Miami, 1993. Pgs. 339- 345. Jerlov,N. G. 950!. Ultravioletradiation in the sea. Nature Lond!166: 111 - 112. Jokiel,P, L Hunter,C. L.,T aguchi,S. & Waterai,L 993!. Eooiogicalimpact of a freshwater "reefkilf' in Kane'oheBay, Oahu,Hawaii, CoralReefs 12: 177 - 184. Kneizys,F. X., Shettle,E. P., Abreu,L W., Chetwynd,J, H Anderson,G. P.,Gallery, W, O., Selby,J, E. A. & Clough,S. A. 988!. Usersguide to LOWTRAN7, Air ForceGeophysics LabRept AF6L-TR-88-0177, Hanscom AFB, MA, 131pp. referredto in Mobley,1994!. Kramer,K. J. M. 990!. Effectsof increasedsolar UV-B radiation on coastalmarine ecosystems: an overview.In: 'ExpectedEffects of Climatic Changeon MarineCoastal Ecosystems', J. J. Beukemael al. eds.!. Arnsterdarn;Kluwar Academic Publishers. Pgs. 195 - 210, Kullenberg,G. 982!. Note on the role of vertical mixing in relationto effects of UV radiation on the marineenvironment. In; 'TheRole of SolarUltraviolet Radiation in MarineEcosystems', J. Calkins ed,!, New York:Plenum Press, Pgs, 283-292. Lamed, S. 995!. Effectsof ultraviolet radiation and nitrogen enrichmenton growth in coral reef chlorophytesDictyosphaeria cavemosa and Dictyosphaeriaversluysii. In: 'UltravioletRadiation andCoral Reefs', D. Gulko& P. L. Jokiel eds.!. Hawai'iInsbtute of MarineBiology Technical Report ¹41. UNIHI-Sea Grant-CR-95-03. Lesser,M. P. 995!. Generaioverview of instrumentation,expenmental methods, and attenuation of UV radiation into natural waters. In: 'Ultraviolet Radiation and Coral Reefs', D. Gulko & P. L. Jokiel eds.!. Hawai'i Institute of IVlartne Biology Technical Report ¹41, UNIHI- Sea Grant-CR-95-03.Pgs 15- 18. Levy, H, 974!. Photochemistry of the trophosphere, Adv. Photochem.9: 369 - 524. Madronich,S. 993!. UV radiationin natural and perturbedatmosphere. In: 'UV-B Radiationand Ozone Depletion', M, Tevini ed.!. Boca Raton, FL: Lewis Publishers. Pgs. 17 - 69. Mobley,C. D. 994!. Light and Water: RadiativeTransfer in NaturalWaters. San Diego,CA: Academic Press. 34 Moore,J. G., Clague.D, A., Holcomb,R. T., Lippman,P. W.,Norrnark, W. R. & Torresan,M. E. 989!. Prodigioussubmarine landslides on the Hawaiianridge, J. Geophys.Res, 94: 17465- 17484. Patterson,K. W. f995!. Attenuationin Kane'ohe Bay as interpretedfrom PUV profiles. /n: 'UltravioletRadiation and Coral Reefs', D. Gulko8 P. L. Jokiel eds.!. Hawai'iinstitute of Marine BiologyTechnical Report ¹41. UNIHI-Sea Grant-CR-95-03. Pgs. 35 - 39. Roy,K. J. 970!, Changein bathymetricconfiguration, Kaneohe Bay, 1882 - 1969.University of Hawaii Sea Grant Publication 70-6, HIG 70-15. Schaefer,V. J, 8 Day,J. A. 981!. A fieldguide to the atmosphere. Boston: Houghton Mifflin Company. 359pp. Smith,R, C. 8 Baker,K, S. 979!. Penetrationof UV-B and biologically effective dose-rates in natural waters, Phofochem. Photobiol. 29, 311 - 323. Smith,S. V., Kimmerer, W. J., Laws,E. A., Brock,R, E, & Walsh,T. W, 981!. Kane'oheBay sewagediversion experiment: perspectives on ecosystemresponses to nutritional perturbation. Pac. Sci. 35 !: 279 - 395. Universityof Hawai'iDepartment of Geography 983!. Atlasof Hawai'i,2nd ed. Honolulu,Hl,. Universityof Hawai'i Press. 222 pp. Yoshihara,T. 8 Ekern,P. C. 977!. SolarRadiation Measurements in Hawaii.Honolulu, Hawaii: HawaiiNatural Energy Institute, University of Hawaii.94 pgs. 35 UltravioletRadiation and Coral Reefs. 1995. D GulkeL P L Jokiel eda} HIMBTech RepOrt¹41. UIVlkl-SeaGrant-CR-95-03. Attenuatian in Kane'Ohe Bay as interpreted frOrn PUV prOfileS Karen W, Patterson Institutefor ComputationalEarth Systems Science Departmentof Geography unrversityof California Santa Barbara SantaBarbara. California 93 f06 ABSTRACT:During the Summer Of 1994, optiCal prafileS Were made in VanOue regions of Kans'ohe aay TheaeprOfiles had a speCtral range includingthe ultravioletand visible portionsof the spectrum The profilesdiscussed in this paper werecollected wilh a eiOSphenCalInStrumenta puV-500 and are grOuped intc tWO tranaeofS, One alang the mafdr axia Of the bay and One alOng the minor axiSof the bay. In general,attenuatiOn increaaed within a fewmeters of the bcttOm Of the bay. PrafileS COlleCted fuet~ thebay hadmarked! y lowerattenuation than those inside the bsy which can in partbe attributedto thebath~ configurabonof the area. INTRODUCTION Toassess the effectsof ultravioletradiation on marineorganisms, including corals, and howvariations in ultravioletradiation might effect these organisms in nature,it is necessaryto quantifythe optical environmentin whichthe organismslive andto knowhow this environmentaffects the spectralquality and quantityof radiationreceived by the organisms.Unlike many other marine organisms, corals are fairly stationary which simplifies quantification of the incident spectral radiation corals receive, Radiometricmeasurements assist in the quantification of the radiation environment in which these organisms live. Optical data can be considered in relation to other environmental constituents and variablesto obtaininsight inlo the majorfactors influencing the radiationenvironment of the organisms and howthe radiationenvironment changes as thesefactors change. Inthis paper,I wiltdiscuss the general attenuationcharacteristics of Kane'ohe Bay, Hawai'ias a result of profile measurementsmade in the ultraviolet and visible portions of the spectrum during the summer of 1994, Results are also discussed in relation to the general bathymetry of the area. METHODS During the summer of 1994, an optical survey of Kane'ohe Bay was performed. Included in this survey were profi e measurementsmade with a Biospherical InstrumentsPUV-500 filter-based radiometer, The PUV-500makes instantaneous measurements in fivespectral bandwidths as wellas measuring temperature and pressure depth!. Four of the five spectral channels are -10nm bandwidths in the ultraviolet portion of the spectrum centered on 305, 320, 340 and 380 nanorneters. The fifth channel is a broadband PAR 00-700 nm! cha~nel. Kirk et al. 994! provide a more detailed explanation of the spectral characteristicsof the PUV-500 channels. The profiles were made along two main transects which included a range of aquatic environments. Profiles were obtained in areas where the bottom cover was muddy,sandy, and/or coral covered as wellas a coupleof "bluewater" profiles just outsidethe bay, The approximatelocations of the profiles discussed in this paper are shown on Fig. 1. IVlostof the profiles extend all the way to the bottom. The only exception to this is one profile which was taken just outside the bay and is represented by the northernmost S on Fig. t, Rawprofile data are not directlycomparable due to suchthings as varyingdegrees of cloudcover and timeof datacollection. Wind conditions and sea state were similar over the courseof daysthat datawere collected.Because of this, mixingof bottomsediments into the watercolumn by wind-inducedmixing was not considered as a concerning factor, In order to compare the profiles, the following was done. Each profiie was normalized to the surface values to eliminate the effects of varying cloud cover over the course of each profile. These normalized values were fitted with an average curve to remove the scatter in the data due to wave action effects. A profile from the southern portion of the bay is shown in Fig. 2 as an example. The average curves were used to calculate attenuation coefficients for each profile at depth intervals of 0.1 meters, New profiles were then created using these attenuation coefficients and a surface "clear-sky" noontime vaiue. This surface"clear-sky" noontime value is the maximumsurface value from measurements made on July 30th whenthe PUVsurface unit was placedon the roofof the Pointt ab on CoconutIsland collecting data every several seconds. This maximum surface value was multiplied by,95 to account for attenuation through the air/water interface. The processed profiles are shown in Figs. 3 and 4. For further discussion, the transect running along the minor axis of the bay will be referred to as transect S and the transect running along the major axis of the bay will be referred to as the transect T. 37 t57~ So' 46' 30' 2tO 25' Figure1.A mapolKane'ohe Bayshowing theapproximate ~ ot theProfiles discus inthis ~r. 6'sm~ " transectanrt rs ie~ the~ along themaIor a olthe bsy DISCUSSION In general,each profile exhibitedfairly uniform attenuationdown to within a few meters of the bottom, In the few meters near the bottom, attenuationgenerally increasedas shown by an increase in the spacing between meter numbers at depth on Figs. 3 and 4. This is most likely due to an increase in suspended sediments in these deepest few meters. Sufficien data was not collected as to the type of bottom cover to compare the increasedattenuation at depth based on the type of bottom cover. However, I would suspectthat the profileswith greater increasesin attenuationat depth are the ones with a primarilymuddy bottom cover rather than a primarily sandy or coral-covered bottom. The greatest attenuation of al! collectedprofiles was found in the second and third profiles of transect S, which were collected in a shallow muddy area. With the exceptionof the two transect S profilesjust outsidethe bay Fig. 4!, irradiance at 305 nm fell below the threshhold of detection of the instrument at about 6-9 meters depth, shallower than the depth at which the near-bottom increases in attenuation begin. Irradiance at 320 nm generally fell below detection limits at about 1-3 meters from the bottom and irradiance at 340 nrn generally fell below detection limits within a meter of the bottom. With the exception of one profile, irradiance at 380 nm and PAR remained at detectable levels at the bottom. 305nm 320nm 340 nrem 4 4 4 E E E 6 ~ 6 6 O O 0 10 10 10 12 12 0.01 0.1 0.001 0.1 0.001 0.1 380 nrn PAR 4 4 E E 6 ~ 6 Q C3 10 10 12 12 0.01 0.1 0.1 1.0 Figure2, An examplertormalized curve-fitted piofile. This is the southernmostprofile iri Figure1. marxedby a T. NOtethe deoieaaeih Soatterdue tOWave aotibn With irioreaairig depth At transect T Fig. 3! there appears to be a trend of increasing attenuation progressing from the more southern profiles to the more northern profiles. The reason for this is not known at this time, The eight profiles represent a range of environments and bottom cover. Without knowing the factors leading to the increase in attenuation in the more northern profiles along this transect, it can not be concluded that this cross-baytrend is an actual cross-bay trend in the bayand not just the coincidental result of otherfactors whichdo not vary in a southto northtrend in thebay. Thereis significantly higher attenuation inside the bay than outside the bay. The fourth and fifth profilesoftransect S {Fig.4! werecollected just outside the bay while all others were collected inside the bay.Irradiances ata depthof13 meters for these two profiles were, in most cases, at leastone to two ordersof magnitude higher than the bottom depths ofprofiles inside the bay Fig. 4!. Thewaters inside andoutside the bay are fairly well separated bathymetrically bya shallowregion. The second and third profilesof transectS werecollected in thisshallow region. The marked difference in attenuationbetween profilescollected inthe bay and those collected justoutside the bay can, in part, be explained bythis bathymetricbarrier with the outer profiles having a muchgreater deeper water oceanic influence. 305nm 320nm 340nm 380nm PAR B s+ E~ C. &CV fi Csa C C s E E fi 3 cb lJ 4 4 e r T- T! T41~ I h 'ii, s T- T~Ti Y~ia h tii h rg 'It T47 305nm 320 nrn 340nm 380nm PAR tt 4 L tt C.C ca C4 fi E fi4 s s ca C s! s4 5~ st sp s~ $~ si 52 sg Q s, Figure4. pUVprcfilee fcrthe S transeCtatthe fcur UV waVelengthS SndpAR. The approximate locations fOrthe prafilee are ShOWn in Fig.1. EaChCOlumn S.thrcugh S,! resxeei~a ~ Prafife,inOrder, frOm ineide the bey tO luet outside. The amcunt Of radiaSOnieShcwn on the y-axie The nurnberS platted iepreeent depth in meterS NOte S, and S, Were taken in the ~ Channel wheredepth was lessthan 4 m. Thescatter in thedata due to waveaction was removed to aidin thecomparison of profiles;however, it shouldnot be neglected. The scatter due to waveaction decreases with increasing depth Fig.2!. The scatterin the PAR 00-700 nm! figureis greatermainly due to a widerspectral bandwidth. The other figures represent a 10 nm bandwidth rather than a 300 nrn bandwidth. This decrease in wave action effectswith increasing depth may be animportant factor to considerwhen analyzing the effects of ulbavioletradiation on corals in someexperimental setups, Tanks are generally shallower than the depths at whichcoral specimens are collected. Tanks also generally have water flowing through them which can create ripplesacross the surface of the tank sirniiar to small ripples on the sea surface. Also, some experimentshave been done where coralsare lransplantedfrom a deeperto a shallower depth for the 40 purposeof assessingthe effectsof increasedultraviolet radiation on corals Gleason& Wellington,1993i Gleason,1993!. Whencorals are movedfrom deeper to shallowerdepths, spectral quantity and quality change.This can be compensatedfor by usinga combinationof filters. Waveaction scatter is greaterat shallowerdepths and the magnitudeof variation in very short-term radiation doses increases. Such wave scatterradiation is generallynot compensatedfor, norgenerally addressed as a concerningfactor, in the interpretation of experimental resufts, ACKIVOWLEDGEIHEAITS:I wOukf like to thankPaul JOkiel and everyOne at H!MBfOr their helP white l waSat Hlkiis.I WOuldalsO like tOthank Ray Smith and Paul JOkiel fOr making thiS trip pOSSible. This wOrk waS Suppetted by RaySmith, IceCOIOrS '93- NSFNPP- 9220662. LITE RATURE CITED Gleason,D, F, & Wellington,G. M. 993!. Ultravioletradiation and coralbleaching. Nature 365: 836- 838. Gleason,D. F, 993!. Differentialeffects of ultravioletradiation on greenand brownmorphs of the caribbeancoral Porites astreoides, LimnoL Oceanogr 38: f 452 - 1463. Kirk,J, T. 0, Hargreaves,B, R., Morris, D, P Coffin, R. B David, B., Frederickson,D., Karentz,D., Lean, D. R, S., Lesser,M. P., Madronich,S., Morrow,J. H., Nelson,N. B. & Scully,N. M, 994!. Measurementsof UV-Bradiation in two freshwaterlakes: an instrumentcomparison. Arch. HydrobioL Beih. Ergebn. LimnoL 43: 71 - 99, ultravioletRadiation and Coral Reefs. 1995. D Giilko8 p L Jokiei eds I HIIABTech. Report ¹41 UNIHiSea GrahhCR-95-ff3 'The influence of Solar UV-8 Radiation on Copepods in the Lagoon at Coconut Island, Hawai'I kiroaki Saito and Satoru Taguchi HOkkaidONabOnal Fiahenea ReaearCh lnatitule, Katsura-koi 116, KuahirOeSS. JaPan ABSTRACT The in¹uenoeOf SOlar uttravioiet radiatiOn On copepOdS Waa determined in the lagOOnat COCOnutISland, Hawai'i TheinfluenCe Of UV-B On egg hatching rate waa determined uaing LaCvdtcoera rnadurae. The eggS were incubated in quartZ bottlesunder natural solar radiation. The dose of UV-Bwas controlled for the duration of theinvestigation by covenngthe bcttfeswith a lumiiarSheet whiCh abscrbed SOlar radiaticn ShOrter than 315 nrn. The influenceOf UV-B On SurVrval rate Waa determinedusing nauplii of ~ spp Survivalrate of thenauplii did not decrease with UV-B radiahon. Nauphi in the expenmentalbottle with UV-B radiationmolted to ~id stagesat a ratesimdrar to the those in the controlbottle ~ UV-B.Theae reSulta indiCate that INTRODUCTION Copepodsare the mostdominant zooplankton and playan importantrole in marine ecosystems,It is known that UV-B radiation influencestheir survival rate Dey et ai., 1988!, fecundityand egg hatching rate Karanaset ai 1981!. In oceanicwaters, many copepods may avoidUV radiationthrough diet verticalmigration behavior. They dtstribute throughout the deep layersduring the day and migrateto the food-abundantsurface layer during the night. Copepods can not, however, avoid UV radiation by vertical migrationwhile in shallow environmentssuch as lagoons,estuaries, or tidal ponds. It would,therefore, be expectedthat copepoddistribution in shallowenvironments would be eitherseverely influenced by solarUV-B radiation or theywould be well-adaptedto the photo-environmentand littleinfluenced by present-daylevels of UV-B. In thisstudy we determinedthe influenceof UV-Bradiation on copepods using egg hatching rateand nauplii survival rate in the shallowlagoon at Coconutisland, Hawai'i. Animals in theirearly life stagesare usuallymore vulnerable than their later stages Damkaeret ai., 1980!, The results obtained, therefore, were expectedto be indicative of the most sensitiveeffects of UV-B radiation relating to copepod distribution in the lagoon at Coconut Island. MATERIALS AND METHODS Experimentswere carried out in the lagoonat CoconutIsland, Hawai'i N 22 26', W 157 47'!. Duringthe experimentsunderwater photosynthetically active radiation PAR! and UV-Bradiation were measuredcontinously with a BiosphericalInstruments underwater spectroradiometer PUV- 500. TheP UV-500 measured UV-B radiation at 305nm, 320 nm, 340 nm, and 380 nm spectra. PARand temperature were also measured. The sensor was set at a depthof 15cm in thelagoon. h hin r f i m r Labidoceramadvrae, the dominant copepod species in the lagoon,was used to investigate the influenceof solarultraviolet radiation on the egghatching rate. A pumpwas usedto collect zooplankton during the night of July 28, 1994 and L. madurae females were sorted out under the microscope.The females were individually placed into 30 ml vialsfilled with filtered ,2 pm Nucleoporefitter! seawater and maintained for 8 hours.Before sunrise on July 29, the produced eggswere sortedand threegroups of 50 eggswere each put into individual250 ml quartzbottles filled with filtered .2 atmNucleopore filter! sea water, piaced just before sunrise into a water table containingflow-through seawater, and maintainedfor 48 hours. The hatchingtime of L. madurae was between 30 and 36 hours at ambient temperature, UV-B dose was controlled for the durabon of the investigationby coveringthe bottleswith a lumilarsheet TableI! whichabsorbed wavelengthsshorter than 315 nm. As a control,a darkbottle was preparedand all threebottles werecovered by the lumiliar sheet throughout the experiment. Nauplii and unhatched eggs were counted after incubation. 43 Ta5ei UV-8dace to the eggS Of LSbaddrerrr readurm Dose mW crn'nm' Ex .bottle Duration of UV-8 ex osure Stan End 305nm 320nin Control-light 0,0 0.0 0,0 Control-dark 0.0 32. 5 El 14:00 19:30 15. 3 42. 1 E2 i 3:00 19,'30 19. 8 56.2 E3 12:00 19:30 26.4 E4 11:00 19:30 34,5 73.4 E5 10:00 19:30 40. 8 86,8 E6 5:55 19:30 47. 0 100,0 Surfacewater was collectedwith a bucketand filteredwith 100 pm nylon mesh to exclude the largerzooplankton. Filtered sea water was placed into quartz bottles and kept under natural solar radiationin watertables with flow-through sea water. Six experimentalbottles and three dark controlbottles were prepared and covered with a lumiliarsheet. The experimentwas started at 6 am on July 27, 1994, Twoexperimental bottles and one control bottle were recoveredevery 24 hours for threedays. Waterin the bottleswas concentrated to 20 ml with a 20 pm nylon mesh and put into 20 rnl glasstubes. A lampwas placedat the top of eachtube to help distinguish living- active from living-inactive and dead animals. Living-active animals concentrated toward the lamp by their phototaxic behavior while living -inactive or dead animals sunk to the bottom of each tube. The bOttorn5 ml Of water in each tube waS plaCedintO a glaSSpetri diah and living-inaCtive animate were distinguished from dead animals under a dissecting microscope, 'The rest of the water from each tube was also placed into petri dishs to which was added 1 ml of formalin, and the animals werethen counted. Mostof the animalsin the sampleswere naupliar and earlycopepodid stages of Oithona. RESULTS Dielchanges in UV-8radiation at each wavelength measured on July29 areshown in Fig.1. Thedaily solar ultraviolet radiation during the experiment was approximately 90% of thatobserved on July 28 when there was little cloud cover. Temperature during the experimentwas between 26.PC and 29.0'C. Hatching rates in the experimental light! and control dark! regimes were higher than 98%. No significant effect of solar UV-B radiation onLabidocera madurae egg hatching rate was observed Fig. 2!. Dailysolar ultraviolet radiation dunng the experiment isshown in Fig.3, Solarradiation onJuly 28 was less influenced byclouds and the daily radiation was hi h t t h gt o UVradiation and PAR during the three experimental days. Ambient water temperatureduring the experiment was between 26.6'C and 29.0'C. Dunngthe experiment,survival ratesof naupliidid not decrease with time whether expased t0 UV-8 or not Fig.4!. Survivalrates of copepodids i decreased after an incubationperiod lan ger ours.. NegativeN influences of UV-8 -8 an survival, however, were not observed during the o ays. he contnbution of Oif n Oiffronato thetotal number of copepodidsincreased in b o th thee experimentalbottles and in the controlss, andan the percentageof Oifhonato copepodidsin the n eso were similar to those of the two co controlbottles at eachsampling time. Thus it erre a -B radiationhad little influence onthe molting ofnaupliar to copepodid 29 tnp-r ae rrrt ~l tL II~ R ~ Ie I 27 26 0:00 3:00 6:00 9:00 12:00 15 00 18:00 21:00 0:00 ~ 2000 E 1000 CL 0 I IIII II III II 11II II II II II III II III III II II I II nIt ItIP 0:00 3:00 6:00 9'.00 12:00 15:00 18:00 21:00 0:00 5.0 4,0 3.0 PS ~ ~IP e 2.0 ~ re~ EP P'$PLS a ~ ~ ~ 1,0 p ~ ~ e re rn I t I It 0.0 I II II III II III II III I I In 'I It I IIII I II II I II III II II ~ 0:00 3:00 6:00 9:00 12:00 15:00 I 8:00 21:00 0:00 Figure 1. Diel Changmin temperature,PAR, and ultraviOletradiatian in INavetertgthOt 305 nrn, 320 nm, 340 nm, and 380 nm at t 5 cm depth in the lagoonof CoconutIsland. 100 80 70 40 30 o 20 10 I 0 I III Iiiililaeliiiil' '- III III IiII I II IIl I III I IIS 0:pp 3:00 6.'00 9:00 12:00 15:00 18:00 21:00 0:Pp 100 80 E 70 60 50 40 0 I I II I III I I II I I I II I I II I I I I I11 I I I~ 0:00 3:00 6:00 9:00 12:00 15:00 18:00 21:00 0:00 100 80 I ~~ E 70 ~ la I Ira 60 ~ 5 a ~Isa ~ 50 40 sa Paa ~ a ~ ~ ~ 30 I ~ a ~ aa a 20 I ~ I j ~ ra 10 '«r~ 0 II III IIIII IIIII I IIII I' ill+ L- IIII nII I III I III II I I' 0.00 3:PP 6:00 9:00 12:00 15:00 18:00 21:00 0:00 Figuret cont.!. Diet changes intemperature, PAR,and u~ radiationin~ngth af305 nm, 320 nm, 340 nrn, and380 nm at t 5cm depth in the lagoon at ~ Island. 100 80 60 40 20 10 20 30 40 50 UV-8 DOSF.: 305nm rnW cm' nm'! Figure2. The irttruertCaOt UV-BdOSe 05 rirrt! Onthe hatChingrata or Lar&rOCimnmkpm. SquareSindiCate experimentalbottle anal open circraSindiCate COntrOI bOttle. DISCUSSION In this studythe egghatching rate of Labidoceramadurae was not notablyinfluenced by solar UV-8 radiation. The daily UV radiation during the present experimentwas approximately90% of that on July 28 Fig. 3!. The daily UV radiationat the summersolstice would be higher than those observed during the present experiment. We are not sure whether solar UV radiation on a clear day around the summer solstice would influencethe hatching rate of L, maduraeor not. However, the eggsof L. maduraehave a negativebuoyancy and are adhesive. Thesecharacteristics of the eggs would suggest that the eggs may sink quickly to the bottom of the calm lagoon and become attached to the substrate. UV radiation at the bottom of the lagoon the depth is 0.5 rn to 3 m! is lower than the amounts employed in the present experiments, This suggests that naturally produced eggs are usually exposed to weaker Uv at the bottom of the lagoon than the ones ln the present experiments,and in vivo hatching may be not be influenced by UV-B. No influence of solar UV radiation on the survival rate of Oithona nauplii was observed in this study. It was obviousthat solar UV-8 radiationdid not lethally influence the naupliarstages of Oithona . While we did not examine the influenceof UV-8 on the ingestion and assimilationof food or growth rate of Oithona nauplii, becausesolar UV-8 radiation had little, if any, influenceon the molts of naupliarand copepodid stages, the results suggest that ingestion and growth of Oithona nauplii under UV-8 would be similar to that of naupliiwithout exposure to UV-8. In this study no influence of solar UV-8 radiationwas observedon copepods in the lagoonat Coconut Island. However,it is known that the present-daylevels of solar UV-8 radiationcan cause serious damage to copepods in temperate and boreal areas. Hatching rates of the copepod Paracaianus sp. were decreasedby soiar UV-8 radiation in October Saito, unpublisheddata!, Solar UV-8 radiationin April and June lethally affectedthe copepod Calanus sinicus in the inland Sea, Japan Uye et aL, personalcommunication! and UV radiationin those areas is lower than that used in the present experiments, Therefore, we can say that copepods in the lagoon at Coconut Islandare well-adapted to theirphoto-environment and they are less influencedby presentlevels of UV-8radiation than Paracalanussp. and Calanussinicus, These differences in sensitivityto UV between copepods may be due to differences in protection from UV radiation. Formsof UV protection in copepods are not wel! known. Pigments and their concentrationare importantfor protectionfrom UV in copepods Rrngelberget ai., 1984! as in the other marine 1500 8 1000 a- soo 0 305 320 340 380 Wavelength nm! Vy 1500 C 1000 500 320 340 380 Wavelength nm} 1500 1000 cit 500 0 305 320 340 380 Wavelength nm! Figure3 DairyeOiar ultraviOlet radiatidn in the~rigth Ot305 nm, 320 nm. 340 nrn. and 380 nm et 15cm depthin thelagoon at Caxnut Islandbetvvetm tuty 21 and July 29, t 994. 48 N applies 100 0 80 60 + COI TROL 40 0 EXP. 20 24 48 72 TIME ttr! Copepodid f00 0 0 0 80 0k 60 j, CONTROL 40 0 EXP. 20 0 0 48 72 TIME IIr> Copepodid/Cope podi d+nauplius 90 rl CONIROL 0 EXP. 60 -o 50 o. 40 <80 0 > 20 k 0 I00 0 0 0 24 48 72 TIME hi Figure4. Influenceof ultravroletradiation on Offrrorfaspp.Top and middle figures show suhnval rates of naupliusand copepodro Stagea,raepeCtiVdy BOttam fig~re ShOwS Copepodid percentage tO naupls pluS COpepedrdS. Crrdee anCI tnang lee rndiCale experimentaland controlbottles, re~ly. animals Chapman and Hardy, 1988; Shick etal., 1992!, Eggsof Paracatanusare transparent while those of Labidaceramadurae have a green pigment. It is speculatedthat the green pigment in L. madufaeeggs may block UV radiationand thus serve as oneof the factorsthat makes L. maduraeeggs less vulnerableto UV than eggs of Paracatantjs. While we have no informationon UV protectionin Oifhona, the results of the present study indicatethat this organism is well-protectedfrom solar UV-B radiation. Mycosponne-likeamino acids are possible candidatesto serve as protectivecompounds in this organismas such compoundshave been found in manymarine organisms in the worldocean Karentzef al., 199t!. DeterminatiOnOf SuChUV-prOtective COmpOundS ln COpepOdsadapted tO the photo-envirOnment in the lagoon at Coconut island should be made in the future. 49 ACIrfvottILEDGItfElirrS.Wewould like to thank Dr. paul Jokiel forhis wndness inorganizing thisexperiment andDrs. Robert KinZieandMai LopeZ fartheir dfaouMicns withuS that aided ourunderstanding theenVirOnrnental featureSOfthe lagOOn, TttieWOrk waSSuprOrSXI pari!ybythe Pauley FOundatian inCalifcrnia andthe GIObal Envircnment ReeearCh Program Of theEnvironmental Agency,Japan. Inaddrbon. support was provided bya SasakawaScientific Research Grant from The JapanScience Society awarded toH. Saito. Contribution B-538} from Hokkaido National Fishenes Research Instrtute. LITERATURE CITED Chapman,J.& Hardy, J.T. 988!. Effects ofultraviolet radiation onmarine fishes. Final report, Oregonstate University, USEPA Coop. Agrmt. CR-812686-02-0, 31pp. Damkaer,D.M., Dey, D. B, Heron,G. A. 8 Prentice,E,F, 980!. UV-Bdose/dose-rate responsesofseasonally abundant copepods of Puget Sound. Oecolagia 76; 321 329. Karansas,J.J Worrest, R.C. & VanDyke, H. 981!. Impact ofUV-B radiation onthe fecundity of thecopepod Acartia clausii. Mar, Biol, 65: 125- 133. Karentz,D.,McEuen, F.S., Land, M. C. & Dunlap,W,C. 991}. Survey ofmycosporine-like aminoacid compounds inAntarctic marine organisms: potential protection from ultraviolet exposure. Mar. Biol. 108: 157 - 166. Ringelberg,J. Keyser, A. L.8 Flik,B. J. G, 984!. Themortality effect of ultravioletradiation in translucentandin a redmorph of Acanfhodiaptamusdenficomis Crustacea, Copepoda! and itspossible ecological relevance. Hydrobiol. 112: 217 - 222. Shick,J.M., Dunlap, W.C., Chalker, B.E Banaszak, A.T. 8 Rosenzweig,T,K. 992!. Survey of ultravioletradiation-absorbing rnycosporine-like amino acids in organs of coral reef holothuroids.Mar. Ecol. Prog, Ser. 90: 139- 148. 50 ultravioletRadiation and COralReefa, 1995, 0 G lko& P L Jck elleds !. HIM6Tech. Repen ¹4l UNIHI-SeaGran', CA 95-03. A biological weighting function for phytoplankton growth inhibition P. J. M. Peterson, R. C. Smith, K. W. Patterson Inebtutefor CcmputabOnalEarth Syateine SCienCe Universityof California,at Santa Barbara SantaBarbara, California 93106 P. L. Jokiel Hawai'iInSbtute Of Marine BiOIOgy P. O. Box 1346 Kana'ohe.Hawa 'i96744 ABSTRACT There is concernthat reducedstratospheric ozone, snd the subsequentincrease in ultrav otetradiation at the surfaceof the ocean.could have an adverseeffect on phytoptanktonEvaluabng the possible influence of ~ stratosphencozone on an organismrequires an accurateestimabon of the biolog callyweighted fluence rate, that is, the effecbvedose rate. The effecbvebiological dose is the integralof the ~ spectral rrad anceand an organism's wavelengthdependent biological response to this irradiance.Currently there is no generallyaccepted txolog calwe ghbng functionfor assess ngthe influenceof ultravioletradiabon on phytoplankton.We discussa procedurefor esbmabngthe b o!og catweighting funcbon for observedgrowlh inhibibon in phytoplankton,et! !, given the inhibrledresponse of several culturestos light regimes: PAR 00-700 nm!; PAR+ Uv A 20-400 nm!; and PAR+ Uv A+ Uv B f280-320 nm! Workby Jokieland York 984! provides%e irradiance parNions and growth inhib bondata used. An atrnosphencmodel Green,1980! is usedto reconsbtutethe spectralquality of the intxdentirradiance. Our resultsshow that some pubbshed biologicalweighting functions DNA setlow, 1974!,generabzed plant fCatdwelh1971!, Erythema NAS, 1979, 1982!,and Cullenet al. 992!! areinconsistent w thcomputed chant!as in dosevs. observed changes in b oit9calresponse. A funcbonalform suggestedby Rundel 983! of c i! = e ' was investigated. Forthe cufture Syrnbrcxfin/tan micros~ an a, of 4.45 nrn '] produtxidthe best rit Althoughthe ex sbngdata does not allow for a precise esbmationof E !.!,the workpressn~ hereprovideS a methadfOr dentrlyingb OIOg Ca!we ghbngfunCticnS whiCh are ncons stentwith the experimentalresults of Jokiel and York. INTRODUCTION The determinationof an appropriateweighting function is a crucialcomponent in the quantitative estimation of a possible ecological impact of increased Uv-B, caused by decreased stratospheric ozone, on phytoplankton tNAS, 1979, 1982, 1984; Rundel, 1983; Caidwell et al., 1986!. Biological weighting functions also called "action spectra"! are used, along with spectral irradiance, in calculating biologically weighted fiuence rate or "dose"!. Q=f c kiE kidi. where 6 Cl Cf 0 Q. fft Cl K fg V Dl 0 a Cl Cf 'Uf: I Q. I Q Df c Cl Cl Cf ftf Ct K 280 300 320 340 360 380 400 Wavelength I'nm] Figure 1 Fourst SChemabCof ~ frrad'anra.' at theearth'S Surfaoe COmpared with an aCbcn spectrum tO illuStrate OppOSing ~ as a funcbmof wfa~~ 52 1987; Haeder and Haeder, 1988; Smith et al., 1992!. UV-B penetrates natural waters to ecologically significant depths Jerlov, 1950; Calkins, 1975; Smith and Baker, 1979, 1981! so there is a potential to adversely influence phytoplankton productivity. The estimation of dose as a function of depth Smith and Baker, 1979! is strongly dependent on the spectral shape of e !. There are several potential action spectra which have been suggested as biological weighting functions for describing the effects of enhanced UV-B on phytoplankton productivity. In spite of the importance of accurate action spectra, there is considerable uncertainty with respect to the primary target for inhibition and the corresponding z !,l. Smith 989! and Smith and Baker 979! have reviewed the problems associated with these uncertainties. These authors point out that usual "C techniquesfor estimating phytoplanktonproductivity in which relativelyshort-term incubations provide an estimate for the biological weighting for photoinhibition e X!, may be inadequatefor assessmentof possible longer term UV-B damage that may be ecologically significant, As an alternativeto "C productivityexperiments, Jokiel and York 984! carried out long term -2 weeks! monoculture expenments with natural sunlight and combinations of UV filters and neutral density filters to determine the relative importance of present day levels of solar UV radiation and PAR in causing rnicroalgaegrowth inhibition. These workers found that tong-term growth photoinhibition when observed! was "due almost entirely to UV radiation." They used various radiation regimes which included PAR 00 - 700 nrn!, UV-A 20 - 400 nm! and UV-B 80 320 nm! and naturalsolar radiationwas filtered into 3 light regimes; PAR, PAR + UV-A, and PAR + UV-A + UV-B. Each regime was then reducedto 92'/o,38/o, 20'/o and 6'/o of its intensity using neutral density filters, giving twelve radiationtreatments for each experiment. We use their data in the following. MATERIAL,S AND METHODS Abridged spectrophotometry Nachtwey and Caldwell, 1975; Smith and Baker, 1982! is a method which can be used for estimating e X!. This involves; 1! hypothesizing a possible action spectrum, 2! calculating dose rates for the several treatments using this action spectrum; 3! plotting the computed dose rates for this hypothesizede X! vs. the observed growth inhibition and 4! checking these results for internal consistency. If the trial c X! has the correct spectral weighting,then all the data points would fall along a curve describing the organism's response to varying dose rates. Using this technique, we tested several published action spectra Figure 2!; DNA Setlow, 1974!, generalized plant Caldwell, 1971!, Erythema NAS, 1979, f982!, Photoinhibition Jones and Kok, 1966; Smith ef al., 1980!, and Cullen et al., 992!. As discussed below, none of these biological weighting functions produced results consistentwith Jokiel and York's 984! data. In contrast, reasonablyconsistent agreementwas achieved by generating an action spectrum of the form e'>' as suggestedby Rundel 983!, To test the sensitivity of the methods, we also investigated action spectra of the formsI'e'<" andk"e'~' N =0,1,2,3 and a, varyingso as to producea widerange of results. By making use of the relationship Rundel, 1983! between differential biological response and differential biologicai effective dose, ~~ = f AE X!c X!dX ! it is possible to associate observed fractional growth reduction with the correspondingchange in dose rate, Jokiel and York 984! observedthat only UV-A and UV-B caused long-termgrowth inhibition, so we took the growth rate for PAR at a particular intensity to be our baseline. The fractional growth reductionfrom this value for a giventreatment is plotted against the difference in calculateddose. No assumptionis made a priori aboutthe shape of this curve; all that is required is a relative agreementfor the effects of the various light regimes consistent with the E !,! being tested. 53 Someexisting action spectra nor O c 0 Q. Ol O 5l IO 0 0 0 4P Cl 300 310 320 330 Wavelength fnrn] Figure 2 Figige2.Vanpue b'0!og~Wiegnttng ~, nOnrialzgd IO290nrn, verSus wavelength. Genera!IZeCIplant Ca 4tell. 1971!,CullenDNAat al, 1992!.ssthw, 1974!,Phoirsnnibition JonesKKok, 1986!, E~ As,1979, 1992!, Theequipment necessary toaccurate! y determine thespectral irradiance inthe ultraviolet portionofthe spectrum wasnot available duringthese experiments. Asa consequence, surface irradiancevalueswere estimated usingan atmospheric model Green efal., 1980! with atmosphericparameters chosento match thelocation andtime ofJokiei and York's work. The modelallows a good firstorder estimation ofthe incident spectral irradiance, andprovides an accurateestimate ofthe spectral shape inthe ultraviolet region ofthe spectrum. Ourabsolute abridgeddoseratesspectrophotometry. will be in error, butthe relative values areaccurate andsuitable forthis technique of DISCUSSION Wefound the 'best fit'of observed growth inhibition tocalculated doserates was p'i.! = e ~ ! whereX isthe wavelength innanometers!. Figure3a,b,c shows thesteps involved informulating thisconclusion. InFigure 3a,the relative action spectrum isshown with respect tothe various 54 filtered radiation regimes. The various irradiance regimes are products of the incident solar irradiance and the transmittance of the filters [E X! = E ., X! T X!]. We show data from 280 - 450 nm Sinoe fOr X < 280 nm E ., k! iS inaignlfiCant and far k > 450 nm c X! iS inaignifiCant. Figure 3b shows the integrand of Eq. f!, and the biologicallyweighted fluence rate as a functionof wavelength spectral dose vs, wavelength!. Figure 3c is a plot of the observed fractionalgrowth reduction versus changes in dose for the four neutral density filter treatments in the PAR + UV-A + UV-B light regimes. E X! is reduced through the use of neutral density filters which changes the magnitudeof the dose, calculated as shown in Figure 3b, The difference in dose due to the use of neutral density filters is what is being referred to as "change in dose" on Figure 3c and in later figures. In contrast to the relative agreement shown in Figure 3c, the results for non-consistent action spectra;a! c,~ ., b! Epi, c! EpNpand d! r~~ areshown in Figures4 and5. Thedivergence in Figure 5 between the calculated dose rates for UV-A 's! and UV-B 's! give a clear indication that the action spectra used does not represent an accurate description of the biological weighted response to spectral irradiance. These four action spectra are insufficiently weighted in the UV-A region to agree with the results of Jokiel and York 984!. There are at least two reasons why the dose versus effect plots are inconsistent: first, the weighting function under test is incorrect for the biological effect studied; second, the assumption of reciprocity i.e., dose is the product of intensity times time! may not hold for the range of doses given in the experiment. We have no information with respect to dose versus response for the growth inhibition observed here. Assuming reciprocity holds, we conclude that biological weighting functions like s . , which shows a relatively high weighting in the UV-A region of the spectrum, is required for consistency with the long-term growth inhibition results observed by Jokiel and York 984!, If these results prove to be of general validity with respect to UV effects on natural phytoplankton populations, then the predicted influence of enhanced UV-B on these populations will be less than that predicted for co~ but greater that that for E Effective irradiance and Relative Action Spectrum satmsvslenq& tnmt Figuren Figuresa. IncidentSpeotral irradianoe fcr Severaliadiatiori regimeS and the UV-A RtmdeilaCtiOn SpeCtrum verSua WaVelerigth 55 Dose Rate Per Wavelength c5 ~ & 3IIo I Clqnrn3b Frgure3b. Ehotoocally wtaghted ftuenca rate dose! versus wavelength forthe radra0on regimes shown in Ftg.3a. Syme Odimum miCrOadrratiCum nt O n O s a 0 'Gn ~nI 3 2 Chnnqsin annn Figure3c. Free tonalgrowth ntductlon versus dtange in ologicaidose for the Uv-A radration regtrna 's! andthe Uv-B radlesollregime s!. 56 Dose Rate Per Wavelength Dose Rate Per Wavelength o aP o o o K o o i-. X o 260 290 300 310 32D 33G 340 28G 290 300 310 320 330 340 Erythema Action Spectrum Plant Caldwell! Action Spectrum Figure 4a Figure 4b Dose Rate Per Wavelength Dose Rate Per Wavelength Ie o o o o o o o 280 290 300 310 320 330 340 280 290 3DO 310 320 330 340 DHA Action Spectrum PhaeocfactylumAction Spectrum Figure 4c Figure 4d Figure4. Biologicalweighted iluenoe versus wavelength for severalradiation regimes and for various action spectra. 57 Symbiedirrirjm miCrOadriatiCum Symbiodinium microadriaticum o ErythemaAction Spectrum Plant Caldwel I! Action Spectrum Irr aal O 0 o 6 o ol o 6 c o C o o irr o < o 0.0 0 1 0.2 0.3 0.4 0.5 0.0 0.05 0. 10 0.15 Change In dose Change In dose Figure Sa Figure 5b Symbiodirrium microadriaticum S ymbiodini um microadriati curn DNA Action Spectrum c Phaeodactylum Action Spectrum cu o o O Irr er a o si o o o o or c o 0 o o ÃI riru u. 0.0 0.01 0.02 0.03 0.04 0.05 0.0 0. t0 0.20 0.30 0.40 Change in dose Change in dose Figure Sc Figure Sd FigureS ReSpOnSeverauS dOSe tcr uv-A 'S}and uV-8 ZS!radiation regimeS for vanOuS schon spectra ACKNOWLEDQEMENTS Thiswork was supported by Ray Smith,Icscolors '93- NSFNPP-9220952. LITERATURE C ITED: Caldwell,M. M. 971!. Solarultraviolet radiation and the growthand development of higherplants. In: Giese,A. C. ed,! Photopysiology,Vol 6. AcademicPress, New York, p. 131 - 177. Caldwell,M. M., Camp,L. B.,Warner, C, W. 8 Flint,S. D. 986!. Actionspectra and their key rolein assessingbiological consequences of solarUV-B radiationchange, In: 'StratosphericOzone Reduction,Solar UitravioletRadiation and Plant l ife', R. C. Worrestand M. M. Caldwell eds.! Spnnger-Verlag, Berlin. NATO AS> Series, Vol G8. pp. 87 - 111. Calkins,J. 975!, Measurementsof the penetrationof solarUV-B intovarious naturalwaters. Impacts of Climate Change on the Biosphere, CIAP Monogr. 5: 267 - 296, Cullen,J, J., Neale, P, J. & Lesser,M. P, 992!. Biologicalweighting function for the inhibition of phytoplankton photosynthesis by ultraviolet radiation. Scrence 258. 646 - 650. Green. A, E. S., Cross, K. R, & Smith, L. A 980!, improved analytic characterization of ultraviolet sunlight. Phofochem. and Phofobiol. 31: pp. 59 - 65. 58 Haeder,D, P. 984!. Effectsof UV-Bon motiiityand photoorientation in the cyanobacterium, Phormidium unci natum. Arch Microbial. 140: 34 - 39. Haeder,D. P. 985!. Effectsof UV-Bon motilityand photobehavior in the greenflagellate, Euglena gracihs, Arch Microbto/, 141: 159 - 163. Haeder, D. P. {1986!. Effects of enhancedsolar UV-B radiation on motile microorganisms,ln: 'StratosphericOzone Reduction,Solar UltravioletRadiation and Plant Life', R. C. Worrest and M. M. Caldwell eds.! Springer-Verlag,Berlin. NATO ASI Series, Vol GB.pp. 223 - 233. Haeder, D. P. {1987!, Photosensorybehavior in procaryotes, MicrobiologicalRev, 51; 1 21. Haeder,D. P, 8 Haeder,M. A. 988!. Inhibitionof motilityand phototaxisin the greenflagellate, Euglena gracilis, by UV-B radiation. Arch Microbiol. 150.' 20 - 25. Jeriov, N. G. 950!. Ultraviolet radiation in the sea. Nature 166: 111 112. Jokiel, P. L. & York, R. H. Jr. 984!. Importanceof ultravioletradiation in photoinhibitionof microalgalgrowth, LimnoL Oceanogr,29: 192 - 199, Jones, L. W. 8 Kok, B. 966!. Photoinhibitionof chloroplast reactions. 1. Kinetics and action spectra. Plant PhysioL 41: 1037 - 1043. Nachtwey,D, C. 8 Caldwell,M, M. [Eds.] 975!, Impactsof climatechange on the biosphere, Climate ImpactAssessment Program Monogr. 5, Part I, Ultravioletradiation effects. Departmentof Transportation,Washington, D, C,: PB247 724, NTIS,Springfield, Virginia. 674 pp. NAS.National Academy of Science,National Research Council 979!. Stratosphericozone depletionby halocarbons:chemistry and transport. Committee on Impactsof Stratospheric Change, NationalAcademy Press,Washington, D. C. NAS.National Academy of Science,National Research Council 982!. Causesand effects of stratosphericozone reduction: an update.Committee on chemistryand physicsof ozone depletionand the Committeeon Biologicaleffects of increasedsolar ultravioietradiation, NationalAcademy Press,Washington, D. C. NAS. NationalAcademy of Science, National ResearchCouncil 984!. Causes and effects of stratosphericozone reduction: update 1983. Committee on causesand effectsof changesin stratosphericozone, National Academy Press, Washington,D. C. Rundel,R. D. 983!. Actionspectra and estimation of biologicallyeffective UV radiation.Physiol. Plant, 58: 360 366. Setlow,R. 974!. The wavelengthsin sunlighteffective in producingskin cancer: a theoretical analysis. Proc. Natl. Aced. Sci., U. S. A. 71:3363- 3366, Smith, R. C. 989!. Ozone, middle ultravioletradiation and the aquatic environment. Photochem. Photobiol. 50: 459 - 468. Smith, R. C. 8 Baker, K, S. 979!, Penetrationof UV-B and biologically effective dose-ratesin natural waters. Photochem. Photobiol. 29: 311 - 323. 59 Smith,R. C. & Baker,K. S. 980!. Stratosphericozone, middle uitravioiet radiation and 14C measurementsof marine productivity. Science 208: 592 - 593. Smith,R. C. & Baker,K. S. 981!. Opticalproperties of theclearest natural waters 00-800nm!, Applied Optics 20: 177 - 184. Smith, R. C. & Baker, K, S. 982!. Assessment of the influenceof enhanced UV-B on marine primaryproductivity. In; 'The Roleof SolarUltraviolet in MarineEcosystems', Calkins, J. ed!. Plenum Press,New York. p. 509 - 537. Smith, R. C, & Baker, K. S. 989!. Stratosphericozone, rniddle ultraviolet radiation and phytoplanktonproductivity. Oceanography2; 4 - 10, Smith,R, C., Prezelin,B. B.,Baker, K. S Bidigare,R. R.,Boucher, N. P., Coley,T., Karentz,D., Maclnlyre,S., Maffick,H. A Menzies,D., Ondrusek,M., Wan, Z. & Waters,K. J. 992!. Ozonedepletion - ultraviolet radiation and phytoplanktonbiology in Antarcticwaters. Science 255: 952 - 959. Worrest,R. C. 982!. Reviewof literatureconcerning the impactof UV-Bradiation upon marine organisms.In 'The Roteof SoiarUltraviolet in Marine Ecosysterns',Calkins, J, ed,!. Plenum Press, New York. p, 429 - 457. Worrest,R. C. 983!. Impactof solarultraviolet-B radiation 90-320nm! uponmarine microalgae. Ptrysiol. Plant 58: 428 - 434. Worrest,R. C BrookerD. L &Van Dyke,k. 980!. Resultsof a primaryproductivity study as affectedby the type of glass in the culture bottles. Limnot. Oceanogr.25: 360 - 364. Worrest,R, C Van Dyke,H, & Thomson,B. E. 978!. Impactof enhancedsimulated solar ultravioletradiation upon a marine community. Photochem,Photobiol. 17: 471 - 478. Worrest,R, C., Wolniakowski,K. U., Scott, J. D., Brooker,D L., Thomson,B. E. & Van Dyke,H. 981!. Sensitivityof marinephytoplankton to UV-B radiation:Impact upon a model ecosystem. Photochem. Photobial. 33: 223 - 227. 60 UltravioletRadiattos and CoralReefs. f995. G.Gulkc S P. L Jckiel eds,!, HIMB Tech. Report set. UNIHI-SeaGrant-CR-95-03. Physiological and biochemical effects of UV radiation on the marine phytopiankton ItIannochioropsis sp. and OunaIiejla sp. Barbara J. Butowt and Terner Fisher t KinneretLimnotoqicail Laboratory, israel Oceanographic and Drnnological Research, POB 345, Tibenas, Israel. 2 Departmentof LifeSciences, Bar-lian University, Ramat Gan, 52tco, israel. ABSTRACT:The effectsof UV-A and UV-B radiationon photosyntheticparameters and antioxidatfvemecharvsms in NennaohfOrcpaiS Sp.and DunafieifeSp. Were inVeStigated. BOth UV-A and UV-B radetian CeuaeddeCreased growth ratesin Nannochforopsisbut didnot significanttyeffect fauna/ieffa growth. Photosynthesistn Wannoch!oropsiswas adversely affected pnmarily by UV-B as reflectedin the reductionof Rubisco and LHCP!I levels. After 48-hours, photosyntheticparameters such as lightutihzabon efficiency and quantum utiTization recovered from the initial shock of exposure to UV radiation. ln addison, there was a decrease in stress-respondingantioxidattve enzymesand correspondingincmases in Rubiscoand LHCPltlevels. UV-B causeda significant~ase in both Mn SOD, Cuzn SOD and Catalaseacfiviftes after 4-hours. Overall,Dunaliefla was lesssensitive to uv Streas.Possibly this was due to a wefl~vetoped oOntinuouacell repair mecharasmWhiCh preVenbad U Vqnduceddamage under these expenmentafconditions. INTRODUCTION Current concern over the world-wide effects of solar ultraviolet UV! radiation has led to biological investigationsspanning the molecularto comtnunity levels of ecological organization. As watercovers about two-thirds of the earth'ssurface, the importanceof understandingand predictingthe influence of UV-A and UV-B radiationon both freshwater and marine environments cannotbe underestimated,especially in view of their high penetrationin seawater Smithef al., 1992!. Phytoplankton,an integralcomponent in the biogeochemicalcycling process,are susce tibleto hotochernicaldamage due to UV radiation Jokief, 1984!, Any profoundeffect on this groupo s couldpossibly cause an imbalancein globalsupplies of photosynthetically fixedcarbon and disruptionof trophicdynamics. Oxidative stress and photosynthesisare intrinsicallyinterconnected. An oxidant is produced as a result of illumination, Suboptimalconditions for photosynthesis,such as high iight intensity OrIOw CO2 COnCentratiOnS,Cauae a deCreaaein the effiCienCyOf energy tranafer frOm phOtOnStO CO2 and reault in damage tO the ChIOrcplaetS. PhOtoaeneitiZere, SuCh aS ChlOrcphyll and ffavinS, absorb the UV wavelengths of solar irradiance, and form reactive molecules such as triplet excitedchlorophyll and active oxygenspecies e.g., 02, H202, and OH ! whenexcess light energy is not dissipated as heat or fluorescence Asada & Takahashi, l 987; Asada, !994!. Furthermore,these active oxygen species can oxidize membrane lipids and proteins, and cause generalcelt destruction Fridovich, 1986!. Both plants and algae possess complex antioxidative mechanisms which counter the adverse effects of oxygenradicals. These mechanismsbuffer energy transfer limitations which would ultimately lower photosyntheticactivity and prevent deleterious oxidation reactions within the cells. The superoxide 2 ! scavengingmetallo-enzyme, superoxide disrnutase SOD!, exitsin three mainforms: CuZn SOD chloroplasticand cytostolicj,Mn SOD mitochondrial!and Fe SOD. The disrnutationof 02. to Hzoz is rapidly followed by inactivationof this product by ascofbate peroxidaseor catalaseby reductionor disproportionationrespectively Asada, 1994!. Recent researchon endogenousprotective mechanisms in the zooxanthellaeof Aiptasia pa/lida, demonstratedsignificantly increased Super Oxide Dismutase SOD! and catalase activitiesafter exposureto UV radiation Lesser et al., f 989!. Cftaetocerosgracllis, a marine diatom.wassimilarly exposed to UV irradiationand while there was no significantchange in SOD activityover 48-hours,catalase activity did increaseon acclimationto PAR + UV Hazzard, 1994!, probablydue to an increasein UV-inducedoxygen radical production, In thisresearch we aimedto elucidatethe natureof definedUV-induced changes in SOD andcatalase activitiy in diferentsize marinephytoplankton. The relevanceof speciesand size differenceswas illustratedby Karentzef al. 991! who showed differentialcell survival characteristicsand photo-enhancedrepair under conditions of UV irradiation. Thereforeit is relevantto investigatethe effectsof differentUV wavelengthson defensemechanisms which couldaffect repair mechanisms in differentspecies. In addition,the site of the specific UV- inducedchanges is indicatedby the relativechanges in CuZnSOD and Mn SOD. In orderto gainan insightinto the effectsof UV-A and UV-B radiationon photosynthesis,we established Photosynthesisvs Irradiance P vs I! curveswhich providedinformation on the physiological statusof the cells and the potentialeffects of verticalmixing in the water column. To date, most of the work on photosynthesishas concentrated on the light intensity- dependentregulation of ribulose-1,5-bisphosphate carboxylas~xygenase Rubisco! and the light harvestingpigment protein complex of photosystemII LHCPII! Morttain-Bertrandet at 1990; Falkowski& LaRoche,1991!. Rubiscois the mainenzyme participating in photosynthetic carbonfixation and LHCPII is the main proteinconnecting the lightharvesting pigments of photosysternII PSll!. A positivecorrelation between photosynthetic parameters and Rubisco was reportedby Falkowskiand Laroce 991!. Also, changes in the amountof LHCPII and Rubiscowere found duringacclimatization to bothhigh and lowlight levels. It was concludedthat such modificationsin each protein pool play an important role in algal cells acclimating to changesin ambientlight regimes. Few reportshave examinedchanges in Rubiscoand L.HCPII levels underUV-A and UV-B stress,so this workalso givesan initialinsight into molecularphoto- acclimation due to UV radiation stress. MATERIALS AND METHODS Culturesof the marine phytoplankton Nannochjoropsissp. Eustigmatophycae! and Ounaliellasp. Chforophycae!were obtained from the culture collection of theHawai'I Institute of Marine Biology HlMB! and grownunder Westinghouse "Coot White"fluorescent lights. The cultureswere inoculatedin nineliters of F2 mediumin orderto obtaincells in a logarithmicgrowth phaseprior to day 1 ofthe experiment.Duplicate flasks of cultures for eachtime period!were incubatedin an outdoorwater bathand exposedto three differentirradiation treatments: T ryan @mtej ~FI!te Full Solar Spectrum UVT! AclavS 33c Ruoropolymerfilm UV-B+ UV-A+ PAR! No UV-B UVA! MylarS Type D Ruoropolyrnerfilm UV-A+ PAR! No UV UVO! 100'%%dClear Acrylic Safety Glazing sheet PAR! Thecells were analyzed after 4-, 24- and48-hours exposure to thedescribed conditions. Thenatural irradiation was plotted during the experimentsto insure that there were no major differencesinradiation dosage throughout the ~ay experiments.Rgure 1 showsa typical24. hourradiation profile as measuredand recordedat the weatherstation located at the Point Laboratory,HIMB See Gulkoef al., thisvolume!. Ateach time period a sampleof eachduplicate treatment was fixed in Lugol's solution and thecell number was determined using a hemocytometercounting chamber and a light microscope.Chlorophyll a and carotene were calculated for each treatment after filtration on a GFC filter and overnight extraction in90'%%d methanol Jeffrey & Humphrey,1973!. 62 P t nth tic res nse A Photosynthesisvs Irradiance P vs I! curve was establishedfor each of the treatments using a YSI oxygen rnicroelectrodewhich measuredevolution of oxygen during photosynthesis. Theoxygen released was converted into an electronicsignal and the data was collectedusing an analog system. Photosynthesiswas then calculated per unit cell for chlorophyll Dubinsky et al, 1987!. The photosyntheticparameters were calculatedaccording to Fisher 987! and included derivationof the optical cross section. The optical cross section is defined as the in vivo, spectral average, chlorophyll a-specific absorption cross section a ! as calculated from each culture's absorbance and chlorophyll a concentration Fisher, 1987!. t 600 f400 i200 tooo 4 oL' 800 5 0 N. 6oo 2 400 200 8 8 8 8 8 8 8 8 8 8 8 C3 C4 $ g Ijg 0 A 0 4> 00 0 Time Figure t. A typical natural surface rad ation environment protila over a 24 hour period showing photosyntheticallyaosve radiation PAR! and total ulti avioiat radiation UV ~4oo nrn!. ntioxidativ act lv The cells were concentrated by centrifugationat 5.000 rpm and homogenateswere prepared usingglass beads and a Teflonshomogenizer. The resultingsuspension was spundown at 14,000 rpm and the supernatant used as the crude enzyme extractafter filtration on a 0.45 p.m Milliporets filter. Catalase activity was measuredspectrophotometrically according to Beers and Sizer 952!. CuZn SOD and Mn SOD activities were quantifiedby a modiTiedend-point method Oyanagui, 1984!. Total SODwas assayed in the absenceof KCNwhereas Mn SOD wasmeasured in the presenceof 3 mM potassiumcyanide. CuZn SOD KCN-sensitive!was calculatedas the difference between total SOD and Mn SOD. Total protein was estimated according to Bradford 976!, The hornogenatedescribed above was further puriTiedaccording to Sukenik et al., 992!. The resulting protein extract was analyzedfor Rubiscoand LHCPll content using sodium dodecyl sulfate SDS! polyacrylamide-gelelectrophoresis PAGE! describedby Laemmli 970!. After separation, 10 pg protein was loaded onto each fane and the gel was stained with Coomassie Brilliant Blue. The positionsof the Rubisco and LHCPll proteinswere verified by comparison with peroxidase-stainedgels which were reacted with antibodieslo Rubiscoand LHCPIIusing the Westernblot procedure, Polyclonal antibodies against LHCPil and Rubiscofrom Nannoch/oropsiswere kindlysupplied by Dr. A. Livne,I.O.I.R., Haifa, Israel. A densitometer scannerwas usedto calculatethe amountsof Rubisco largeand smallsubunit! and LHCPII accordingto theirarea on the gel. Followingtheir staining,the gels were photographedusing a video camera. Beforethe opticaldensity was measured Image Pro Plus8 program!,the gel was calibratedagainst the backgroundusing increasing concentrations of protein.Values presented are averagesof duplicatesamples and were calcuiatedas L area/10 Ii.gprotein; the relative amountsof Rubiscoand LHCPIIwere determinedfor each treatment. The Lowrymethod 956! was usedto determineprotein concentration. RESULTS The cloudynature of the weatherin Hawai'icaused fluctuations in the radiationprofiles, but the overallradiation intensity PAR + UV! did notvary significantlyduring the courseof the two, 3- dayexpenments. Midday PAR values, however, varied from &00-3400 pmol photons s' m 'and UV valuesvaried from 3% mW cm', The patternof incidentUV radiationwas alwaysparallel to that of PAR alone, elf ro I Both Nannoch/oropaisand Duna/ie//ashowed an increasein cell numberduring the courseof each ~y experiment Figs. 2a and 2b!. Dunalielleshowed little differencein growthbetween the three radiationtreatments although at the endof 4&-hours,both UVA and UVT gave a slightly highercell number,Conversely, Nannoch/oropsis showed a weIIMefinedresponse to PAR radiationonly; the number of cellswas higher at 24-and4&-hours than underUVT or UVA treatments. Chlorophylla contentof Nannoch/oasis decreasedon exposureto all radiationtreatments exceptinitially to UVO!. Thiswas also true for Dunaliellaalthough there weresigns of a recoveryin chlorophyllcontent after 48-hours. This decreasemay have beendue to the sudden exposureto naturalhigh irradiancesas comparedto the Iow laboratorylighting. The ratioof caroteneto chlorophylla increasedwith time forall treatmentsfor Nannoch/oropsis,while this valuestayed constant throughout the experimentfor Dunalie//a. P t nthe ' me r Typicalhyperbolic curves were obtained when the photosynthetic potential of the two species was measuredunder increasing irradiation intensities; representative P vs I curvesfor Nannoch/orapsis after 4-hour exposure! and Dune/iella atter24-hour exposure! are shownin Figs.3 and4, Photosynthesisincreased up tc a saturatingirradiation intensity I ! between60- 240@mole photons rn ~s ', andthen either remained stable or decreased with further increases in irradiationintensity. There was a decreasein P when exposedto UVTfor both NannochioropsisandDunalie//aon a per unit chlorophyll and per cell basis. In Nannoc/i/oropsis, P per unitchlorophyll increased under a UVAregime; the opposite was true for Dunalie/la, whereUVA inhibitedphotosynthesis more than UVT. Oeteilsof the effectsof UV radiationon differentphotosynthetic parameters are shownin TablesI andII forNannoch/oropais and Dunaliella, respectively On a percell basis, the radiation utilizationefficiency of Nannochloropsisdecreased steadily after 24-hours of exposureto UV-A andUV-B radiation, as comparedto PARonly. UV-A + PARresulted in a iessdramatic decrease in photosyntheticefficiency after 24-hours. After 48-hoursof exposure, all Nannoch/oropsis samplesshowed similar photosynthetic efficiencies per cell!. The UVT treatment also adversely affectedthe optical cross section a'! afteronly 4-hours, but this recovered' after 24-hours. The maximaquantum utilization forboth UVT and UVA treatments were both much higher than the control UVO! value after 4-hours of exposure. However, 24-hours of exposurecaused a great declineinthe quantum utilization and by 48-hours, cells exposed toUVT gave only half the value of cells exposedto UVO. Unlike Nannochlorapsfs,the photosyntheticefficiency of Dunaliel/a increasedafter 4-hours underUVT and UVA treatmentscompared to PAR-onlycells This situation was reversedafter 48-hours. The a values for Dunaliellacells exposed to UVT and UVA were greatly reduced after 4-hours relative to the UVO treatment. After 48-hours, values were more comparable with UVT-treatedcells having the highest opticalcross section. Quantumutilization valuesfor Dfjnaliefta show that UVT caused an immediate decline relative to UVO treatment and that this effectwas sustainedthroughout the 48-hourtreatment. Althoughno clear patternwas shown, therewas a generalincrease in respirationafter 4- and24-hours for both algae, which then decreased after 48-hours. a!.11 1070 510 Time h! 0.9 0.8 0.1 0.6 8 0,5 8 0.4 0.3 0.2 10 Time b! Figure 2. Changes in cell numbem over e 48hour period of exposure to UVO PAR only!, UVT PAR+UVB+UVA! end UVA PAR+UV-A! for a! hlarjnochioropeisand b! Dunefie/fe. UVO E 4.5 8x »10 3.6 6x »10 3 2,6 4x10« 1.5 2 x s310 O 0.5 Al -0.5 0x10-10 -2 O -1. 5 -4x a-» 200 400 600 800 1000 R I rra dian ce E 4.5 ex »10 3.5 6x m« 8 Z5 4x1010 1.6 2x »10 'OC4 0 0.5 -0. 5 0-2x10 10O -1.5 -4x10-10 0 200 400 600 800 1000 I rr a di an ce UVA E 4.5 Sx %10 3.5 6x»10 CJ 2.5 4x 101 0 1.5 2x %10 O 0.5g g -05+ 0 -2x10100 -1,5 4x10 10 0 I rra di en ce Figure3. Photosynthesis vs.Irtadssrce curve after 4h exposure forIVerxrochtoropsiia Photosynthesis expressed perunit chIorophyll{tt!and per cel {F!. Irradiance unit~ol photonsrn2 s 1. UVO 12 2x10-8 E 10 86 1x10 8 c7 4 0.5x10 8 0 0 0 CV %.5x10-8 2 O -1 xt~ 400 600 1000 f rradia nce UVT I 12 2x10+ E 10 'tx10 8 8 6 is 0.5x10 8 cu 2 0 0 cu -2 0Sx10-8 -0. 0 I -6 -1x108 200 400 600 1000 I rradi a nce UVA 12 10 8 6 1x10 8 Ie 0.5x10 8 Al 2 0 0 0 io -2 -0.5 x10-8 -1x10 8 200 400 600 SOQ 1000 irradiance Figure4. Photosynthesisvs frradiancecunre after 24h exposure for Durialia/la.legend as in Fig.3. 67 UVO E 8 x 1010 E 4.5 6 x 1010 3,5 4x1010 2,5 2 x10'0 g 1.5 O 0 0.5 -2 x10 10 O -0.5 4x10 10 a -1.5 0 200 400 600 I rra diance E Bx 1010 45 6x 1010 3.5 4x 1010 8 25 2x 1010 1.5 0 0 0.5 -2x 10-10 g -05 4 x 10-10 -1.5 0 200 400 600 I rradi an ce UVA E 8x 1010 4 5 6x 1010 3.5 4 x 1010 2.5 2x 1010 g 1.5 0 05 0 -2x1010o I -0.5 4x10-10 cu -1.5 0 I 0 200 400 I rra d i an ce Figure3.Photosynthesis vs.irradiance curveafter 4h exposure forNannochloropsis Photosynthesisexpressed perunit chlorophyII ! andper cali e!. Irradiance units=ttrnol photonsrn 2 s-t. D> 0 RRR RR RR IZ 0 U CDN 0 IA d D CA IA d N 0 cCp p~p p p pp 0 NIA 0 N ID NN z CDZ N ~ IA 'N ID CA CD IA OO~ O IA 000 'QO ID IA N CDQQ CACAN CDNO O 0 Z CDIDN CDCD CD CDCA CD CA CD OO CANIA CDOO O 0 IA N ID w CDID CA N DIN 0 W CIcCD P cn CDw ~ l IAW CDOCD CAIA CD O 0 Cfl 00 0 OO 0 Cfl O. 0 0 ID CD C7INN CDgDI OQ MID IA g! CN ON 8! .IA Z CA' ecA' IA ZcA OoocD 0 0 Z0 UC c g CA CAcDN CA .c Dc IAIA 0 2 1.8 c 1.6 1.4 h. 1.2 E .4 0.8 ~ 06 0.4 0.2 0 14 108 6 c 4 0 Houa Figure5 The effeCtOf UV radiatiOn on CuznSOD and Mn SODaottVity in NannOCniaropeiSUnitS are abSOlute vnnS Of active. 69 0.6 0.5 0.4 O 03 0.2 0.1 24 Hours 3.5 c 2,5 2 r50.51.50 Hours Figure 6. The effeCtOf UV radiatiOnOn Cuzn SOD and Mn SOD aCtivityin Ounafiella. Units are absoluteunitS Of activity SOD activity in both species showed a general decrease with time; different irradiation regimes apparently altered the dynamics of SOD activityfor each time and for each species, Both Nannochloropsisand Dunaliellashowed an increase in Mn SOD and CuZn SOD-specific activity after only 4-hours exposure to UVT UV-A+ UV-B + PAR! as compared to UVO PAR only! or UVA PAR + UV-A! Figs. 5 and 6!. There was a drop in OunallellaCuZn SOD act~vity after 48-hours for all treatments, although no notabie differences were observed between the treatments when measured after 24- or 48-hours, There was a significant drop in both NartnochloropsisCuZn SOD and Mn SOD activity after 24-hours for all treatments, althoughthe differences between treatments was slight. After 48-hours, the only discernible difference in activity was slightly higher CUZn SOD in UVT and UVA treated algae. Overall, SOD activity in 70 Nannochforopsiswasgreater than that of Dunalieila, and Mn SOD activity was higher than CuZn SOD for both algai species. 80 C 70 60 50 40 30 20 10 0 24 Hours Figure7. TheeffeCt Ofuv radiaticnoncatafaSe aCtiVity inNarrnachlOrapeis. 14 12 10 T5 8 84 6 24 Hours Figure8 Theeffec! of UV radiation on catalase activity in Dunaliefla Narrnochloropsis catalase activity in both the UVO and UVA treatment increased relative to the UVT treatment after 4-hours Fig. 7!. There was no significant difference between treatments after 24-hours; after 48-hours,there was a discernible difference in actrvityunder UVO conditions when compared with UVT, UV-A + PAR UVA! caused a significantdecrease in catalase activity relative to PAR-only UVO! in Dunaliellaafter 4-hours Fig. 8!, but this changed after 24-hours to prompt a significant ~ncreasein activity relative to UVO and UVT treatments!, After 48-hoursexposure, both UVT and UVA treatments gave higher catalase activities than UVO in Dtjnali ella . Rubisco and LHCPII Rubiscoand LHCPII for Nannochloropsiswere analyzedfrom stained PAGE gels Fig. 9!, and the results detailed in Table III. Afterexposure to UVA and UVT treatments for up to 24- hours, Rubiscolevels in Nannochloropsisdecreased, respectively,by factors of 1.6 and 2.0. In this alga, LHCPII levels also dropped after 4-hours; by a factor of 3.0 for UVA and 1,7 for UVT treatments. After 24- and 48-hours,there was no significant change in LHCPII levels. No clear trend was discerned in the effects of UV radiation on Rubisco and LHCPII levels in Dunaliella. a! 4 hours b! 4 hours c! 48 hours Figure9, 12 5% SDS-PAGEOf NannooftlarcpSia extraCts showing Aubisco and ! HCPliproteins after a! 4h, O!24h, and c! 48h exposureto UVO,UVTand UVA. 000 000 000 CG 0 00 0 lh D 000 g a 0 Cl 000 000 CIIn ID noDC 0 0 ICIn DC II3n CII e D .o ODCn 0 One 440 Z~ CCI ZZ 0 00 0 0 0 I 0 0 0 D CCI< hl CCIn 0 ~ze 0 0 CD CCI Z Z IC O OOO 0 .O 0 I ID e E z OZID LL Z Z CD CCt O gOCD E - e I- cc K n D e ZZe 03 c CD c 0 Co O 0E E E lh 0 CflCll bl O ~ G3 ggO G3 C O I 4 ZZw CD C CCC D3 0E C0G3 O 0 0 n 0 0 Ch Ch G3 CD 4 ON CDc CDh Al n~e Ch CCI 0 CU 03 " E wchn I D E CllCC + DCAl DC 0IO OL 0c «33Om 0 D 0 G3 c 0 Ct3 CD0 0 Ih n DC V «D 0 D W IDI Dl O ctnc Q. Ch 000 0 0 0 0 0 IG3 0 OO0 OO0 OOO 0Ch D D V3 0 0 D D E e 0 'tl CD cDp! e 03 0CV E Cb C4Cil 0 0 0 000 OOO 000 E c c O OG3 OG3 000 000 0 0 Cl. 0 D3 Q Ch D CD 0 Gl 0 0 Ch n «D 0 E «D new CDCll 0 O Ch C0 C6$ lh N IU E0p w CDc PV D 0. Pl Q O C03 0 CD Ch 0 g 0 nOO 000 O coo CD 03 Cc« 0 03 E Q ID ~ e c p. CD c c 0 c CD G3 0 0 CD 0 D 0 CD c O G3 C D «3 «D 0. CT G3 0 D D O c «D 0 E D 0 0 0 E C CD E 3C3« Y CD CD D Ct ZO E U 0 D DISCUSSION This set of experiments set out to discern the potential physiologicaland molecular damage caused by natural radiation to the photosyntheticmachinery in two marine algae; Nanochtoropsis and Dunaiiella. The importanceof antioxidant enzymes in the detoxificationof intracellularfree radicals formed by UV-A and UV-B was inferred from the results of these investigations. Despite a fluctuating radiation environmentand limited time for research, it was shown that ultraviolet radiation adversely affected the ability of two diverse marine algae to fix carbon. UV-B radiationsignificantly inhibited Nannoch/oropsis'photosynthetic paraineters, probably as a result of the inhibition of Rubisco which directly controls P, . A similar trend was also shown in Chaetoceros Hazzard, 1993!. In both Nannoch/oropsisand Dunalieiia,partiai destruction of LHCPII was caused by UV-A and, to a much lesser extent, by UV-B radiation. It appears that there is a differential sensitivityto UV-A and UV-B by photosyntheticallyimportant proteins,as well as by different algal species. UV-A is also invoived in photo-enhancedcell repair and actually assists in cell survival Karentz et al., 1991!. Both Nannoch/oropsisand Duna/iet/ahave the potential to counter the increased risk posed by UV radiation. This was suggested by the rapid increase in SOD Mn and CuZn! levels in both species and catalase in Nannochioropsisexposed to UV-A + UV-B + PAR UVT!. It is our conclusion that UV-B radiation alone caused an increase in 0, radicals and H,O, which, in turn, induced higher activities of SOD and catalase production. During this early period of antioxidative activity,there was a simultaneousresponse in certainphotosynthetic parameters indicating a weakeningof the systemdue mainly to the UVTtreatment. For example, after 4-hoursexposure to UVT, Nannoch/oropsisshowed decreasesin radiation utilization efficiency, optical cross section, quantum utilization, and overall photosynthesis. The simultaneous increase in catalase and SOD activity, however, may have aided in the eventual recovery of photosynthesis. Additionally, in Nannochtoropsis, there was an overall increase in carotenoids and in the ratio of carotenoidsto chlorophyll a, which would also have afforded additionai protection to the cells. It is possiblethese algae have UV-protective pigments i,e mycosporine-likeamino acids! which could also aid in the photoadaptiveprocess Smith et al., 1992!. Another indicator that the cells wereless stressed after 48-hours, possibly due to photoadaptationand repair of cell damage, was the decreased respiratory rate for all treatments. Besides the obvious morphologicaldifferences between the two species that were chosen, it was shown that they also have characteristicantioxidative mechanisms. For example, Nannochtoropsls which is 40 times smaller than Dune//ella!had much higher SOD and catalase levels at time 0!, possibly because it is more readily swept to the water surface, and is less able to avoid UV radiation than the denser flageltate. Alternatively,the higher protective activity may have been a local response to the initial switch from indoor growth at moderate irradiationto high outdoor irradiation intensities. The resultsalso indicate that the turnoverof Mn SODand Cu/ZnSOD in Nannoch/oropsisis perhaps slower than that of Dunatiettasince, after 24-hours exposure, SOD activity still fluctuated under the different irradiation treatments, It is also possible that the DNA repair mechanisms in Nannoch/oropsismay be more severely affected by UV-B present in the UVT treatment, hence the relativedecrease in SODatter 24-hours. These conjectures require further in-depth research not only on a physiologicallevel, but on a transcriptionallevel as well. An overall comparison of the effects of UV radiation on Nannoch/orapsisand Dunalielia indicatedthat the largeralga, Dunall ella, showed superior photosynthetic ability and irradiation utilizationefficiency, and wasbetter adapted to long-termstress. Fewersignificant physiological or biochemicalchanges were seen. It is suggested that the smaller Nannoch/oropsisis more sensitive, yet better adapted to sudden, short-term exposures to UV radiation stress. This was indicatedby the relativelyhigh turnover and reconstitutionof Rubiscoand LHCPII by IVann och/oropsis. TableIV: Theeffect of differentuv regimes on the Rubisco and LHCP11 proteins SDSPAGE analysis!. Values expressed in %%d ofarea protein according to densitometer/10pgprotein. The standard deviation for duplicatetreatments varied +/- 5 - 10o%%dof the average given values. ACKNOWLEDGMEhlTS:We thank Ors B. Kirtzie ill, P.Jokiel arid G. Grau for their help, care arid use ot facilities; Or.M. Lesser foruseful discussiorts andericouragemerit, aridF. Te. S. Saritos and Bernie for their techitical support Wealso thank Or. Z OubirtskyThis research was supported bythe Edwin W. Pauley Foundation and the Lhited States-israelBiNaooitaf Science Fourtdatiori L ITERATU RECl TED Asada,K. 994!. Productionand action of activeoxygen species in photosynthetictissues. In: 'Causesofphotooxidative stress and amelioration ofdefense systems inplants', Foyer, C. J. & Mutfineaux,P. M. eds.!. CRCPress inc., pp. 77- 104. Asada,K., Takahashi, M. 987!. Productionand scavenging ofactive oxygen fn photosynthesis. Irr.'Photoinhibition', Kyle, D. J., Osmond, C. B.,Arntzen. C.J. eds.!.Elsevier, Amsterdam, p. 228 - 287. Beers,R, F. Jr. K Sizer, I. W. 952!. A spectrophotometricmethod for measuring the breakdown of hydrogenperoxide by catalase,J. Eliol,Chem. 195: 133 - 140. Bradfbrd,M-M. 976!. A rapidBnd Seneitive methOd far the quantifiCatiOn of miCragrarn quantitieS of proteinutilizing the principle of protein-dyebinding. Anal, Biochem. 72: 248 - 254, Dublnsky,Z.,Falkowski, p.,post, A. F. g yanHenf, U. M. 977!. A systemfor measuring phytoplanktonphotosynthesis in a definedlight field with an oxygen electrode, J. PlanktonRes. 9: 607 - 612. Falkowski,p.tf, LaRoche, J. 991!. Acclimationtospectral irradiance inalgae i. Ph1rcol.27:8 - 14. 75 Fisher,T, 987!. The effectof photoadaptionon photosynthesisand growth of some rnicroalgae. MSc thesis. Bar lian University, Israel. pp 1 -86. Fridovich, I. 986!. Biologicaleffects of the superoxide radical. Archs, Biochem. Biophys, 247; 1 - 11. Hazzard,C. 993!, Acclimation of marine phytoplanktonto ultraviolet radiation. PhD thesis. University of Hawai'i. pp. 47- 83. Jeffrey,S. W. & Humphrey,G. F. 975!. Newspectrophotornetric equations for determining chlorophylla, b, c andc2 in higherplants, algae and natural phytoplankton. Biochem, Physiol, Pfanz, 167: 191 194. Jokiel,P, L, 984!. Importanceof ultravioletradiation in photoinhibitionof microalgalgrowth. LimnoL Oceanogr. 29; 192 199. Karentz, D., Cleaver, J. E. 8 Mitchell, D. L. 991!. Cell survival characteristicsand molecular responsesof Antarctic phytoplanktonto ultraviolet-B radiation. J. Phyco!. 27: 326 - 341. Laemmli,U. K. 970!. Cleavageof structuralproteins during the assemblyof the head bacteriophage T4. Nature 227: 680 - 685. Lesser, M. P. & Shick, J. M. 989!, Effects of irradianceand ultraviolet radiation on photoadaptationin the zooxanthellaeof Aiptasiapallida: primary production, photoinhibition, andenzymic defenses against oxygen toxicity. Mar.Biol, 102:243- 255, Lowry,0, H., Rosebrough,N. J., Farr,A. L. 8 Randall,R. J. 951!. Proteinmeasurements with the folin phenol reagent. J. Biol. Chem. 193: 265 - 275. Morttain-Bertrand,A., Bennett, G. 8 Falkowski,P. G. 990!. Photoregulationof light harvesting chlorophyll protein couples associatedwith PS II in Dunaliella. Plant Physi ol. 94: 304 - 311, Oyanagui,Y, 984!. Reevaluationof assay methods and establishment ofkit forsuperoxide dismutase activity. Analyt. Biochem. 142: 290- 296, Smith,R. C., Prezelin,B. B.,Baker, K. S Bidigare,R. R.,Boucher, N. P., Cofey, T Karentz, D., Maclntyre,S Matlick, H. A Menzies, D., Ondrusek, M., Wan, Z. & Waters,K. J. 992!. Ozonedepletion: ultraviolet radiation and phytoplanktonbiology in Antarcticwaters. Science 255: 952- 959. Sukenik,A Livne, A., Neori,A., Yaakobi, Y. Z. & Katcoff,D. 992!. Purificationand characterizationof a light-harvestingchlorophyll protein complex from the marine EustigmatophyteNannochloropsis sp. PlantCell Physiol.33: 1041- 1048. UltravioletRadiation and Coral Reefs. 1995. D Gulko& P L Jokiel eds! HINIBTech Reporte41 UNIHI-SeaGram CR-95-03 Distribution of mycosporine-like amino acids in the tissues of Hawaiian Scleractinia: A depth profile I. B. Kuffner, M. E, Ondrusek, and M. P. Lesser Hawai'ilnebtute Of Marine BiOIOgy, P O BOx1346, Kans'Ohe. Hl 96744 2 univeraityOtHawai'i, Department ofOceanography. 1000Pcpe Road, HOnclulu, Hl 3universityof New Hampshire,Department of Zoology,Durham, NH 03824 ABSTRACT:The bssues of fivespecies of Hawaiianhermatypic cora la were found to containconcentrabons of MyCOSPOnne-likeaminO aoidS MAAs! inveraely COrrelated todepth and dOSe Of UV radiabOn FOur Of the Six depthprOfileS COnduCted reeulted in highlySignifiCant p< 01! linearrelabcnahipS between MAA COnCernrabon and UVradiabcn level MOntjeravenuCOSa, iMOnfiipcra palula, pcnteS COmpreSSa, Pociifopora damiCOmiS and Pocrffcpora~ria werecollected at a seriesof depths in andoutside of Kans'oheBay, 0'ahu, Hl and analyzedusing HPLC to idenbfyand quarrbfythe UV-absortxngcompounds. Eight knownand two unknown compoundswith absorption maioma ranging from 313 to 360nanometers were separated and quantrlied. Spectroradiometncmeasurements were made simultaneously dunng sampbng to quanbfyand analyze the light regimeat the collectionsites. INTRODUCTION Hermatypiccorals have evolved to flourish in environmentscharacterized by clear, oligotrophicwater. The transparencyof this waterresults in an extremelylow attenuation coefficient,and organismsliving there are exposedto largefluxes of UV radiation Jerlov,1950!. Uv-A wavelengths 320 - 400 nm! and Uv-B 80- 320 nrn! radiation has been found to cause substantialphysiological damage to organismsexposed at irradiancelevels commonly emitted by the sun Caulkins, 1982!, especially at low latitudes where the ozone layer is thinner allowing higherlevels to reachthe Earth'ssurface. Targetsof adverseeffects in biologicalsystems include the informationcarrying nucleic acids and proteins. The recognition of UV radiation as a seiective pressurewithin a coralreef community has been welldocumented Jokiel 1980!. Behavioral adaptationssuch as avoidance,diurnal migration and cryptic lifestyles are not availableto sessile organisms,specifically corals that needto be exposedto the sun for their photosynthetic zooxanthellae symbionts. This lifestyle presupposes the existence of a protective mechanism against the sun's damaging rays. First discovered in corals by Shibata 989!, a group of water soluble compounds since termed Mycosporine-like Amino Acids or MAAs! absorbing in the 320 nm range have been found to perform this function. These compounds have been identified and quantifiedin many marine and terrestrial organisms Chalker 8 Dunlap, 1990!. Dunlap et al. 986! conducted a study of the bathymetric distribution of MAAs in corals of Australia and found a considerable decline in the concentration of S-320 compounds in Acropora spp. with depth. This pattern was presumed to be an adaptation to the amount of UV radiatio~ found at those depths. In this study, quantitative analysis of light regime has been correlated with concentrationof seven described MAAs in five species of corals. Corals were collected along a depth gradient at two sites varying drastically in spectral quality, one inside of a bamer reef and one on an open-ocean island. A comprehensivebaseline survey of the MAAs occurring in Hawaiian corals was also conducted in this study. MATERIALS AND METHODS Corals were collected using snorkeling and SCUBA techniques on the windward side on 0'ahu, Hawai'i 1o N, 157 W! during the summer months of June and July. Corals were collected at two sites; inside Kane'ohe Bay at a site called the Sliver Reef, and outside of the bay in open ocean water at Moku Manu Island. The conditions inside the bay are characterized by a high attenuationof light due to a high level of particulate matter. The open ocean site at Moku Manu displays relativelytransparent, oligotrophic water with a low level of attenuation of light through the water column. The spectral irradiance 00-700 nm! was quantitatively maes~red at both sites ualng a UCOrLI-1800UW SCanning SpeCtraradiameter LiCOr, LinCOln, Nebraska!. The cosine- corrected collector and sensors were programmed to scan from 300-700 nrn in 2 nm intervals. All measurements of ambient soiar irradiance were made at approximately 12 pm. For each depth 77 threescans were taken and the mean reported inunits of rnW m' nm'.The instrument was deployedand coral samples simultaneously collected using SCUBA. Montipora verrucosa was collectedinside Kane'ohe Bay Sliver Reef! at 1.5 m, 3 m,4.6 m, 6.1 m, 7.6 m and9.1 rn, and at MokuManu at9.1 m, 12.2 m, 15.2 rn, 18.3 m, and 21.3 rn, Pocilloporameandrina andMontipora patulawere collected at6,1 rn, 9.1 m, 12.2 rn, 15.2 m, 18,3 m, and 21.3 m atMoku Manu. Porftes compressaand Pocillopora damicomis were collected at1.5 m, 3 m,4.6 m, 6,1 rn, 7,6 m and 9,1 mat Sliver Reef, Branches ofeach cofony were harvested andplaced inplastic bags for transport backto the lab where they were immediately frozen at -50 C untilthe time of extraction. Coralsamples were thawed, cleaned ofepiphytes, broken into small pieces, and placed ina knownvolume of100 /o HPLC grade Methanol toextract overnight at-20'C, The extraction and analysisofMAAs were performed according tothe procedures inDunlap and Chalker 986!, Dunlapetal. 986!, and Shick efal. 992!. Samples were extracted in5 cm'HPLC grade 100/. methanol.Individual MAAs were separated byreverse-phase isocratic HPLC on a BrownleeRP-8 columnprotected withan RP-8 guard, inan aqueous mobile phase including 0.1'/o acetic acid and 45'/omethanol. Detection ofpeaks was by UV absorbance at313 and 340 nm. Identities ofpeaks wereconfirmed byco-chromatography withstandards ofMycosporine-glycine, shinorine, porphyra-334,palythine, asterina-330, palythinol, andpalythene. Peaks were integrated and quantificationofindividual MAAs was accomplished usingthe quantitative standards listedand by on-tinediode array spectroscopy. AllMAAs were normalized toprnoI MAA/mg protein using the solubleproteiri from an aliquot ofthe extracted sample. Protein was analyzed using the proceduredescnbed in Lowryet al. 951!. Linearregression analysis was conducted toassess thepossibility ofan explanatory- responserelationship between MAAconcentration andUV radiation level, A linearrelationship betweentheexplanatory variable, UV radiation level, and the response variable, concentration of MAAs,was hypothesized. Itwas hypothesized tobe linear because UVradiation and light! has beenshown to attenuate exponentially with depth, and the hypothesized notion that MAA concentrationwouid also decrease exponentially with depth. Since these two variables are hypothesizedtovary together inan exponential manner with depth, it follows that there would be a linearrelationship between the two variablesthemselves. RESULTS Theconcentrations ofMAAs inaff five species ofcoral analyzed inthis study clearly display an inverserelationship withdepth. In all samples, there was a single MAA high in concentration relativetothe others detected which showed thisdecreasing pattern most vividly. Montipora verrucose,thespecies that was collected atboth Kane'ohe Bay and Moku Manu, revealed the samepattern at both sites Fig. 1!. TheMAA highest in concentrationinthis species was palythine,which showed a decreaseofaround two orders of magnitude from the shallowest to deepestcollection. Concentrations ofMAAs inPocillopora meandrfna Fig,2! and Montipora pafula Fig, 3! from Moku Manu were plotted both against depth m! and UV dose W rn '!. ConcentrationsofMAAs decreased withUV dose asthey did with depth. Mycosporine glycine wasfound in highconcentrations inP. meandrina, whereas inM. pafula, palythine was the most abundantMAA. podtes compressa collected inside Kane'ohe Bay Fig. 4! showed a two orders ofmagnitude decrease inasterina, butan unknown MAA showed anunusual pattern peaking in concentrationat 5 m approximatelymidway between surface and bottom!. Thespectral data collected atthe two sites Figs. 5 &6! show that radiation inthe lower PAR wavelengthsandthe UV portion ofthe spectrum are attenuated atmuch shallower depths inside thebay than outside, The amount ofintegrated energy reaching thebottom ofKane'ohe Bayat 10m was0.59 W m-,and at Moku IVlanu the value at this depth was 15,8 W m. AtMoku Manu 1 m!there was 6.86 W mz oftotal UV reaching the bottom on the day of collection, The attenuationof light through a depthof 21 m atMoku Manu seems to besimilar to theattenuation oflight in only 10 m ofwater column inside Kane'ohe Bay. Theresults of the linear regression analysis of therelationship between MAA concentration andUV radiation level Table I!are highly significant forfour of the six depth profiles: M. patula 78 p<,001!, P. meandrina p=.003!, M. verrucasa at the Kane'oheBay site p=.005! and P. cornpressa p=.005!. R-squared values were also very high, signifying a strongcausal relationshipbetween the explanatoryvariable, UV dose, and the response variable, concentration of MAAs. Table I. Linear regression analysis. '= P valueof less than .01 significancelevel DISCUSSION The concentrationof MAAs in the tissues of four species of corals have been found to be inverselyrelated to the amountof UVradiation reaching the coralcolonies. Montipora verrucose sampledover a 23m depthgradient outside the bay showed a similartwo-orders of magnitude decreasein concentrationof palythineas the samplecollected inside the bay wherethe wateris only 10m deep.The spectral irradiance data, showing a similarlevel of attenuationof UV radiation at bothsites even though the water column was twice as deep inside the bay,supports the speculationthat the corals are responding to somesort of cueassociated with light regime, and not simplydepth. Thisstudy supports the ideathat MAAs are indeed acting as UV radiation blockersin coraltissues because corals of thesame species were found to containvery different concentrationsof MAAsat similardepths when sampled from areas differing drastically in light regime. Theresults of thelinear regression analysis between MAA concentration and UV light level are verysupportive of the hypothesisthat this is indeedan explanatory-responserelationship. Thisstudy supports the hypothesisthat the coralsare respondingto the levelof UV radiation, resultingin the accumulationof higherconcentrations of MAAsin highUV environments,There is needfor caution,however, because many parameters of the manneenvironment decrease with depth,and pinpointingwhich one is explicitlyresponsible will be quitedifficult. It has been suggestedthat increased water motion and its associationwith increased metabolism may act as a cue to increase IVIAAproduction Jokiel & Lesser, 1994!. However,it still remainsto be confirmed thatthe coral is harvestingMAAs from the symbiotic aigae, and not either obtaining them through dietor producingMAAs themselves. Presently the only known origin of MAAsis theShikimate pathway,a complexmetabolic tree that produces the aromaticamino acids, plastoquinones, vitaminsE andK, and many more compounds in photosynthetic microorganisms and higher plants Bently,1990!, Consideringthe diversityof non-symbioticorganisms that contain MAAs, they mustbe obtainingthese compounds through diet, just as mostanimals obtain the essential aminoacids. A non-symbioticcoral, Tubastraea coccinea, does contain high levels of MAAs, 79 A. 'I I c !OO 92l I 'I Rlythbe l 1 1 921 IO Depth m! My~lyanr. iso UNKlI 0 Dq& m! ~ Vaa 0m'~!hymy01y ~ orK0fy00!m Bay 80 My~ycins 6. OcE 30 10 E 0-, 17.5 110 2.373 My~ rre1yane Porrkpa33r @Nrem,0 PelyrraL. 10 10 15 Deptb m! Rare2, PbgicpoianmnWne MAA concenrrebons fromMcku MarW pothsd bolrr agalnet deptrr rn! and VV dose PVm !. c 100- 0- I 17.5 129 10 7.5 UVdeme ~! 0.. - -a"-. CI IXI Patythttt Ptttyttttn! 10 Depth m! Rgttnt 3. hdn1 mrsptatia MAAot;e0enttzttnne ttnm Maku Manuwee ttt04edboth a !atnM depth m! and UV doaa tnt~. Myooeparhcglydiac 10 Depth m! Rgura4, Perkscori preeaa MAA concentralkms vafsuedepth in Kana'ohe Bay. 1.000&01 1.000&00 'E ! .OOOE-01 'Ev 1,OOOB02 1.000E03 k 1.000EO4 1.000E-05300350 400 450 500 550600 650 700 Wavclcagtb nm! Rgure5. Spectral data coNecM at the Sliver Reef, Kanrr'ohe Bay. 1,000E+01 1,000E+00 1,000EOI 1,000E42 1,000EO3400450500550 600 650 700 Wavelottgth{tttn! ptgureS, ~ data oattaOtedat MOku Manu, supportingtheidea that corals can obtain MAAs through diet.The mechanism thatis responsible forstoring appropriate concentrations ofMAAs in response tolight regime iscompletely unknown.Ifcorals harvest MAAs from their zaoxanthellae, itis possible that increased concentrationsofMAAs are a resultofan increased rateof photosynthesis dueto higher PAR andhigher water motion lower dNusion factor!, andit is simply coincidental thathigh levels of MAAsarefound inthe tissues ofcorais thatare found closest tothe surface inhigh water motion, highUV environments. Stototty:UVReNatke OnCoral Reefe atthe HEN ~ of MarineetOIOgy. Weweutd tiketO thanTr Dr.paul L,Joklel foraraeNng thiereeeeroh opportunity, DavidGusto forhelp durtrg collect toneand kfentittoalon ofoorets, and all Ihe perttdtpanteofthe eunvrrer preyrem feretepOrt during thereeearoh. UTERATURE CITED Bentkky,R. 990! The Shikimate Pathway- A Metabolic treewith many bnttnChes. In;Critioal ReviewsinBiochemistry andMolecular Biology', Vol.25, issue 5,pages 307-384. CRC Press,Inc. Boca Raton, Rorlda, USA. Chalker,B.E., Dunlap, W.C. 990!,UV-8 and UV-A light absorbing compounds inmarine organism.Proceedings ofIAe Worirshop: Response ofMariine Phytaplanldon toNatural VariationsinIJV-8 Flux, Scripps Instrlution ofOceanography. Duniap,W.C.,Chalker, B.E.,Oliver, J.K. 986!. Bathytnetric adaptationsof reef-building coralsat DeviceReef,Great Barrier Reef,Australia. III.UV-B absorbing compounds. J.Exp. Itrfar. Biol. Ecol.104: 239-248, Jerlov,N.G. 950!.Ultraviolet radiation inthe sea. Nature 116: 111-112. JokielP. L. 980!. Solarultraviolet radiation and coral reef epifauna. Science 207:1069-1071 . Karentz,D., McEuen,F. S,, Land,M. C. & Dunlap,W. C, 991!, Surveyof mycosporine-like aminoacid compounds in Antarticmarine organisms: potential protection from ultraviolet exposure.Marine Biology 108: 157-1 66. Lowry,O. H., Rosebrough,N. J Farr,A, L. & Randall,R, J. 951!. Proteinmeasurement with the folin phenol reagent, J. Biol. Chem 193: 265-275. Maragos,J. E. 972! A studyof the ecology of Hawaiian reef corala, PhD dissertation, University of Hawaii, 290 pp. Shibata,K. 969!. Pigmentsand a UV-absorbingsubstance incorals and a bl~reen alga livingin the GreatBarrier Reef, Plantcel/ PhysioL 10:325-335. Shick,J, M.,Dunlap, W, C., Chalker,e. E.,Banaszak, A. T. & Rosenzweig,T. K. 992!. Surveyof ultravioletradiation-absorbing rnycosporine-like amino acids in organsof coralreef holothuroids,Mar Ecol. Prog. Ser. 90: 139-148. tfkravtoiatRadiation asa CoralRssfa. 1ttt6. D. Gulkod P. L. Jokel ede.!.HIRIB Tech. Report eeh UNlHI-SeaGrant UV-absorbing compounds in the coral Poci]/opora cfamicornis: interaction effects of light, water flow and UV radiation. Paul L. Jokiel Universityof Hawai'i, Hawai'l l~ of Manna Biology, P.O.Box 1346, Kaneofm, Hawai'i 96744 Michael P. Lesser Universityof New Hampsfvre,Department of Zoology,Durham, New Hampshire 03824 Michael E. Ortdrusek Universityof Hawai'iat Manoa,Department of ~raphy, Honolulu,Hawsri 86822 ABSTRACT: A direct refaftcrv&lpsxieta betwetm dOSS Of SOlar u travicka UV! ~ and concentrabcnOf rnlrccsporinekke tsnino eckts MAAs!in the Hawaiianreef coral Rbcrsoporadsmirxxnis. ~r, MAA aoncentratkmis sfsoinfluenced by 5ow regime and flux ol phokxvrnthsacaliyecsve radiason PAR!. High fluxes of UV radiatkmrn reef environmentsare correlatedwith rrigh PAR and 11igh water velcoity, ~ all threeparamshm diminish exfmnenlatly with i~ depth.Thkr Ccrr83alkxl ls further ~ensd alcnghorfzÃTlal gradients on reefs.The ctsafera water Is typkxtdy found On outer reefsgrowing in oceanicwater. Theseocean reefstfttlcally experieres highwakrr~ due lc oceanswell when comparedto the moraturbid lagoonreefs expo@ only lo small winddrtvsnwaves, ~xtufaisd pathetic rate appearsto be a maiJorfactor eHecbrgthe ~ changre in MAA concsntra5cewhen this coral is grownunder ~ flOwregknea and ~ fluence ratesof UV and PAR ~. High PARanCttcr high walar vetoaly signi5cenaly enhanoethe elfeCtOf inC~ UV ~ OnMAA Ccnoentraticn.Thua, Obeyed dif!erersaraSnd Changrm in adAAS under differenterwironmenlal condi5ons might notdirec5y rellect differsrncesor chsngre in UV radiate 5ux unlessall other parametersare squivalsnL Unpublishedmanuscript. Preprints available from the primaryauthor. UltravioletRadiation end Coral Reefs, 1995. D. Guliro4 P. i.. JokielIedrr.i. HIMB Tecrr. Report e4t. UNIHi-SeaGrsrrt4:R-tf543. Response of a Pacific stony coral to short-term exposure of ultraviolet and visible light Sarah Lewis l~ of Erxtlogy,University of Georgia,Athens, GA SOS02 ABSTRACT: Coloniesof the Pacificstony coral Qontfporaverrucose were transrplantedfrom 10m depthto an ~ respiromsterat 0.5 m depth. The corelswere ~ for 1 to 2 daysto fullitsy or 3P%%dsun, withoutUV-A or UV-B,with UV-Abut not UV-B, or wtlhboth UV-A and UVW. Metallo measuremerrhwere taken oon6~ for eachcoral and leveteOf ChtrXOphytl Snd MAAwere determinedat the CulmineticnOf the experiment No significantrnterw:Son ~ ultraviolet UV! radiaticneffects and vtrdbleirradiance photcrsIrrnthettcsllyactive radiation= PAR! eflects was ~. Coralse~ to full sun ~ eig~ lower maximumnet prhotorsyrrtheslsndes, respirationrates, and photoernthetic~, but net P:R ratios,ccmpeneabon point, end saturedonpoint were uncfxtnged.These results ~ that i~ visible irradiancewas detrrmentalto both the phototerrntheficalgae and to lhe coral lissue. Maxirrum net photosynthesisrates and rdiorophylle levelswere lowerIn corals ~ lo UV, but respirationrahss remained the same. Thie mey indiCatethat UV wss ~ng tOthe phOSXryrrrtheboalgae but not the coral tissue. There was nc slgnlrroantd~ ~ effect of UV-A and effeCtSOfNrr4, + UV-8 for any ~ variable. These resullsere importrmtbecause they indicatethat coralerespond duty even to very short-term~ lo boih i~ visible irradianceand to i~ UV i~. INTRODUCTION Tropical coral reefs are regularly exposed to high leveis of visible irradiance, or photosynthetically active radiation PAR, 400 nm - 700 nm!, and ultraviolet radiation UV-A, 320 nm- 400 nm and UV-B, 280 nm - 320 nm! Cullen & Neale, 1993; Gleason, 1993!. While it has long been recognized that high visible irradianm reaches these reef organisms, historicaliy It was believed that UV radiation was not a significant Influence. Although high levels of UV reach low latitude ocean surfaces due to the thinness of the ozone layer and the low zenith angle of the sun Baker et al., 1980!, it was believed that these short wavelengths were attenuated rapidly and efficiently by the water and, therefore, did not reach reef organisms Smith & Baker, 1979!. However, it is now well known that UV radiation penetrates to considerable depth in tropical oceans Jerlov, 1950; Jerlov, 1968; Smith & Baker, 1979; Fieischmann, 1989!. Concern is mounting over the potential increase in UV radiation reaching coral reefs as ozone depletion continues Hader and Worrest, 1991!, Reef organisms may not be able to adapt quickly enough to survive the changing conditions. On shorter time scaies, episodic events such as unusually calm periods may result in dramatic water column clearing as witnes:ed at bieaching locations in the Caribbean in 1987 and 1990 Goenaga et al.. 1988; Gleason & Wellington, 1993!, These water column clearing events can provide for greater exposure of reef organismsto both UV radiation and visible irradiance. Reef organismscan employ three main defense mechanismsagainst UV radiation: avoidance, protection, and repair. The sessile nature of stony corals coupled with the dependence of the coral-zooxanthellae symbiosis on solar radiation necessitates that corals be exposed to UV radiation. Therefore, corals are ieft with two options: protect themselves, and be capable of repair shoukl damage occur. In shaliow water marine environments,it is beffevedthat many sessile invertebratesempioy UV absorbing compounds to protect themselves from the damaging effects of UV radiation. These compounds, formerly known as S-320" Shibata, 1969!, are collectiveiy known as mycosporlne-likeamino acids MAAs! with abscrrpffonrnaxirna in the 310 - 360 nm range Hirata et al., 1979; Tsujino et al., 1980; Karentz et al., 1991!. It has been suggested that hermatypic corals synthesizeor accumulatetheir own suites of MAAs as protection against this radiation Dunlap & Chalker, 1986; Dunlap et el., 1986!. Joldel and York 9&2! observed a decrease in these compoundswhen UV was blocked from Pocilloporadamicomis, and Maragos 972! observed decreased concentrations as depth increased. UV radiation has been implicated in damagingorganisms both in terrestrial Bnd aquatic systems Harm, 1980; Wood, 1987; Cullen & Neale, 1993!. Worrest et al. 981a; b! correlated altered species compositions and standing crops of algae with increased long-term UV dosage, Lesser & Shick 989! reported 30'/o lower growth rates in zooxanthellaefrom Aiptasia pallida acclimated under high visible tight conditions with UV radiation than those acclimated under high visible iight conditions without UV or acclimated in iow light conditions. Jokiel and York 982; 1984! also found reduced growth rates in a number of algal species, including zooxanthellae, when exposed to visible light with UV-A + UV-B radiation. There is also evidence of UV induced 89 photosyntheticinhibition Sisson, 1986; Lesser & Shick,1989!. Studies of this photoinhibition suggestUV damagesor destroyschlorophyll and/or chloroplasts Gessner & Diehl,1951' ,Smith et af 1980; Hader8 Worrest,1991!. I easerand Shick 989! foundreduced levels of chlorophyllinAiptasis pallida in the presence ofultraviolet radiation, lt has been suggested that increasedUV radiation has been instrumental incausing widespread bleaching observed in tropicaloceans Fisk & Done,1985; Harriott, 1985; Oliver; 1985; Goenaga etal., 1988!. However, theevidence is strictly correlational and is confoundedbyincreases in visibleirradiance. Theshorter wavelength, higher energy UV-B radiation, isconsidered more biologically damagingthan UV-A Cullen & Neale,1993!. Bothwell etaf. 994! discovered that UV-B disrupts manyphatosynthetic proCeSses inCiuding pigment stability, eleCtrOn tranSpOlt Syatem, and photosystemII reaction centers. Despite the belief that UV-B is more damaging than UV-A Calkins& Thordardottir,1980! ~ numerousstudies of UV effects have not investigated these componentsindependently but, see Jokiel & York,1984!. It is importanttoconsider that while UV-Bphotons may be more damaging perphoton than UV-A, there are much greater fluxes of UV-Ain the ocean than UV-B. Bothwell etal. 994! concluded that although UV-B is more disruptive,higher photon flux in UV-Ausually produces the majority ofinhibition of photosynthesisin algae. Thereis some debate regarding the relative contribution ofvisible irradiance and UV radiation indamaging reef organisms. Brown etal. 994! speculate that the bleaching patterns observed incorals inThailand result from longer wavelength, photosynthetically activeradiation PAR! and thatUV radiation played a nominal rote,if any at all. They contend thatthere isincreasing evldercethat high levels of PAR negatively affect algal photosynthetic systems review in Powles,1984!. Contrastingly, Jokiel and York 984! consider that the role of PAR in photolnhlbitionhasbeen overestimated andthat long-term photoinhibition effectsareprimarily causedbyUV radiation. They discovered thatalgae intheir study could rapidly photoadapt to increasedPAR 92% surface irradiance!, butthe addition ofUV resulted ingrowth photolnhlbltfon.Theirstudy agreed with previous studies Steernann-Nielsen, 1962;Steernann- Nielseneta/., 1962; Prezelin & Matlick, 1980! that showed thatsome rnicroalgae canrapidly photoadapttohigh levels ofvisible light «24 hrs!.This debate canonly be settled byfurther, non-correlationalresearch onthe relative impacts ofthese two light components. Inaddition to theimpact ofUV radiation orvisible irradiance onreef organisms, theremay be an interaction betvwlenthesetwo effects. Itis possible thatthe combination ofthese two factors produces greaterdetrimental effects than either ofthe two acting alone. Itis equally possible thatone factor may amelioratethe effectsof the other, Theexperiments inthis study were designed totest for acute effects ofshort-term exposure ofthe Hawaiian stonycoral, Mont/para venuamr toecologically realisticlevels ofincreased visible Irradiance,increased UVradiation bothUV-A and UV-B!, and the interaction ofthe two. The specificquestions addressed were: ! Does the metabolic abilityof the Hawaiian stonycond, fi/fonttpuaverrvcosa, changewith increased visibleIrradiance and/orUV-A and/or UV-B radiation? !.Does chlorophyll contentchange with increased visible irradiance and/or UV-A and/or UV-B radiation? !,Do MAA levels change withincreased irradiance and/orUV-A and/or UV-B radiation? MATERIALSAND METHODS Thisstudy wasconducted atthe Hawai'i Institute ofMarine Biology HIMB!, Coconut island, Kane'ohe,Oahuduring thesummer of1994. All coral pieces were collected offthe Coconut Island'sLighthouse Dockfrom large colonies ofptating Montfpora verruccmat a depth of10 rn. A pairofcoral pieces weretaken from thesame location oneach colony; onepiece ofthe pair was usedinthe experimental treatmentswhile theother piece ofthe pair was used immediately forlab analysistoobtain initial estimates forchlorophyll a leveLs,and MAA levels. A total of36 pieces from18 colonies was used for this experiment. Thecollected piecesof M. verrucose weretransported approximately 300m inshaded fresh seawatertothe site of the in-situ respirometer inthe evening before each experimental run.The 90 respirometerwas locatedan a suspendedplafform at a constantdepth of 0.5 m. Six piecesof M. ~rruccea were collectedat a time. One piecefrom each pairwas randomlyplaced in each of the three chambersof the respirometer,while the otherpair-member was takento the lab for immediateprocessing, The treatmentswere establishedin a 2 PAR! x 3 UV! x 2 Days!incomplete factorial design. The three UV treatmentswere establishedby placingfilters over each respirometerchamber. One filteronly allowed PAR to pass,one fiiterallowed PAR+UV-A to pass,and the thirdfilter allowedPAR + UV-A + UV-B to pass, Two visibleirradiance treatments were crossedwith this set of three UV treatments.Neutral density screening was used to createtwo PAR levels:full light intensityand simulated10 m lightintensity 0% surfaceintensity at the coralcollection site!. The factorialdesign is incompletein thata secondday of treatmentwas appliedonly to the corals exposedto the fulllight PAR treatment. All combinationsof treatmentswere replicated3 hmes. Photosyntheticmeasurements were made usingthe suspendedrespirometer. The experimentalcorals were placedinside sealed 2.3 liter plexigiasschambers for the duraffonof the experiment.The chamberlids were quartzand thereforetransparent to UV-A and UV-B radiation. Eachchamber was connected to a submersedimpeller pump which fully exchanged ali thewater in the chambersevery hour. Temperaturereadings taken periodicallyinside the chambers showedthat this flushing rate prevented significant heating inside the chambers < 0,5 'C higher than surroundingwater!. Uniformityof oxygenlevels throughout the chamberwas achievedby rotatingstir bare beiowa perforatedpedestal that heldthe coral. Oxygenproduction photosynthesis!and consumption respiration! were measured by YSI oxygenprobes and recordedevery 4 minutesby an Omnidatadatalogger. Ught was measured by a UCorlight meter and 4pi steradianspherical sensor attached to the respirometerand recordedevery 4 minutes, Allthe oxygendata was downloaded from the datalogger to a computerimmediately after eachrun, The recordedvoltage readings were converted into oxygen pprn! and light uE rn' s'! units.Rates of oxygenconsumption and production were calculated and plotted against the irradiancevalues to develop iight saturation curves, The curves were fit to thedata using the followingmodel: P = R + P - R! - e~! Thismodel yielded the foiiowing response variables: P, R, a, i and I . P is themaximum net photosynthesisrateachieved by the coral. It is measuredas the horizontal asymptote ofthe light saturationcunIe. R is thenighttime respiration rate for the coral. The initialslope a! at the compensationirradiance Ig of thelight saturation curve is termedthe photosynthetic efficiency. It is theirradiance level at whichthe coral produces enough oxygen to compensatefor its respirationand thereis a nst productionof oxygen. I is the saturationirradiance. It is the irradianceat whichthe coral reaches its maximumnet photosynthesis P~. Threemetabolic response variables I I ,net P:R! were independent of normalizations. Threemetabolic response variables R, netP, alpha!were normalized inthree ways: per cm' surfacearea, per gram wet weight, and per microgram chlorophyll a. I Afterremoval from the chambers,each conaIwas taken to the labin seawaterand surfacearea was determined,Each coral was videotapedand projectedsurface areas were then calculated. This valuewas then multipliedby 2 to determinethe surfacearea of the coralInvolved in photosynthesisand respiration because M. verrucormhas tissue on both the top and underside of the plate. Onepair-member was analyzed for chlorophyll immediately after collection from the field. As thispiece was taken from an areaof the colony immediately adjacent to the experimental cord piece,its chlorophyll level served as an estimate of the pre-treatment chlorophyll level of the pair member. Chlorophylllevels were determined photcxtx~ly. A smallplug was taken from the middle ofeach piece using a 1cm diameter cork borer and then ground in 90% acetone. The ground coraland solvent were placed ina darkrefrigerator toextract overnight, The tubes were then spunin a refrigeratedcentrifuge at3500 x gand the absorbance ofthe supernatant wasthen measuredona scanningspectrophotometer at150 nm, 663 nm, and 630 nm. Chlorophyll a valueswere determined using the equations ofJeffrey and Humphrey 975!. hS &men MAAlevels were determined using HPLC. A smallplug was taken from the center of each coralwith a 1cm diameter cork borer and then placed ina -5PCfreezer. When the experiment wascompleted, allfrozen samples were sent to Dr, Michael Lesser University ofNew Hampshire! wherethe samples were then extracted using methanol. The extract was separated using an HPIC andthe peaks were quantified and identNed using standards. All the MAAs were riormalizedbyprotein. Protein values were determined using the Lowery method Lowrey, 1951!. Theresponse ofan individual coral piece to a treatmenteffect may be influenced by pretreatmentfactors. Hence, a covariateanalysis with these factors ANCOVA! might provide a morepowerful testthan a simplenoncovariate parametric test ANOVA!. Covariates were only usedwhen the covariate model was significantly different than the reduced, nonwovanate model. Thechoice between differen significant covariate models follows the method outlined byC. L. Mallows 973! also see the description under PROC REG In SAS/STAT User's Guide, Volume 2, 1990!.Indspendencs ofthe covariates was tested by a linearcorrelation procedure Dilorio, 1991!. Comparisonsoftreatment means were tested using t-tests, but only if thetreatment effect firsttested significant under an F-test. The signNcance level for all tests was 5%. Allstatistical analyseswere camed out using a PCSAS package. RESULTS Theset of potential covariates were: MAA, chlorophyll, respiration normalized bysurface area,respiration normalized bychlorophyll, and respiration normalized bywet weight. Each of thesecovariates represents a "before treatment factor. The respiration rates of each experimentalcoral were measured during the night prior to exposure tothe UV and PAR treatments.The chlorophyll andMAA estimates for each experimental coral were obtained from thebioassay ofthe corresponding pairmember, ChlorophyllandMAA were evaluated ascovarlates forall metabolic response variables. The respirationrates were only used for the metabolic response variables with the same normalization. TableI shows thecorrelation analysis forthe covanates. The only covaiiates that showed a signNcsntcorrelation were R bywet weight and R bysurface area correlationcoeNcient = 0.73, p=0.001!.Since these two covartatss were always used separately inany analysis, allcovariate modelstested used a setof independent covariates, Tables II, III,and V showthe covariates used lnthe analyses. Of all the covariates tested, only two were used: Chl-before and MAA-before. An ANCOVAp~ure wasused to ansiyze the following response variables: R normalized byChl usingChl-before ascovariats, net P normalizedby Chi using MAA-before ascovanate, and net P normalizedby surface area using MAA-before and Chl-before ascovariates. All other responsevariables were analyzed using an ANOVAprocedure. Withe10 m lightcorals - UVeffects: Coralsunder shaded condwons were run for 1 day.For the MAA analysis,the beforetreatment chlorophyll level was a signNcantcovariate, but there was no significant Uveffect p=0,58!, For the chk rophyII a analysis,nocovariate tested significant, and therewas no significant UVeffect p=0,61!. 92 Withinfull lightcorals - UV effects: Coralsexposed to fullvisible light were runfor 2 days. No covariatestested significant in eitherthe MAAanalysis or the chlorophylla analysis.There was no significantUV effect for totalMAA levels p=0.16! or chlorophylla level p=0.08!, The covariatesused and the significancelevels of the treatmenteffects are summarizedin Table Vll, andthe meansand standard errors are providedin TableVill. 'n 1- P There was no observed interaction between the UV treatment effects and the PAR treatmenteffects for any of the metabolicresponse variables. SignNcance levels for both unnormallzedand normalizedresponse variables are providedin TablesII and III, respectively. The meansand standard errors are providedin Table IV. UV Effects: Compensationirradiance I J, saturationirradiance I J, and net P:R ratiodid not show a significantUV effect p=0.59, 0.39, and 0.52, respectively!.There was no signNcant differencesbetween the meansfor each of these responsevariables among the different UV treatments Table IV!. Of the metabolicresponse variables normalized by surfacearea, wet weight,and chlorophyll,the oniy significant UV effectoccurred with net P normalized tosurface area p=0.02,Table III!. The coralsthat were shielded from UV had a highernet P than thoseexposed to UV-A Table IV, Figure1!. Mowever,this design could not detect a significantdifference between those exposed to UV-A + UV-8 fromthose shieldedfrom UV or thosereceiving UV-A-only meansand tgroupings- TableIV!. The lightsaturation curves shown in Figure2 providean overallview of the UV treatment effects on the rnetabolisrnof M. verruimsa The higher net P for corals receivingonly visible light is evident. PAR Effects: There was no signNcantPAR effecton I I ,and P:R pW.57, 0.12, and0.46, respectively,Table il, means- TableIV!. Therewas a highlysignificant PAR effect for each ol the metabolicresponse variables for each of the three normalizations.The 10m light coralsshowed significantly higher net P, R andalpha values all p c 0.05 - TableIII, meansand t-groupings - Table IV!, Figure1 showsmean valuesand 95% confidence intervalsof net P normalized by surface area, The same trend was evident for the other twometabolic response variables normalized by surface area, as wellas for allmetabolic responsevariables normalized by wet weightand chlorophyH. Figure3 showsthe light saturation curves for the 2 PARtreatments, irrespective of UV treatment.They clearlyindicate the effectsof increasedvisible irradiance on the photosyntheticability of M. verrucosa, inF V There was no interaction observed between the UV treatments and the day of exposure for any of the metabolicresponse variables TableV!. 93 UV Effects: Whenfull light corals from the three UV treatments were compared forthe first and secondday of exposure, saturation irradiance IJ andnet P: R ratiowere not different amongthe UV treatments pW.72, and 0.33, respectNely, Table V!. However,there was a significantUVeffect for compensation irradiance p=0.01, Table V!. Coralsshielded fromUV had significantly lowercompensation pointsthan those exposed toUV-A means andt-groupings, TableVI!. However, itwas not possible todistinguish thecompensation pointof corals exposed toUV-A+ UV-8 from that of corais exposed toUV-A or shielded fromUV TableVl!. Figure 4 shows the lower compensation irradiance forcorals shielded fromUV. Maximum netphotosynthesis, respiration, andphotosynthetic efficiency normalizedtosurface area, wet weight, and chlorophyll didnot show significant UV effects pvalues -Table V,means and standard errors - Table Vl!. Figure 5 shows thelight salurationcurves for the full light corals for both days of exposure tothe different UV treatments.Although corals shielded from UV appear tohave a higherphotosynthesis, thiswas notsignNcant. Day Effects: Therewas no significant difference forsaturaff onirradiance ornet P; R ratiobetween thefirst day and second day of exposure forthe full light corals pW.14, and 0.70, respectively,TableV!. However, thecompensation irradiance wassignificantly lower duringthe first day of exposure and increased during the second day of exposure p&.002,Table V, means and t-groupings - Table Vl, Figure 4!. Ofthe three variables normalized tosurface area, wet weight, and chlorophyll, only respirationnormalized tosurface area showed e significant dayeffect ~.02, TableV, meansand t-groupings, TableVi!. Figure 6 shows thatrespiration rateswere significantly higheron the secondday of exposure. DISCUSSION UV Effects: Thelack of a UVeffect for chlorophyll ortotal MAA levels forfull light or 10 m light coralsshould beconsidered inthe context that the exposure timewas only two days and oneday, reel:iectively. Further studies using larger sample sizes may determine whether ornot chlorophyll e levels and total MAA levels change during short-term exposures to IncreasedUV irradiance. Previousstudies have found that corals shielded from UV for an extended time tend tolose their MAAs, while corels exposed tohigher levels of UV for an extended time tend toincrease their MAAs Jokiel ti,York, 1982; Scelfo, 1985!. Kinzie 993! found that M. verruoosaacclimated inpAR + UVhad higher levels of these compounds than those acclimatedinPAR oniy. Although these changes occurred after multiple weeks of exposure,itis not yet known how quickly corals ofthis species wiil change MAA levels. Thisexperiment didnot uncover anychanges inMAA levels in2 days. i 1- Theobservation thatno interaction occurred ~n UVradiation and visible irradiance afterone day of exposure suggests that the detrimental effects ofeither treatment were not exacerbatedorameliorated bythe other treatment. A previous study with freshly isolated zooxanthellaefrom the zoanthid, p~ can5aeorumhave indicated that there can be a synergisticeffectbetween these two factors Lesser etal., 1990!. UV Effects: Inthis experiment, onlyone of the metabolic response variables, the maximum net photosynthesisrate,showed a significant UVeffect after one day of exposure. The observationthatnet p washighest inthose corals shielded from UV suggests thatUV radiationmey be damaging thephotosynthetic components ofzooxanthellae, These resultsare consistent withprevious studies, For example, Kinzie 993! found enhanced 94 photosyntheticability in fullsun by Monfiporaverrucosa acclimated to PAR + UV comparedto coralsacclimated to PAR only. Lesserand Shick 989! foundUV exposure decreasednet P in freshlyisolated zooxanthellae but not culturedzooxantheliae from Aipfasia pallida. The inabilityto detecta differencein net P between coralsreceiving only visible lightfrom those exposed to UV-A + UV-B allows2 interpretations.First, increased levels of UV-B may amelioratethe effectsof increasedUV-A. Second,the experimentaldesign was not sufficientto detectthe difference,The firstinterpretation seems unlikely,and perhapsa follow-upstudy with an increasedsample size would be able to makea distinction. The lack of a UV treatment effect on the respirationrates indicates that UV is not affectingthe coraltissue and is consistentwith results obtained by Kinzie 993!. One day of exposureto increasedUV did not significantlychange the irradiancenecessary for the coraisto reachcompensation Ig or to achievesaturation I j. It is irnfxxtantto consider thatthe UV effectsobserved occurred after very short-term exposures to naturally occurring levels of UV radiation. PAR Effects: Powles 984! providesa reviewof evidencethat highievels of PAR affectalgal photosyntheticsystems, causing photoinhibition and subsequentlyphotoxidation at elevateddoses over prolongedtime. In thisexperiment, similar detrimental effects of increased PAR were observed after only 1-2 days of exposure. Net P, respiration rates and photosyntheticefficiency were all significantlylower in coralsexposed to fullvisible irradiance.These resultssuggest that significantincreases in PAR perhapsdue to water columnclearing events! may interruptthe properfunctioning of boththe hostcoral and the zooxanthellae. These resultscontrast with previous work by Jokieland York 984!, whofound remarkablyhigh tolerances to PAR in the dinofiagellate,Syrnbiodinivm microadria5cum a symbioticcoral zooxanthellae!. This alga demonstratedgrowth photoinhibition to increasedlevels of UV but,even at fullsurface intensity, visible irradiance produced no inhibitoryeffects. I i F I lh The coralsexposed to fullvisible irradiance were runfor a secondday to allowcomparison of changesfrom the firstday of exposureto the secondday of exposurefor the differentUV treatments. UV Effects: Sincecorals exposed to UV-A had highercompensation irradiance than those shieldedfrom UV, it suggeststhat UV-A is stressfulto corals. However,the lowsample size of the experimentdid notallow any distinctionto be determinedbetween UV-A effects and UV-B effects or between PAR only and UV-A + UV-B. Day Effects: I speculate that the higher compensation irradiance and higher respiration rates observedduring the secondday of exposureare due to cumulativestress from the high levels of visible irradiance. CONCLUSION Exposingcolonies of Monfiparaverrucose that were photoadaptedto lightlevels at a 10 m depth,to dramaticallyincreased visible irradiance, appeared to detrimentallyimpact both the photosyntheticzooxantheliae as wellas the coraltissue. These coloniesexhibited decreased maximumnet photosynthesisrates, respiration rates, and photosyntheticefficiency. Coionies exposed to dramatically increased UV irradiance did show a metabolic response, but did not respond to the same degree as to the increased visible irradiance. The response to the increased UV appearedto be limitedto the symbioticalgae. Coralsshielded from UV had highermaximum net photosynthesisrates but no otherdifference in metabolicresponse variables were observed. Significantly,the treatmenteffects observed in this exper mentoccurred following exposureto naturallevels for only 1 to 2 days. TableI. Peeraoncorrelation ooeflldenta for anafysafor oovariatss ' i~ signtfioanfiyconsisted, p < 0.05!. TablevtsRAe 11,~ fight~ data affects- corntrartaon fornonnatbaffoncf first day ofinde ~ pendent for ~ afi treatments. ' ~ Sgnlficancep <0,05!. levels from ANOVAsfor UV and Tableill. ~ data- comparisonoffirst day of exposure for al treatments.Sgrillicance levels from ANOVAs or ANCOVASfcr UV and vtelbtai light ~ effects for~ ncrmrditadtO Surface area SA!,wet vrelght Wgt!, and chlorotrhyfi Chl! ' i~ p < o.as!. TablelV. Metabolicdata - comparisonof first day of exposurefor afi treatments. Mean values for metallic respond~ ~ sizes, andstandard errors letters repretxmt t-test groupings, Values In difterent gnxfixt are significantly different at p c 0.05!. 97 TableV, ~fc date- comparisonof first day versus second day of UVerrpoeurs by full light corels. SignfTicancs levels from ANOVAefor ~ reeporm vaiabiwfor VV treatment sflect and day effect ' ' rffceteep c0.05!. 98 TableVl. Metabolicdata - ocrntxafsonof first day cf exposureand secondday of expo>ereby shadedcorals. Meanvalues, SampleSlaea, ~ srrorsfor metabc icresponSe vartableS letterS regweeatt t-teat groupinga. Valuea in ~ groupsare sign~ differentat p < 0.05!. Table Vli. Significancelevels from ANOVAsand ANCOVAstor UV treatmenteffects for chlorophylla ~ cm ! and total MAA nmol mg ' protein!for 10 m fightcorals after t day exposuresnd for full light coralsaffsr 2 days exposure. TableVIII. Sean and ~ errcrS forChtcrzrptrtrS a ugcm'! ccnSSnt andfctai ~rfne-like aminO acids nmcUmg protein!fcr 1 0mNgtrt Corals and trff llgrrt Ccrals. CV 8 w 20 0 00 ~ I 8 la+ UVA+ -Igm Light Fili Light Rgbsurfacel. Sgniflcsntarea. Masre UVandand PAR95% ~~e effectslevee.afterone day of~ for mrodmumnetphctoeynthssis normalizedby 100 25 ~ 20 15 10 5 0 -5 z 0 Irradiance uE! Vis. only Vis+ UVA ~ Vis+ UVA+ UVS Figure2. Ughteaturatrort cuwes tor ell corelstor ttrstrtay of expaam to the three W trestmretta. 30 25 20 15 0 M 5 0 H 5 0 -10 z 0 Irr ad i an ce u E! Rgure3. Lighterrturetion cvrvm tor ttrrrt rtay of errtrceureto the two~ PAR treatmerrts. 101 Fleischmann,E.M. 989!. Themeasurement andpenetration ofultraviolet radiation into tropical marine water. Limnol, Oceanogr. 34: 1623-1829. Gessner,F. & Diehl,A. 951!. Diewirkung naturticher ultraviolettstrahlung aufdie chloraphyllzerstorungvcnplanktonalgen. Arch. Mikrobioi. 15: 439%54, Gleason,D.F. 993!. DiffererItialeffects of ultravioletradiation on green and brown morphs of theCaribbean coral Pontes astrsoicres Umnol. Qceanogr. 38 !: 1452-1483. Gleason,D.F. & Wellington,G,M. 993!. Ultravioletradiation and coral bleaching, Nature 365: 836-838. Goenaga,C Vicente, V.& Pumstrong,R. 988!. AposymbiosisinPuerto Rican zooxanthellate cnidarians.Proc. Aswc. Is. Mar.Lab. Canbb. 21: 49, Harm,W. 980!. Biologicaleffects of uitaviolet radiation. Cambridge University Press, London, Hamott,V.J. 985!. Mortalityrates of scieractiniancorals before and during a massbleaching event, Mar. Ecol. Prog. Sar,21: 81-88. Hoegh-Guidberg,O.& Smith, G.J. 989!.The effects ofsudden changes intemperature, lightand salinityon the population density and export of zooxanthellae from the reef corals Stylophora p'sNIataEsper and Serietopora hyatrix Dana. J. Exp.Mar. Biol. Ecol. 129: 279-303. Jeffrey,S. W. 8 Humphrey,G.F, 975!. Newspectrophotometric equations for determining chlorophylisa,b, c andc2 in higher plants, algae and natural phytopiankton. Biochern. Physiol.Plf, 167: 191-194. Jerfov,N. G. 950!. Ultravioletradialion in theaea. Nature London! 166: 111-112. Jerlov,N. G, 968!. OpticalGeography. Elsivier,Amsterdam. 194pgs. Jokiel,P. L, 8 York,R. H. 982!. Solarultraviolet biology of thereef coral Pocillopora damicomis andsymbiotic zooxanthellae. Bull. Mar. Sci. 32 !: 301-315. Jokiel,P. L &York, R. H. 984!. Importanceofultraviolet radiation inphotoinhibition of microalgal growth,Limnol. Ocaanogr. 29: 192-199. Karentz,D., McEuen, E. S., Land, M. C. & Dunlap,W. G. 991!. Surveyof mycosporine-like aminoacid compounds inAntarctic marine organisms: potential protection from ultraviolet exposure. Mar. Biol. 108: 157-168. Kinzie,R. A. III. 993!, Effectsofambient levels of solar ultraviolet radiation on zooxanthellae and photosynthesisof the reefcoral Montipora verrucara. Mar, Biol, 116: 319-327, Lesser,M. P. & Shick,J, M. 989!, Effectsof irradianceand ultraviolet radiation on photoadaptationinthe zooxanthellae of Aiptash pallirta: primary production, photoinhibition, andenzymic defenses against oxygen toxicity. Msr. Biol. 102: 243-255. Lesser,M. P., Stochaj, W. R., Tapley, D. W. & Shick,J. M. 990!. Physiologicalmechanisms of bleachingincoral reef anthozoans: effects of irradiance, ultraviolet radiation and temperature on the activitiesof protectiveenzymes against active oxygen. Coral Reefs 8: 225-232, Maragos,J. E. 972!, A studyof theecology ofthe Hawaiian reef corals. Ph, D, Thesis, Dept. of Oceanography,Univ. of Hawai'i, 290pgs. Oliver,J. 985!. Recurrentseasonal bleaching and mortality ofcorals on the Great Barrier Reef. Proc. 5th Int. Coral Reef Congr. 4: 201-206. Powles,S. B. 984!. Photoinhibitionof photosynthesis induced by visible light, A, Rev, Plant Physiol, 35; 15-44. Prezelin,B. B. & Matlick,H. A. 980!, Time-courseof photoadaptationinthe photosynthesis- irradiancerelationship of a dinoflagellateexhibiting photosynthetic periodicity. Mar. Biol. 58: 85-96. Scelfo,G. 985!, Theeffects of visibie and ultraviolet solar radiation on a UV-absorbingcompound and chlorophylla in a Hawaiianzoanthid. Proc. 5th Coral Reef Congr. 6: 107-112. Shibata,K. 969!. Pigmentsand a UV-absorbingsubstance in coralsand bluegreen algae living in the Great Barrier Reef. Pf, Ceil Pysiol., Tokyo 10: 325-335, Siebeck,O. 988!. Experimentalinvestigation ofUV tolerance inhermatypic corals scleractinia!. Mar. Ecol. Prog. Ser. 43: 95-103. Sisson,W, B. 986!. Effectsof UV-8 radiationon photosynthesis. Irr. Worrest,R. C. andM. M. Caldwell eds.!; Stratospheric ozone reduction, solar radiation and plant life. Springer-Verlag,New York. p. 161-169. Smith,R, C. & Baker,K. S. 979!. Penetrationof UV-Band biologically effective dose rate in natural waters. Photochem. Photobiol. 29: 311-323. Smith,R. C. & Baker,K. S., Holm-Hansen,O., Olson,R. 980!. PhotoinhibNonof photosynthesisin natural waters. Photochem, Photobiol. 31: 585-592. Steemann-Nielsen,E. 962!. Inactivationofthe photochemical rnechanisrn in photosynthesis as a meansto protectthe cellsagainst too highlight intensities. Physiol.Plant. 15: 161-171. Steemann-Nielsen,E., Hansen, V. K. & Jorgensen,E, G. 962!. Theadaptation to differentlight intensitiesin Chlorella vulgaris and the time dependence on transfer to a newlight intensity. Physiol.Plant, 15: 505-517. Tsujino,I.,Yabe, K. & Sekikawa,I. 980!. Isolationand structure ofa newamino acid, shinorine, fromthe red alga Chondrus yendol Yamada et Mikami. Bot, Mar. 23: 65-68. Vareschi,E. & Fricke,H. 986!. Lightresponses of a scleractiniancond Pierogyra slnuosa!. Mar. Biol. 90: 395%02. Wood,W. F. 987!. Effectof solarultra-violet radiation on the kelp Eckioniaradiiata. Mar. Biol. 96: 143-150, Worrest,R. C., Thomson,B. E. & Van Dyke,H. 981a!. Impactof UV-B radiationupon estuarine microcosrns. Photochem. Photobiol. 33: 861-867, Worrest,R.C., 'Thomson, B.E, & VanDyke, H. 981b!.SensNvity ofmarine phytoplankton to UV-B radiation:Impact upon a modelecosystem. Pftofochern.Photceiol, 38: 233-227. MATERIALS AND METHODS Theexperiment wasconducted between July 12 and July 26, 1994, at the Lighthouse Point LP!and Bridge toNowhere BTN! sites onCoconut Island, Hawai'i. Fifteen coral colonies ofthe platingmorph ofMonfipora ventfcrms weretagged ata depthof10 m atthe LP site. Two fragmentswithminimum dimensions of15 cm2 were broken offfrom each of these parent colonies.One fragment was transplanted toa depth of1 mat the BTN site treatment fragment! andthe other fragment remained directly beside the parent colony as a controlfortransplantation controlfragment! 1!. Fig.Transplanted andcontrol fragments wereaffixed toa uniformsubstrate piasticmesh plafform. Treatment fragments wereinduced tobleach byexposing them to increasedsolar irradiance viatranspfanhttion fmma low light LP site at 10 m! to a highlight site BTNat1 m!for 4, 6 or14 days. Transplanted coralfragments wereexptmed toa 70%%d increase in averagetotalirradiarce overthat observed at10m based onintegrated lightmeasurements made every2 nmusing a IjCor Li-1800UW underwater spectroradiometer between300 - 700nm!. IntegratedUVB 00- 320 nm!, UVA 20 - 400nm! and photosynthetically activeradiation PAR 400-700 nrn! levels were approximately RP%%d,93%%d and 66% greater at1 mthan at 10 rn respectively.Treatment fragments weretransplanted tothe BTN site to better replicate thelow waveaction conditions present at 10m atthe LP site LPsite is beside a dockwith high boat tra5c!,These sites are separated byapproximately 100m, occur inthe same small lagoon and haveSimiiar Sedimentatian andtemperature regimeS. HOuify temperature Values were recorded bothat the 1 mBTN and at the 10 m LPsite using two HOBO brand miniature data loggers. The twosites differe by less than 1.0oC on average and neither temperature regime was high enoughtoinduce bleaching Lpaverage temp. = 26.5+ 2,0oC and BTN average temp, = 27.02 1.0oG! Jokief8 Colas, 1977!. acheduh.Ini5ally Idetr0!, two ~»nte ascherere brcken Olrof escf Of15 parent coionk» st10 m ertrern» = i»renteokerk»! Ore~ wse trenepktntedR 1m arete= tnSSrnsnt ~! andthe other ~ wae piggy' dksrStrbeside 5»parent ceteny ssa CcntrctfOrtrenrSrtenteticn Itnanyke= oorrkol fneynenlei. Afler4 dayS, 5 ~ Chosen~~e serestLLrr»dd to1Q rn. The esn» process wserepeakrd ondey 8 snrtdsy 14. At eaoh 5me ~, bahksgmenkr endthe trarent artonkre wereeernpkrd andCtVoroprryl s sndtotal lipid levels were determined. Twosubsamples fromeach fragment andparent colony were taken by drilling with a 1.25cm corkbere~ thrcugh theCOraf plate. One subsample waeanalyzed forChlorophyll a COnCentrations andthe other for total lipid leveLs inorder todetermine initial levels ofboth of these parameters. Sincelipid and chlorophyll a levels didnot vary significantly withina coral plate determined priorto conductingtheexperiment!, a singe sample from each fragment andparent colony adequately 108 representedtheconcentrations ofthese parameters F = 2.167,n =6, p 0.08;F =1.596, n =6, p < 0.194respectively!. Afterfour days July 16!, all ofthe treatment fragments, control fragments and the parent colonieswere again sampled and anaiyzed for total lipid and chlorophyll a levels, Five of the fifteentreatment fragments were randomly selected, returned totheir original sites at 10 m and subsequentlymonitored forsigns of recovery. This procedure was repeated again on days 8 and 14 July20 and Juiy 26! of the experiment thus returning alltreatment fragments totheir original depth Fig. 1!. Chlorophylla and lipid analyses were performed asfollows: Chlorophyll a was extracted from fresh,finely-ground samples according tothe method described byJeffrey and Humphrey 975! andreported in ug/cmz. Lipids were extracted from finely-ground samples samples had been frozenat - 500Cfor 1- 2 weeksprior to extraction!in a chloroform;methanol :1,v:v! solution. Extractswere then washed once with 0.88'A potassium chloride solution, three times with a methanol:watersolution {1:1,v:v! and dried at QPCfor 24 hours before weighing. Animal tissue biomasswas determined following lipid extraction byburning the skeleton and remaining tissue residuein a mufflfurnace at 45 PCfor 6 hours.Lipid content in corals was reported as 4 lipidper gramdry tissue weight. This method differs slightly from Harland efsl. 992!where samples weredecalcified prior to lipid extraction which can result in lipid loss during the decalcNcation process triglycerides canhydrolyze in acid solutions and the glycerol component of the molecule is then soluble!, Thedata were analyzed by pairwise comparisons between parent colonies and control fragmentsaswell as between control fragments and treatment fragments tadetermine rflipid and/orchlorophyll a levels had changed intreated fragments. The null hypothesis was that the differencebetween the abovementioned pairs was less than or equalto zero and was rejected at analpha level of 0.05 by means of a pairedANOVA on each sampled date. Comparisons ofthe lipidand chlorophyll a levels in the treatment fragments that had recovered for 10, 6 and0 days wereexamined by means of an ANOVA, The relationship between lipid and chlorophyll a levels was assessedby meansof a correlationanalysis. RESULTS Boththe parent colonies and the control fragments were observed to havenormal coloration throughouttheexperiment. Their chlorophyll a levels were not significantly different from each otherat anytime Table l! Fig.2!. Treatmentfragments were initially observed tohave similar pigmentationasthe controls but began to pale on the third day of exposure tohigh light Table l!. Theybecame progressively paler with ircreasing exposure time. Chlorophyll a levels were signNcantiylower in treatment fragments than in controlfragments on days 4, 8 and14 Table l! Fig-2!- Duringthis interval, lipid levels in the parent colony, control fragment and treatment fragment samplesdid notdiffer Table 1! Fig.2!. Furtherindication that bleaching,as indicatedby decreasedchlorophyll a, was not accompanied by a decreasein lipid levels was revealed by a lack ofa significantcorrelation between chlorophyll a and the percent lipid per gram dry weight F=1.734, n=17l, p .190! Fig. 3!. Recoveryinbleached fragments was assceaR by directly comparing lipid and chlorophyll a levelsin fragments that had bleached and recovered for 4 and10 days,8 and6 daysand 14 and0 days,respectiveiy. Lipid levels and chlorophyll a levels were not signNcantly different between treatmentfragments that had recovered for 10, 6 and0 days F=0.124,pc0.884, n=15; FW.243, p<0,788, n=15 respectively!. 109 Tebteb Resultsofpetnrrtse oornperlsons. CHLe ~chlorophyll e,P-CT = peirwisecomparison ~ parent ootontsssndoontmt trtrgmsnte, CT-T=psirwise acrrrpertson besrrteen control fragments endtreatment Aayrents, n number ot semtses. DAY 0 DAY 4 DAY 8 DAY 14 P< n P n P n P< n CHL a P~ 0.78 14 0.28 15 0.91 1 0 0.44 4 CT-T 0.38 14 0.00 15 000 10 0.04 4 %LIPID P~ 0.98 14 0.83 15 0.26 1 0 0,55 4 CT-T 0.53 14 0.33 15 0 30 10 0.19 4 DISCUSSION Theupper surface of all treatment fragments appeared paler in color after 3 daysof bleaching andremained pale throughout the experiment. This overall paler appearance was reflected in lowerchlorophyll e levels. The decrease in pigmentlevels did not recover to pre-bleachlevels duringthe course of the experiment irrespective ofthe length of exposure to highlight. 4pids didnot decrease in bleachedfragments of M. verrucoseover the course of twoweeks. Rather, a generaltrend towards increased lipid levels in theparent colony, control fragment and the treatmentfragment was observed. M. venucosa has a naturallunar cycle to itslipid levels that correspondstospawning Stimson, 1987!. Since the experiment was initiated immediately foliowingthe July spawning, the observed trend in increasedIipids seems to bea reflectionof this naturalcycle of repmluction.The lack of a significantcorrelation between chlorophyll a and the percentlipid per gram dry weight is consistent with the observation that decreases inchlorophyll a werenot accompanied bydecreases in lipidlevels Fig. 3!. Studiesby Fitt et aL 993! showed thatlipids in 3 bleachedMontastJea annularis colonies were lower than in 3 unbleachedcolonies 6 monthsfollowing bleaching. There are several possible reasons for the discrepancy between this studyand that of Filtet al. 993!. Ijpidsmay be melabolized very slowly in bleachedcorals makingdecreases in iipidlevels apparent oniy long after initial bieaching. Hemmer, shading experimentsbyHartiott 993! indicatedthat lipid levels in the Hawaiian coral Pocillopora damfcomisdecreased injust one week. When cofsils were shaded, zooxanthellae were initially unableto maintainphotosynthesis at the same level as whenin fullsunlight. Under these condIons,lipid stores were metabolized inorder to fulfill the corals' daily energetic demands. Thissitua5on is analogueto bleachingin thatbleached corals suffer from decreased photosynthetically-derivedcarbon as well. Basedon this evidence, one might expect to observe a changeinlipid levels in the first week of bleaching. But M. verruoosa isa muchslower growing speciesthan P.dflrtf'cornls and predictions of a rapidresponse in Morttlpora based on the latter coralmay be unrealistic. Morrfgrora verrucose in Hawai'ilives in a moreextreme habitat than Caribbeancorals. Therefore It may be naturally more capable of coping with streams such as bleachingmaking it difficult to detectany lipid responses after a shortbleaching period. M. irtartsmsttspawns every month during the summer. Eggs released during spawning can be upto 70%lipid by dry weight Arai et al.,1993!. If stressedcorals can resorb lipids from unreleased eggs,or candelay ripening eggs for spawning, then decreases intotal lipid levels could possittly onlybe detectableover a kwtgerperiod of time. Coralsmay increase heterotrophy in orderto supplementtheir nutritional demands. However,it is suspected that heterotmphy may only account for 1 PAof thecorals diet in some species Wellington, 1982!. Lipid levels simply do not change when corals are bieached, In this Studytreated fragments were compared with genetioally identiCal control fragments in a pairwiee fashion.This is a veryrobust experimental method because it controls for genetic variation betweencorals. Paired comparisons such as these have not been used in anyprevious experimentswhich examined Iipids in corals.I believethat my results are convincing evidence thatlipids do notchange within the first two weeks of bleaching.The hypothesis that lipid levels in 110 40 Q30 4 20 7.9 6.3 0 5.0C! U 0 4 8 14 TH.'vK DAYS! Figure2, Average%lipid per gram dry Weight + One~ SmN! and ~ chtcrcphrtt e ugrcrn ! a one ~ enor! Onday 0, 4, S andf 4, OpenSquarea, Open iaamande and Sckd CtrCtes reprsisent parent'cotcntea, cored fragnetntsavid esttnsrk fragments,respecavely, and aie otfsetslightly fnxn one arum so thatsnor barsdo notoverlap, The numberof parentcdorim, controlfragments and ~ fragmsias are ~ in panSnthiSSee. 111 50 30 20 K 10 g 5.11 6.68 7.90 9.39 12.43 CHLOROPHYLL a ug t:m >! Rg. s. Conalatton~ 5 Ipkj per gramdry weightend log ohtorophtrtta ugrorn !. recentlybleached corals would be lower, and that iipid content in corals would decrease as the lengthof the bleachingperiod increased,was rejected. No lipid response was detectable in bleachedfnttgments over the course of the initial 2 weeks. Runningthe experimentfor 1, 2 and 3 monthswould determinewhelher or not lipid levels change in bleached corals over a longer periodof time. As well, monitoringgamete productionand release over the same period in bleachedand unblescheclcorais would reveal whetheror not gametes are being resorbed or being preventedfrom developing during bleaching. After natural bleaching eventssuch as those observed in the Caribbean in 1983 and 1987, somecoral speciesrecovered more rapidly and more frequently than others. The ability to withstandand recoverfrom prolongedbleaching events i.e., several months! may yet be related to the amount of lipid stores, and may yield some insight into why some coral species recover morequioldy from bleachingevents than others. However, lipids do not appear to play a role in short-tenncoral bleachingand recovery. . I woukt Nketo trank Dr. G. M. Wellington,Dr. D. Krupp,Dr. P.L. Jakiel, Dr. D. Gleason,Tom Wilcox end at the pertiotpentsof the HIM8 sunsnerprogram for theirasnetrur~ rrttNsm and supportand Dr, M. Lesser for the teeOf hie temperature dtea teggere end UCOr tetO SprSdrendkknrekV.ThiSreSeareh wae OOehur~ ae partOf the 1994 tttotogyEttwtnW. end Peuteyewe eupporkrd SrsnnsV Proaramby Ae Edwin kr MarineW.Pautey elototno RrundetkrnW ~ endthe on ~ Coral of Reels' Houston at the HsweitLGIAgrant tnstrtute of have LITERATURE CITED; Arai,T., Kalo, M., Hetntratd,A., lkeda,Y. ~Iizuka, T. & Maruyama,T. 993!, Lipidcomposition of posiveiy buoyanteggs of reef buitding corals. Cons!Reetis, 12: 71- 75. Betsy, J,F, 8 Patton, J.S. 984!. A reevaluationof the role of glycerol in carbon translocation in zooxanthellae- coelenterate symbiosis. Mar. Biol. 79: 27- 38. Brown, B.E. 8 Suharsono, T. 990!. Damage and recovery of corai reefs affected by El Nino relatedseawater warming in ThcwaandIslands, Indonesia. Coral Reefs 8: 163- 170. 112 Coles,S.L & Jokiel,P,L. 978!. Synergisticeffects of temperaturesalinity and lighton hermatypiccoral Montipora verrucose. Mar. Biol.49: 187- 195. Cook,C,B, Logan, A., Ward,J., Luckhurst,B, & BergJr., C.J. 990!. Elevatedtemperatures and bleachingon a high latitudecoral reef:the 1988 Bermudaevent. Cora/ Reefs 8: 45- 49. Croffoth,M.A. 985!. Mucoussheet formation on poritidcorals: effects of alteredsalinity and sedimentation,Proc. 5th lnt. CoralReef Congress,Tahiti 4: 165- 170. Fitt,W.K., Spero, k.J., Halas,J White, M.W. & Porter,J.W. 993!, Recoveryof thecoral Montastreaannuiaris in theFlorida Keys after the 1987Caribbean "bleaching event . Coral Reefs 12: 57- 64. Gattuso,J.- P., Yellowlees,D. & Lesser,M. 993!. Depth- and light-dependant variation of carbonpartitioning and utilization in thezooxanthellate sderactinian coral Sty!ophora pistillate. Mar. Ecol. Prog. Ser. 92: 267- 276, Glynn,P.W. 993!. Coralreef bleaching: ecological perspectives, Coral Reefs 12: 1- 17. Glynn,P.W. & O'Croz,L. 990!, Experimentalevidence for high temperature stress as thecause of El Nino-coincident coral mortality. Cora!Reefs 8: 181- 191. Gleason,D.F & Wellington,G.M. 993!, Ultravioletradiation and coralbleaching. Nature 385: 836- 838. Harland,A.D., SpencerDavies, S. & Fixter, L.M. 992!. Lipidcontent of someCaribbean corals in relation to depth and light. Mar. Biol. 113: 357- 361. Harriott,V.J. 993!. Corallipids and environmentalstress. Env. Mon.Ass. 25: 131- 139. Hoegh-Gulberg, O. & JasonSmith, G. 989!. The effectof suddenchanges in temperature, lightand salinityon the populationdensity and exportof zooxantheliaefrom the reefcorais Stytoptrorapistillate, Esper and Seritoporahysterix, Dana. J. Exp. Mar. Biol Ecol.129: 279- 303. Jeffrey,S.W. 8 Humphrey,G.F, 975!. New spectrophotornetricequations for determining chlorophyllsa, b, c, Biochem.Physiol. Pffanzen BPP! 167; 191-194. Jokiel,P.L. 980!, Solarultraviolet radiation and coraireef epifauna. Science207: 1069- 1071. Jokiel,P.L & Colas,S.L. 977!. Effectsof temperatureon the mortalityand growthof Hawaiian reef corals. Mar. Biol. 43: 201- 208. Porter, J.W., Fitt, W.K., Spero, H.J., Rogers, C.S. & White, M,W, 989!. Bleaching in reef corals: physiologicaland stableisotopic responses. Proc. Natl. Aced. Sci.86: 9342- 9348 Stimson, S.J. 987!. Location, quantity and rate of change in quantrly of lipids in tissue of Hawaiian hermatypiccorals. Bull. Mar. Sci. 41. 889- 904. Szmant, A.M. & Gassman, N.J. 990!. The effects of prolonged 'bleaching' of the tissue biomass and reproductionof the reef coral Montastrea annularis, Coral Reefs 8: 217- 224, Wellington,G.M. 982!. An experimentalanalysis of the effectsof lightand zooplanktonon coral zoonation. Oecologia 52: 311- 320. 113 Uttravtof«IRsdlstbn snd Coral itesfa 1N5. O.Gufko 6 P. L. Jokiel eda.!.HIMB Tech. Report «41. UNIHl-Sea Grani-CR-gM3. Uneven bleaching within colonies of the Hawaiian coral Ijilrontipora verrucosa Andrea G, GrOttOli-Everett' & Ilaa B. Kuffnefz UniversityofHouston DepartiraetofBbbgy HousffmTX 77204-5513 ~1 lnstffubof Marine Biobgy, P.O. Box 1346, Kans'ohe, Hl, 98744 ABSTRACT:When a coral cobny undergoesbleaching, the PBsp0%&may not be unfformacross the enbrecobny. In thts study,Ihe bleaching~ of Afcntfpomwrrruco«a ccbnifw varied sigriffcanffydepending on the ~ quantity andquality of soleil~ thateach area of theoobny wes ~ to. Inorder to inducetfeaching, five Af vair@catecobrves were transplantedfrom a bw-light environmentat 10 m to a high light environmemat 1 m, Four dNerentlight treatmentswere concurrently imposed on four disffnctregions of each coral ~. The four treatmentsIncluded 1! e~ levels of I AR photoerntheticalfyactive ~:400 -700 nm!, 2! ~ levelsof PARsnd Uv* 20 - 400 nm!,3! elevatedlevels Of I"AR, UV-A and UV-B 00 - 320 nm!,and 4! a OOntreltrcettment wffh PAR, UVA and UVB ~ to leviesthat mimickedradiable levels found at 10 m, At the comptetbn of the rine day treatmentperiod, bbsching i~ was quanlfied by measurfngthe corvceirfrationof chbrophyll a zooxsnfheffftedereity. and percentfifxd per gram dry weight in ssmpim from each treatnwnt regionof each coral ~. Chbrophyll a per xtioxsnthe«ewas also oalcLfated.Chbropht«l a conoentn8onsdi«oseeed agniffcanffy in the two treatmentsthat included~ levels of PAR and UV p c 0.013!. Neitherxooxanthellae densffi«e ror ffpKI levelsdecreased «lgn~ in any of the treatments. These resultssupport the hypofhceisthat conalsdo not bleach uniformlywhen ffeated with differentff«vela of solar inadianoe. It also appearsthat Af.ierrucoss, when inducedb bleachin this manner,responch by decieaeng ohtoiophyffe cc~ons, and not by expeffingzcoxantheffae. INTRODUCTION Pigment loss in scleractiniancorals due to reduction in zooxanthellae density and/or the loss of photosyntheticpigment per zooxanthella cell is a phenomenon known as coral bleaching. Over the past 15 years, the incidence of widespread bleaching events on coral reefs has increased throughout the world. Elevatedtemperature, ultraviolet radiation, total solar irradiance and sedimentation are among the environmental factors which have been found to cause bleaching in corals Gleason & Wellington,1993;Jokiel & Colas, 1990; Hoegh-Guldberg 8 Smith, 1989!. Bleaching can result in the interruption of coral growth, reduction in reproductive output and, eventually,death Jokiel & Coles, 1977; Glynn & D'Croz, 1990; Szmant & Gassman, 1990!. The gravity of this phenomenon has lead to increased research examining the effects of bleaching on coral biology and ecology. Uneven bleaching within a coral colony has been observed by several researchers Fltt et. ai., 1993; Jokiel & Coles, 1990!, Jokiel and Coles 990! stated that 'portions of coral colonies receiving the highest incident radiation bleach more readily than portions that are shaded.' In a paper by Fitt et al. 993!, a large COIOrphOtOgraph Of a bleaChed Caribbean coral, M'oritfastrea anriularis, clearly illustrates a mottled bleaching pattern. However, these studies did not quantitativelyaddress the issue, We also observed uneven bleaching in experimentally manipulatedMontfpora verrucosacoral fragments. Each fragment had a wire identNcationtag wrapped around it and was induced to bktach via transplantationfrom a iow-iight site at 10 m to the high-iight site at 1 m for one week. Initially, the coral fragments appeared to have bleached uniformly. Closer examinationrevealed that the area shaded by the wire tag was much darker than the adjacent, unshaded area. Following this observation, we designed a study to empirically measure differential bleaching response within a coral colony by simultaneousiy exposing differentareas of the platingcoral, M, verrucose,to varying levels of solar irradiance. We tested the hypothesisthat uneven bleaching within a coral colony occurs as a result of different levels of incident solar radiation. Manipulationswere aiso performed in order to determine which portion of the irradiance spectrum was inducing the response: PAR, UV-A, UV-B, or some combination of the three. MATERIALS AND METHODS The experiment was conducted between July 23 and August 1, 1994, at the Lighthouse Point LP! and the Bridge to Nowhere BTN! sites on Coconut island, Hawai'i. A coral 115 fragmentwith minirnurndimensirxe of 30 cm x 10cm wasbroken off fromeach of five separate coloniesof the platingmorphology of Montiporaverrucose, at a depthof 10 m atthe LPsite. A smallersubfragment with dimensionsapproximating 100 one wasthen broken off eachlarger fragment,tagged and pieced back at the siteof originas a controlfor transplantation.The five largerfragments were exposed to increasedlight levels via transplantion to a depthof 1 mat the BTNsite, and each centraIy placed under a separatetreatment frame for ninedays Fig. 1!. Each framewas 50 crn x 50 cm and consistedof four adjacenttreatment bands Fig, 1!: 1! A controltreatment with integratedirradiance levels between 300 and700 nm, similarto those found at 10 rn 0 cm x 20 cm band of ultravioletradiation- transparent UVT! Plexigias overlain with two layersof neutraldensity filter!. 2! A highPAR + UV-A+UV-B treatment 00 - 700nm! a 50 cm x 5 cm bandof UVT Plexiglas!. 3! A highPAR+ UV-Atreatment 20-700 nm! a 50cm x 5 cm bandof UVT Plexiglasoverlain with UV-B opaquemylar!. 4! A high PAR treatment 00- 700 nm! 0 cm x 20 cm band of UV-opaque Plexiglas!',. TREA'AvK'Ã7 FUMES + TREATMENT FRAGMENTS 1M CONTROL FRAGMENTS ~ >: ~of~~, unewrn~ wesindoorrdin~ trWrepienttngthemfram 10 m t~ to1 m thrgtHiyht!entt centraNy pissing them Lrnder a ~ framefor > Crete- Eaohframe rrsistedof a pAR,pAR+ W-A+ NrB, pAR+W-A banstrerentstrip as well as a ~ controlwhish recusedbrtei irrertbrnce PAR+ UVW+ WB! by70% in ordertO~ thelewer right leveie at iC m. Byplacing each coral fragment under a separatetreatment frame, different areas of eachcoral's surfacewere simultaneousfy exposed to the fourdNerent treatfnents, Transplanted fragments and trealmentframes were affixed to a uniformsubstrate plastic mesh plafform, SeeGulke ef ef. thisvolLsne! for etens ard more inhrme&rn on the rare of these rittere. 116 Averagetotal irradiance atthe high-light site BTMat 1 m!was 70'%%d greater than that observed atthe 10 m LPsite basedon integrated light measurements made every 2 nrnusing a Li-CorLi- 1800UWunderwater spectroradiometer between 300-700 nrn!. IntegratedUV-B 00-320 nrn!, UV-A 20-400 nm!and photosynthetically active radiation PAR 400-700 nm! levelswere approximately99/o, 93%%d and66 /o greater at 1 mthan at 10rn respectively. Treatment fragments weretransplanted to the BTN site to replicatethe low wave action conditions present at 10 rnat theLP site LPsite is neara dockwith boat traffic!. These sites are separated by approximately 100m, occur in thesame small lagoon, and have similar sedimentation and temperature regimes. Sedimentwas brushedoff of the treatmentframes daily for the durationof the experiment,Hourly temperaturevalues were recorded both at the 1 mBYN and at the 10 m LPsite usingtwo HOBOrMminiature data loggers. The two sites differed by less than 1'C, on average, and neither temperatureregime was high enough to inducebleaching LP averagetemp. = 26.47j 1.%PC and BTNaverage temp. = 26.95k 1.01'C! Jokiel& Coles,1977!, Afternine days, eight samples were taken from each of the four treatment bands within each coralfragment for a totalof 32 subsamples perfragment! and from the control fragment by drilling witha 1.25cm2 cork borer through the coral plate. Three sampies were analyzed for chlorophyll a concentrations,three for total lipidlevels and two for zooxanthellaeconcentrations per treatment per coral, Chlorophylla was extracted twice from fresh, finely ground samples in 10 mlof 100%%dacetone at4'C for24 hours,Samples were then centrifuged for 10 minutes.The absorbance of the supernatantwas measured using a spectroradiometerandthe chlorophyll a concentrations were calculatedaccording to themethod described by Jeffrey and Humphrey 975! andreported in ug/cm2. Lipidswere extracted from finely ground samples samples had been frozen at -5K for1-2 weekSpriar tO extraction! in a chlOrOform:methanol :1,v:v! solution, EXtraCts were then waShed oncewith 0.88%%d potassium chloride solution, three times with a methanol:watersolution ;1,v:v! anddried at RPCfor 24 hoursbefore weighing. Animal tissue biomass was determined following lipidextraction by burning the skeleton and remaining tissue residue in a mufflefurnace at 450'C for6 hours.4pid content in corals was reported as %%d perlipidgram dry tissue weight. This methoddiffers slightly from Harland et al. 992! wheresamples were decalcified prior to lipid extractionwhich can resultin lipidloss during the decalcificationprocess triglycerides can hydrolyzein acid solutions and the glycerol component of themolecule is thensoluble!. In order to determine zooxanthellae concentrations,fresh samples were simultaneously decalcifie in 10 rnlof 10'%%dacetic acid, preserved with a fewdrops of 4%%dformalin and statned with a fewdrops of Lugol'ssolution. Once decalcification was complete, sarnpies were centrifuged on "high"setting for 10 minutes,the excess liquid was decanted off and the remainder was homogenizedfor 30 secondsbefore being resuspended into 10 rnlof 4'%%dformalin for long-term preservation,Four subsamples from each sample were counted using a 0.1mm hemocytometer andreported as theaverage number of zooxanthellaeper cm, Theamount of chlorophylla per zooxanthellaewas determined bydividing the amount of chlorophyll a per crnz by the totat number of zooxanthellaeper cm andwas reportedin ng of chlorophylla per zooxanthellae. Themean lipid, chlorophyll a, zooxanthellae or chiorophylla per zooxanthella levels for the transplantedcontrol fragment and the low light control fragment were analyzed using a student'st- test. All ofthe data were then analyzed by pairwise model I ANOVA'sbetween the control and the fourtreatments to determineif eitherlipid, chlorophyll a, zooxanthellaeor chlorophylla per zooxanthellalevels had changed in any of the treatments.In all casesthe nullhypothesis was rejectedat an alphalevel of 0.05, RESULTS On the first day of the experiment,all of the coral treatmentand control fragments were uniformlydark brown in color. The portion of the corai treatmentfragments positioned under the PAR + UV-A + UV-B,PAR + UV-A and PAR treatmentbands beganto visibly bleach after the fourth,fifth and seventh day respectively. At the end of the nine day experiment,the coral area underthe low-light,PAR + UV-A+ UV-B, PAR + UV-Aand PARtreatment bands, were dark brown like the control fragment!, almost white, extremelylight brown and medium brown in color respectively. The control and low-lighttreatment were not sign!ficantlydifferent from each other with respectto any of the variablesmeasured. This indicatedthat brealdngoff a coral fragment had no significanteffect and that changes in the proportionateamounts of PAR, UVA and UVB due to transplantationwere negligible. Only changes in spectral quantityand quality had an effect on the manipulatedcoral fragments. The degree of b!cachingwas determinedby measuring'the chlorophyll a and zooxanthellae concentrations. Relativeto the controland the !ow-lighttreatment, a significantdecrease in chlorophylla was observedin the PAR + UVA and PAR + UVA+ UVB treatments F = 4.894, p 0.013! Fig. 2a!. h 'l.2 0S ~to 4 K 06 g 08 ~ z04 m06 g 0.4 02 $02 CONT LOW PhR PAR PAR C CRT LOW PAR PhR PAR Uvh UVh UVh UVA VVB UVB 10 a 30 5- Ue D Im''a 06 < X20 ag 5 eS 04 JQ ~ to ga 8 02 C ONT LOW PhR PAR PAR C ONT LOW PAR PAR PAR UVh UVh Uvh UVh UVB UVB FtgLrre2: Mean ~ A. chtoroph ie ~!. 8.zooxanthetae x10lorn !, C. chtoophytl a per zooxttnthetla ngr'zoeraarlhettae!andD. percerrt lipid per gram dry weight tevete in eachof the four treelment ragkxtsregions and control tpnrtnteofthe Hawaiian coral, Af~ |errMCOaaafter g days,Treatments underlined with lireS at theSame level werenet Signlrroantlv ~ alPha = 0 05!.CONT = controlfrttgrnenL LOW~ lighttreatrNmt, PAR T~ = PAR 00- 700nm!, PAA + UV-AT~ = PAR+UVW fS20- 700 nm!, PAR + UVA+Uv-8 T~ = PAR+UV-A ~ UV-8 00-700 nrn!. Thesedecreases in pigmentlevels in the PAR + UV-Aand PAR+ UV-A+ UV-Btreatments were notaccompanied by anychanges in zooxanthellaeconcentrations, Zooxanthellae levels were notsignificantly different in thePAR + UVAand PAR + UVA+UVB treatments relative to the controland low-light treatments. However, zooxanthellae concentrations were significantly higher in ihe PAR treatment F = 3.324, p 0.047! Fig.2b!. Consequently,the chlorophyila per zooxanthellalevels were significantly lower in the PARtreatment F = 4,088,p c0.025! Fig,2c!, Energyreserve levels were determined by measuring lipid levels. Lipidlevels did not differ significantlybetween any of the treatments or control F = 0,351,p 0.789! Fig.2d!. DISCUSSION Chlorophylla, zooxanthellae and chlorophyll a per zooxantheila levels varied significantly in responseto variouslight conditions within fragments of the coral,Montfpora verrucosa. The portionof the coralfragments exposed to elevatedlevels of PAR+ UVA+UVB, PAR+ UVA,and PARexhibited high- to low-levelsof bleachingrespectively. There was no bleaching in eitherthe low-lightcontrol or the transplantationcontrol. Theportions of thesolar spectrum which induced the bleaching response were elucidated, Whilepigments levels did decrease in all elevated irradiance treatments PAR, PAR + UVA,PAR + UVA+ UVB!,significant decreases were only detected in the two treatments that included ultravioletradiation Fig. 2a!. Underelevated PAR conditions UV excluded!,M verrucceadid not significantlylose chlorophyll a relative to controls, but the density of zooxanthellae increased, resultingin anoverall decrease in the calculated value of chlorophyll a per zooxanthella Fig, 2a, b, c!. Thisevidence Indeahe that in HawaiianM. verrvcosa,PAR, as weilas UV,causes chlorophyll a to decrease but that under elevated PAR conditions alone, the coral may be able to compensate forthis by increasing the numberof zooxanthellae.Perhaps the increase in zooxanthellae densityin theabsence of UV isa responseto an increasein potentiallight harvest with@A an increasein the biologicallydamaging ultraviolet radiation. Energyreserve levels were determinedby measuringlipid levels, The percentlipid content pergram dry weight did not significantly differ between any of thetreatments or control conditions.Under standard physiological conditions, fatty acids and glycerol are synthesizedby zooxanthellaefrom photosynthetically-fixed carbon and translocated to the hostwhere they are eithermetabolized or transformedand storedprimarily in the formof wax eatersand triglycerides Batey8 Patton,1984!. Whencorals bleach, chlorophyll a levels decrease; hence the amount of carbonfixed also potentially decreases and othersources of carbonhave to be reliedupon. Despitea dramaticdecrease in photosyntheticpigment chlorophyll a! in thebleached portions of the coralcolonies, lipid reserves did notdecrease in M, verrucasa.Olher work by Grottoli-Everett 995! showsthat lipid levels in M. verrucasado noi changeeven aftertwo weeksof bleaching. Perhapsthe zooxanthellae are able to maintaina highlevel of fixed carbon production because at high-lightlevels, the chlorophyll a pigments are being fully saturated. Alternativel, decreased metabolism,increased heterotrophy, gametes resorption or some combinationof thesefactors duringthe early stages of bleachingare mechanisms by whichbleached corals may be compensatingfor decreasedphotosynthetically-derived, fixed carbon. Giventhat varying degrees of bleachingoccurred in the three elevated irradiance treatments, and not in the low-lightcontrol treatment within fragments of M. verrucosa,we acceptour hypothesisthat uneven bleaching within a coralcolony occurs as a resultof differentlevels of solarIrradiance, The resultsaf this experimentsuggest that PAR, UV-Aand UV-B havea synergisticeffect on bleaching in M. verrucosa,as thedecrease in chlorophylla concentration was greatestwhen all three sectionsof the spectrumwere allowedthrough the filters,and respectively less in the treatments where UVB was screened out and the treatments where no UV was allowed throughthe fiiters. Furthermore,this study indicates that bleaching due to increasedsolar irradiancein the Hawaiian coral, M. verrucosa,results from a decrease in chlorophylla per zooxanihella and not from a decrease in the number of zooxanthellae, Differentialbleaching responses within a coralcolony are quantifiabIe. While uneven bleachinghas been mentionedpreviously in the literature,this studyis the first documented empiricalevidence of thisobservation. When conductingexperiments on bleachedcoral, researchers must be careful to take into account the heterogeneity involved in bleaching in order to avoid biased sampling. 119 uttravtoletftadlstiea anrt Carat Reefs. lttt5. 0. Gtrliro5 P.L JoaislIsds.!. HIMB Tech. RePort 441. UHIHI-Sea Grsrtt-CR 9543. The metabolic response of Fungia scutaria to elevated temperatures under various UV light regimes Sophia V. Hohlbauch Clepartmentof BiologiCal Sdanol, UniversityOf California, Santa Barbara, CA 93106 ABSTRACT:photteyntheae andreapiratten of Funy's Sudan@, aootirnattted tc UVO snd UVT trc~, waremeasured ra 27 C Snd29 C. In the summer,average Seawater temperarturea In Kana'ohe Bay, 0'ahu, Hawai'I range ~ 2S- 27 C. At 29 C, thephcrtoejjmthsdo rates tor coral erxlima5md to UVQ,UVT, and UVTiUVO conditions decreased by 71%,, ltd and24tt, res~y, frommeasurements at27 C. Chawophtrtta and o2, tat Svr other Issrd, ~ bothtampsrrattrras and ~ sti three treatments. INTRODUCTION There has been Bn inCreasedCOnCern over the amountS Of Sohr ultraviolet radiation that reachesthe earth'ssurface, in particularat the equatorwhere ozone is naturallythe thinnest. Someevidence implies that UV radiationlevels haveincreased due to a decreasein the ozone layer.Inclear tropical waters UV radiation, especially UV-B, penetrates to deeperdepths due to the Iowlevels of dissolvedorganic matter and chlorophyllfound in the water Smith&. Baker, 1979!, Dueto a reportedincrease in the occurrenceof coralbleaching, which results in a decreasein eitherchlorophyll or algaldensity, researchers have tested the effectsof UV on coral. Althoughtemperature, salinity, and sedimentationare other possiblefactors contributing to bleaching,UV radiationhas been found to affectcoral growth, calcification, reproduction and viability Jokiel & York, 1982; Jokiel & Colas, 1990; Szrnant& Gassrytan,1990; Gleason& Wellington, 1993!. Thereare many possible mechanisms that animalscan protectthemselves from UV damage. Theyrange from behavioral adaptations, such as movinginto low UV environments,to the enhanced enzyme aCtivityol catztlaaeend aSCOfbateperoxidaae in symbiotic algae and superoxidedismutase in boththe hostand algal symbiont Shick ef al.,1991!. ln addition,coral and/ortheir algaemay also show tat increasein the productionandrror accumulation of UV absorbingcompounds like mycosporine-like amino acids Dunlap& Chalker,1986!. For example, Dunlapet al. 986! discoveredthat variousAcrtttpofa species contained more UV-absorbing compoundsatshallower depths than their deeper counterparts. Furthermore, Drollet et al. 993! foundthat Fuftgiaturtgites secreted mucus containing MAAs. The concentrationof these MAAs decreasedwith increasingdepth as the levels of UV radiationdecreased. Thus, these coral appear to be adapting to their shallow environment. Past researchhas also shown a synergisticeffect between manyfactors includinglight, temperature,salinity, and ultravioletradiation Coles and Jokiel, 1978; Hoegh-Guldbergand Smith, 1989;I esser ef a/., 1990!. It appears that a combination of any of these factors generally resultsin a stressresponse that would not be observedif there wereonly one factor involved. The purposeof this Studywas tO determinewhether FIJngfascufarta expOSed to UV radiatiOn wouldbe moresusceptible to increasingtemperatures, and thus causecoral bleaching at a lower temperature. MATERIALS AND METHODS ri n Specimensof Furfgr'afxxffaria, 1-3 inches in diameter,were collectedfrom patch reefs in Kane'oheBay, 0'ahu at depthsranging between 5-10 feet duringthe monthof June, 1994. All individualswere returned to the Hawe'I Insbtute of Marine Biology, Coconut Island, 0'ahu and placedin a runningseawater tank. After adjustingto laboratoryconditions for severaldays, F. sCutsrfawere aCClimatized fOr tWenty-five dayS in treatmentswhich either blOCked or allowedsolar ultraviolet radiation. A sheet of AdarNI Allied Chemical! was used as a ultraviolet-transparent UVT!filter whereas a sheetof 100'A dear Acrylicwas used to blockal solarultraviolet radiation UVO! seeGulko et al.,this volume!. After the twenty-fiveday incubationperiod, one-half of the individualsexposed to sdar ultravioletradiation were transferredinto UVO conditions. These coral,maintained in UVT conditionsfor twenty-fivedays and UVO conditionsfor seven days,will be categorizedas beingexposed to a UVT/UVOtreatment. The temperatureinside the experimentaitank was regulated;however, there existeda regular dailyfluctuation. Thus, the temperaturewas recordedusing a Hobo-Temp-XTData Logger Fig. 1!. Initialtemperature during the firsttwenty-fhre days was aproxirnately27.9'C duringthe day and 26.~ at night Atthe end of Day25, the temperaturewas increased and maintained for seven daysat 29.9'Cduring the day and 27.9'C at night. Rstas 1. ~ %rnporahre neetsrrsttst twskrs~ Irrlsrv& insidetra rarperimrantsltank st mrsHswsi'i InatitLrta4 MarinaBtrx09tr, ~ tstsnd. CrshLrtntrn tuttr 2$, 1994 Ostr2C! ttattushAugtart 8, 1994 Day 34!. Nst ttrtr~rnthsaia sndnl9ht raapkattrtnararS rnrteiaard St OSya25 Snd32. YSI Electro~ Model 5739! and DissolvedOxygen Monitors Model 58! were used in 880 ml volume chambers that were placed within the experimentaI tank. Net photosynthetic and night respiratoryrates were measuredat 2~ for coralexposed to UVO and UVT conditionsand at 29'C forcoral exposed to UVO, UVT,and UVT/UVOconditions. A totalof three coraiswere used per treatmentat asch temperature, Both photosynthesisand respirationat each temperature were measured on the same coral. Data acquisition and analysis were obtained using Sable SystemsDatacan Version 4.0!. After measuringoxygen evolution and consumption,the displacementvolume and wet weight for each coral were determined. T'wosamples taken from each coralwere extractedtwice in acetone and refrigeratedin the dark The chlorophyllextracts were centrifugedfor 10 minutesbefore their absorbanceswere readusing a HewlettPackard 8452A Diode Array Spectrophotometer. Chlorophyll a and c2were calculatedusing Jeffrey 8 Humphrey's 975! equations. The chlorophyllcontent reproted for eacilcoral is an averageof the twosamples taken. 122 0.08 C 'a 0.06 CV di 0 e ~ 0.04 1{+0.02- 0- 26.5 27 27.5 28 28.5 29 29-5 Temperature C! Retieerrpoeeda NtgttttoteeptnttianUV inttteIy torbabnaF. cotttrte being tntnallWradwith inCrsaaW toX temperatore opaque aattrrtiCrne. forCOral Eacheither point e~repeeenta tO eater anUV,~ blockedof threefromCCtttlUV, Or ~ ndbare ~ 894. Chlorophyllaand cz concentrations didnoi vary both between experimental treatments at eachtemperstunt and between temperatures within the same treatment, For chl a, UVO acclimatizedcoral contained 53.2 = 9.6itg and45,7 = 4.9pg at 27 C and29'C, respectively Fig. 5!.Similarly, coralunder UVT corttfttions contained 48.6 = 7.5pg and 48.4 = 4.0itg al 2~ and SPC,resper&mly, aswel as UVT/UVO coral which contained 52.7 = 12.5p.g. Chlc, concentrationswerelower than chl a asis to beexpected Ftg. 6!. At 2TC,UVO and UVT coral contained33.4 = 10.1itg and29.1 = 7.3pg, respectively, whereas st 29C, UVQ,UVT, UVTNVOcoral have 21.2 = 2.8ltg, 20.1= 1.2kg and26.7 = 6.3ltg, respectively, DISCUSSIONThemebsholic response of F, scvfarra seems to indicatethat initially, at2~, UVhas a detrimentaleffeCt On UVT-aCCiimatized cOraldue to the loWer net phOtOSyntherc rate.Since Chl e enclchl ct cOntent oftheee individualS dOnOt vary, UV dOeSn't affect either the a~ty or rasctit:tncanter pigments; however, it does reduce the photosynthetic efficiency ofthe coral, Shicketal. 991! observed a similar trend in the octocoral, CLsvuiaria, inwhich UVT acdimatized alshad a50% decrease in net photosynthesiscompared to individualsacdimatized to UVQconditions even though chlorophyll content remained the same. At~, theeffect ofUV on the coral seems tobe shadowed bythe effect of a ~ increase intemperature. UVOacdimatized coral photosynthesized theleast even though the 124 026.5 27 27.528 28.5 29 29.5 Temperature G! Rgure4. P/R ratios forthethree UV~s atZPCand 29C 100 80 200 27 29 Temperatur~e' C! Figure5. Chrophphyllaconcentrations forcoral acdimstized srste three experirnentat trrs~. Eachvie representserst ~ Of twosarnptes tatran from each corai ~j. BarSrepress' SEM. 125 100 80 60 CL 0 0 0 20 0 I 27 29 Temperature ' C! PrattleS, ChtcrcphrtrC~ccncantrattar» fcrcOral acoftrre5ad to I» thoseaxpartrrentat ~ts. Eachvstue rspnaaanta and aaraga of two aamptastatarn fram aaCh oOral ~!. Bars apraaantSEM. margin of differenc between these coral and UVT and UVT/UVO acclimatized coral was much kIS. COleSif Jakiel 990! alSOObServed a IOWerphOtOSynthetic rate on both rtcrt-aociimatized and2-month acclimatized coral. SinceF. ~ is normallyfound in shaliow 0 feet deep! waler, It may respondmote to changesin temperaturerather than different kfvels of UV. P/Rra5oe deceased mainly due to the reductionin photosyntheticrates. It is interestingto note thai ColasIL Joldel 990! conciudedthat higherrespiration, and not changes in photosynthesis,ls responsible for coral bleaching at elevatedtemperatures due to both the temperaturedependence of bicchemicaf 9actiorts and the inabilityof coralto maintainhigh P/R faunaeatthese temperatures. Respiration at 29 C wasnoi signNcantly different at anyof the three treatments.In additioft, coral bleaching was not observed at anytime during the experiment. Althougha decreasein photosynthesismsy consequently lead to reducedgrowth rates and reproductivecapabilities, it may not indeed be responsible for bleaching. Sasedupon the present results, UV does not appear to make F. scufaria more susceptible to temperatureincreases. This may be duein partto thelifestyle of thisparticular coral. Since it is rtormaliyfound at shallow depths, it must be adapted to iis higher UV environment. Inaddition, it ispossible thatthese corals, whch produce agreat deal of mucus, may also contain UV-abso*ing compoundsintheir mucus as has been found in Furfgia fungfias Drollet et al.,1993!, In order to fullyUnderstand thiscofel, further measurements of photosynthesis andrespiration must be tradedat normal and elevated temperatures toverify these current findings, Mucus and coral Sssuesamples should also be a~rzed for the presence ofany ~rcosprarine-like UV-absorbing compounds, F.~ a»raarraror»ra atHIMB to' ~ of thalrfaciltttaa arrdaqApmant; anttDr. Miohara Lssaar fortaro Of hia ataothamia»rsnclanatoetrtty'tat~box.thank R.K. TmnCh lbrraVirnang thisrr»ntracrfpL f sct~ ttnano4 aaatatsrkar fromHIMB E.W. Psutsy Founrtsttoni. 126 LiTERATURE CITED Coles, S,L, & P.L, Jokiel 978!. Synergistic effects of temperature, salinity, and fight on the hermatypiccoral Monfipora verrucosa Mar. Biol, 49: 187 - 195. Drollet, J.H., Glaziou, P. 8 P.M.V. Martin 993!. A study of mucus from the solitary coral Funyia fungites Scleractinia; Fungiidae! in relation to photobiological UV adaptation. Mar. Biol. 115: 263 - 266. Dunlap, W.C. & B.E. Chalker 986!, Identification and quantitationof near-UV absorbing compounds S-320! in a hermatypic scieractinian. Coral Reels 5: 155 - 159, Dunlap, W,C., Chalker, B.E. & J,K. Oliver 986!. Bathymetric adaptations of reef-building corals at Davies Reef, Great Barrier Reef, Australia, III. UV-B absorbing compounds. J. Exp. Msr. Biol. Ecol. 104: 239 - 248. Gleason, D.F. & G.M. Wellington 993!. Ultraviolet radiation and coral bleaching. Nature 365: 836 - 838. Hoegh-Guldberg,O. & G.J. Smith 989!. The effect of sudden changes in temperature, tight and salinity on the population density and export of zooxanthellae from the reef corals Sfyiophora pisfillafa Esper and Serr'atopora hysfrix Dana. J. Exp. Mar. Biol. Ecol. 129: 279- 303. Jeffrey, S.W. 8 G.F, Humphrey 975!. New spectrophotornetric equations for determining chiorophyils a, b, and cz in higher plants, algae and natural phytoplankton, Biochem, Physiol. Pflanzen. 167: 191 - 194. Jokiei, P.L. & S.L Coles 990!. Response of Hawaiian and other Indo-PacNc reef corals to elevated temperature. Coral Reefs 8: 155 - 162. Jokiel, P.L. 8 R.Y. York 982!. Solar ultraviolet photobiology of the reef coral Pocillopora damicomis and symbiotic zoothanthellae, Bull, Mar. Sci. 32 !: 301 - 315. Lesser, M,P. Stochaj, W.R. Tapley, D.W. 8 J.M. Shick 990!. Bleaching in coral reef anthozoans: effects of irradiance, ultraviolet radiation, and temperature on the activities of protective enzymes against active oxygen. Coral Reefs 8: 225 - 232. Shick, J.M., Lesser, M.P. 8 W.R. Stockaj 991!. Ultraviolet radiation and photooxidative stress in zooxanthellate anthozoans: the sea anemone Phyl lodiscus semoni and the octocoral Clavuiana sp. Symbiosis 10: 145 - 173. Smith, R,C. & K.S. Baker 979!. Penetrationof UV-B and biologically effective dose-rates in natural waters. Phofochem, Phofobiol. 29: 311 - 323. Szmant, A.M. & N.J. Gassman 990!. The effects of prolonged "bleaching" on the tissue biomass and reproduction of the reef coral Monfasfrea annrrlarr's. Coral Reefs 8: 217 - 224. 127 UittstrfofetRsdlstios sett Coral Reefs. 19f6. D. Golkoft P. I.. Jokielletfs.!. HIMB Tech. Report 441. tiNIHI-Sea Grant-CR-95-03. Preliminary report on the occurrence of mycosporine-like amino acids in the eggs of the Hawaiian scleractiniarf corals Afontipora verrucosa and Fungia scufaria DavidA. Krupp', JacquelineBlanckz DepartmentOf Natutsl Sdenoee,Vhnchmrd Community CONage, Kans'ohe, Hl 98744 Hawai'I I~ of Manna Bioktgy,Uriverslby cf Hawal'i,Kana'ohe, Hl 96744-1348 ABSTRACT:'Ihe rrnrtstsporfncdilkeamino adds IMAAsl cf the eggs of the colorNaihermaphroditic coral Asonffpcvaverrucces snd the solitarygancehorfc coral Fungktsade were assayed. The eggs from both speciescontained ~ high concentratktnsof MAAs, but differed from each other in qualityand quantity cf MAAs.These tlference may be ccstelated with dlfitorsncesin their ~ cf spawning. INTRODUCTION During the past decade, much has been learned about the sexual reproduction of tropical BClerBCtinianCOralS. Details regarding the mOdeeand timing of Borne200+ SpeCieSare now available Harrison & Wallace, 1990; Richmond& Hunter, 1990!. Old dogmas describing most scleractiniansas brooders that release planula larvae e.g., Hyrnan, 1940;Vaughn & Wells, 1943} haVebeen rejeCtedafter many Obeervationshave shown mOStCOraiS tO be hermaphrodNc broadcastspawners reviewed by Fadlallah, 1983; Richmond & Hunter, 1990; Harrison 8 Wallace, 1990!, Many of these broadcastingcorals release their gametes synchronously during annual rnuNspeciesspawning events Hamson et BI., 1984; Babcock>et fQI.,1986!. These mass spawningevents are fantasticspectacles of millionsof eggbundles drifting to the surfaceafter emerging from their parent polyps, Yet despite the tremendous amount of information coilected during the last decade, no generalizatiOnsCOnCeming the various modes of sexual reproduction have been COnfirmedby observationand experimental study. Thus the adaptive significance of brooding-versus- broadcasting,hermaphrOditism-versus-gonoChorism, day-versue-night spawning, symbiotic- versus-asyrnbiotic possessing or lacking zooxanthellae! eggs and floating-versus-sinkingeggs in the life histories of stony corals refnain enigmas, Most broadcasting corais spawn at night when released gametes are not exposed to intense solar irradiation Harrison 8 WallaCe,1990!. Thus for most coral species, expOSureOf garnetBS to damaging ultraviolet radiation may not occur. However, since most of these species produce positively buoyant gametes whose early embryonic stages also float, these embryonic stages may be exposed to full solar radiation during the day following spawning. Consequently, positively buoyant eggs, or the floating embryonic stages, may be expected to possess the ultraviolet radiationblocking substances found in adult corals Shibata, 1969; Maragos, 1972; Jokiel 8 York, 1982; Dunlap et al., 1986; Dunlap & Chalker, 1986; Chalker et al., 1988!, especially those coral eggsand embryos possessing endosymbiotic algae, which are sensitiveto ultravioletlight Jokiei & York, 1982!. The Hawaiiancorals, Mont'fporaverrucose and Furigia scutaria, are broadcastspawners that exhibit different patterns of spawning behavior. Montipora verrucose, a coionial hermaphroditic COral,reieaaeS pOSitiVely buayant egg-sperm bundleS between 2030-2230 hours several nights following the new fnoons of June, July and August Heyward, 1986; Hunter, 1989!. These bundles break apart at the surface where fertilization takes place. The eggs possess endosymbiaticzooxantheilae. By the next morning flattened blastulae may be found floating at the surface of the water. Ffjngia scutafM, a solitary gonochoric coral, expels ciouds of slightly negatively buoyant eggs at about 1700-1900 hours several nights following the full moons of June through September or October Krupp, 1983!. Note that at this time of day, the sun is low enough on the horizon to be partially blocked by the Koolau Mountainsthat rise to the west above Kane'ohe Bay, 0'ahu. Thus theSeeggS are Spawnedat a time when they are nOtexpOSed tO full SOlarradiation. Fuffgia egga, which are much smaller than the eggs of Morttipora, lack zaoxanthellae, 129 Atleast some of the differences inspawning behavior among these corals floating-versus- sinkingeggs and symbetic-versus-asymblotic eggs may be related tothe ability ofthe eggs and developingembryos totolerate exposure tosolar radiation, especially thedamaging ultraviolet rays.As suggested above, eggs that float, yielding floating embryos thatare exposed tosolar radiation,must possess adaptations e.g., such as UV-blocking compounds! tominimize the damagesuffered from ultraviolet lightexposure, especially ifthese eggs possess UV-sensitive zooxsnthellae Jokiel ILYork, 1982!. Conversely, negatively buoyant eggs that lack zooxanthellaeandyield negatively buoyant embryos, might be expected to lackthese adaptations.Inthese respects, theeggs and early embryos ofM. verrucose should possess high concentrationsofUV-blocking compounds, while those of F,scutaifa should not. MATERIALS AND METHODS Colonies approx. 20- 40cm diameter! olMontipora mrrvcoea were collected from shallow water less than two meters depth! on the reef surrounding Moku 0 Loe Coconut island! where theHawai'I institute ofMarine Biology islocated. These colonies were either placed in large microixxtmtanks m x1 mx 0.5m, W xL xD! or individual aquaria receiving continuous seawater oneday before the new rnoons inJuly and August 1994. At about 1800 h eachnight, up to four nightsfollowing thenew moon, the water supply toeach tank was tumed off. During spawning, floatingegl~ierm packets were collected from separate colonies using a filtermade from Nitex. planktonnetling 20 izn! mounted toa twoinch segment ofPVC pipe seven inch diameter!. Packetscolkicted this way were transferred toa similarnet filter placed in a nineinch glass culture dishcontaining filtered .22 un!seawater. Those net filters containing egg-sperm packets were transferredthrough three changes of filteredseawater. Tobreak apart the packets, thenet filters inthe culture dishes were penodicaily gently agitated.After the packets broke apart, liberating sperin and individual eggs, the eggs were separatedfrom the surrounding sperm suspension bylifting the net filter from the culture dish. Thisprocedure leftthe sperm suspension behind inthe culture dish, while the eggs were retainedbythe net filter, The net filters containing theeggs were rinsed infiltered seawater three timesbefore preparing for~cesporine-like amino acids MAAs! extraction andanalysis. Indiviidualspecimens ofFungia acutana were placed into glass culture dishes nine inch 5ameter!lna largeshallow tank during the day of the full moon night in July 1994. These corals hadbeen mainta}ned formore than five months inshallow 0 cmdeep! outdoor aquaria receiving a continuousflow of ambientseawater under neutral density shadecioth about 8PYo transmittance!,Priorto spawning, thetank's water supply wae routed through a cartridge filter to removesuspended particulate matter. At about 1530 h, the water level in the tank was lowered to isolateindividual corals In their rejective culture dishes. The spawned eggs were siphoned from eachdish and separately filtered as described above for M. verrucose. Aliquotsofthe fresh egg suspensions werefiltered through glass fiber filters Whatman GFJC! andextracted inmetihmM Fisher HPLC grade!, These extractions were stored at -500 C until HPLCanahitsis, theprocedure forwhich is dta~bsidelsewhere inthis volume Gulko etaL, 1995!. Methanol-solubleproteins inthe extract were assayed byaliquoting samples into glass tubes andevaporating thesolvent before bringing thefinal volume toone mL with distilied water. These sampleswere assayed forprotein using the Lowry procedure with a BSAstandard Hartree, 1972!. 130 RESULTS Theeggs of Montiporaverrucose and Fungla scutaria exhibited marked differences in their respectiveHPLC chromatograms of mycosporine-likeamino acids Fig, 1!. The chrornatograms for M. verrucowrexhibited two prominentpeaks corresponding to shinorineand palythine standards.A thirdmajor peak did notco-elute with any of the standardsand remainsunknown. Severalminor peaks, possibly corresponding to mycosporine-glycine,porphyra-334 and palythinol,were aisoapparent on someof the chromatograms. The chrornatogramsfor F. scutarr'aexhibited a singiemajor peak correspondingto the mycosporine-glycinestandard, Several minor peaks notidentified! were apparent on a fewof the chromatograms. RgUre1. TypisrdHpl.c Ohrrararfagqgnaof mfrcaspsrirrs-ara arrire aCids~ from fhseggs af lurartporav&raema and RargfasoUaafa. The shinorinecontent of M. verrucesaeggs was estimatedat 530 nrnolmg ' protein methanolsolubleprotein!, while the palythine content was estimated at 837 nmoirng ' protein Table l!. The unknownUV-absorbing eiuate could not be quantified,but, judging by its peak heightand width,is probablycomparable to shirorine.The mycosporine-glycinecontent of F. scutariaeggs was variable,averaging 156 nmolrng ' protein. f31 TableI. ~ Myrxetxxtne4ke AminoAcids xt Corttt Eggs DISCUSSION Theeggs of Funya scutaifa and Mant/para trenvccrsa exhibited marked differences inthe lindaand quantmes oftheir rnycosporine-like amino acids MAAs!. The eggs of Mont/para verrucose,which ffoat and poetess zoaxanthellae, contained perhaps 10 - 20xthe concentration ofMAAs found in the eggs of Fvngiasctjfarta, which are negativelybuoyant and lack zooxanlhellae.These values do fallwithin the highside of the rangeof valuesfor MAAsobserved inthe adult tissues of othershafktw water corals Dunlap& Chalker,1986; Joklel et a/.~ MS; Kuffneret rffl.,1995!, halothuroids Shick ef a/., 1992!,and seaweeds Banaszak & I asser,1995!. Interestingly,the palythlne content af theeggs af Monfiporatrenucosa was about 7x the concentrationfound in adult corals living in shallow water Kuffneret a/ 1995!. Theseshallow wateradult corals also lacked substantial quanfNes of shinorineand an unknownMAA found in theeggs Kuffner efal., 1995!. It appearsaa though these MAAs are selectively concentrated intothe eggsof /lrfonit'paraverrvcasa Thelower MAA content of Fungiaeggs, when compared to those of Monfipora,may reflect theabsence of zaoxanthellae in Fvngia eggs. Becausethe only known origin of MAAsis the Shlkimatepathway, a complex metabolic pathway characteristic ofphotosynthetic microorganisms sndhigher plants Bentley, 1990!, the MAAs in the eggs are assumed tobe derived from zaoxanthellae,arpossibly fram foods ingested by the adult corals. This assumption requires that theeggs of Fungia acquire their MAAs during gametogenesis from the adult coral. In contrast, theeggs of Mbnft'pora may acquire some or all af their MAAs from their own zooxanthellae. Thehigh levels of MAAs found in the floatirtg eggs of Monfh'para may protect the developing embryosand planktonic larval stages from damaging UV radiatian.However, Fungia eggs, being negativelybuoyant, would have a ktwerneed for UV protection, The observed differences inegg MAAsof these two species may thus reflect adaptational differences relevant to their modes of reproduction. Highlevels of rnycosporine-like amino acids MAAs! have been reported tooccur in the eggs afthe scieracbnians Acropora frtfifetactra, Favia paNida and Grmiasfrea favu/tjs J~ & Babcock, unpublisheddata, cited by Harrison & Wallace, 1990!. It wouldbe interesting todiscover ifthe MAAcontent of thesecorals could be correlatedwith zaoxantheilae presence/absence ar floating/sinkirtgeggsin these species. Unfortunately, nodetails regarding MAAs in the eggs of these carals have been reported. . Wewould tiler lo thank Mich«et Ondruaek and ttaa Kuflner for c«nying out the HPLG ~. Dave WeGustoappreciaha~ in ttxrsettingopportixrity ~ the ~ curate, to~ gem«tee, conduct tttterace«rcpt and edltlnyto~gtry Drs. Paul Joltel of «ndthe text~ forLeaser, publication «sinwellthis asvolume, try the Hewst Irwtttute ofMaine Biotorry Dr.Gordon Grau, trecckrr!. This ~ was cc~ aspart oi the t994 Edwin W.pauley Summer progrten inM«rins BiOtcgy, 'Uttnevi6et Rattt«Ikon «ndCoral ReefS'. 132 LITERATURE CITED Babcock,R.C., Bull,G.D., Harrison,P.L., Heyward,A,J Oliver, J.K., Wallace,C.C. & VNllis,B.L. 986!. Synchronousspawning of 105 scleractiniancoral species on the Great BarrierReef. Mer. Biol. 90: 379- 394, Banaszak,A.T. & Lesser,M.P. 995!. Surveyof mycosporine-likeamino acids in macrophytesof KaneoheBay. In: "UltravioietRadiation and Cora Reefs", D. Gulkoand P,L. Jokiel eds!. HIMBTechnical Report ¹41. UNI HI-SEAGRANT CR-95-03. Universityof Hawaii. Bentley,R. 990!. The shikimatepathway - a metabolictree with many branches. Cntical Reviews in 8iochernistryand Molecular Biology 25: 305 - 384, Chalker,B.E., Bames,D.J., Dunlap,W.C. & Jokiel,P.L, 988!. Lightand reef-buildingcorals. InterdisciplinarySci. Rev. 13: 222 - 237. Dunlap,W.C. & Chalker,B.E. 966!. Identificationand quantitationof near-UVabsorbing compounds S-320! in a hermatypic scleractinian, Coral Reefs 5: 155- 159. Dunlap,W.C., Chalker,B.E. & Oliver,J.K. 986!. Bathymetricadaptations of reef-buildingcorals at Davles Reef, Great Bamer Reef, Australia.ill. UV-B absorbingcompounds. J. &p. Mar, Biol. Ecol. 104: 239 - 248. Fadlaliah, Y.H. 983!. Sexual reproduction,development and larval biology in sderactinian corals. A review Coral Reefs 2: 129 - 150. Gulko, D., Lesser, M,P, & Ondrusek, M.E. 995!. Introductionto materials and methods commonlyused by participantsin the 1994 H.I.M.Bsummer program on UV radiationand coral reefs.. In "UltravioletRadiation and Coral Reefs", D. Gulko and P.L. Jokiel eds!. HIMB TechnicalReport¹41, UNIHI-SEAGRANT CR-9543. UnlversityofHawaii. Harrison,P.L. & Wallace,C.C. 990!. Reproduction,dispersal and recruitmentof scleractinian corals. Irr."Ecosystems of the World25: CoralReefs", Z. Dubinsky ed.!,. Elsevier, Amsterdam, pp. 133- 207. Harrison, P.L., Babcock, R.C Bull, G.D., Oliver, J.K., Waliace, C,C, & Willis, B.L. 984!. Mass spawningin tropicalreef corals. Science223: 1186 - 1189. Hartree,E.F. 972!. Determinationof Protein: A modificationof the Lowrymethod that givesa linearphotometric response. Analytical Biochemistry 48: 422 - 427. Heyward,A.J. 986!. Sexualreproduction in fnrespecies of the coral Montfpora.In: Jokiel,P.L., Richmond, R.H Rogers, R,A. eds.!, Coral Reef Population Biology. HIMB Tech Rep 37, pp.170 - 178. Hunter,C.L. 989!, Environmentalcues controlling spawning in two Hawaiiancorals Montfpora venucosa and M. dilihta. Proc. SA Int. Coarl Reef Symp. 2: 727 - 732. Hyman,L.H. 940!. The Invertebrates,Vol 1: Protozoathrough Ctenophora. McGram-Hill, New York, 133 Jokiel,P.L. & York,R.H. 982!. Solarultraviolet photobiology ofthe reefcoral Pocillopora dsrrrlccvnfrrand symbioticzooxanthellae. Bvli. Mar. Sci. 32: 301 - 315. Jckiel,P,L Lesser, M.P. 8 Ondrusek,M.E. MS!, UV-absorbingcompounds in thecoral Ploc8fcporadsmiimmfs: effects of light,water flow and UV radiation, Krupp,DA 983!. Sexualreproduction and early development ofthe solitarycoral Funya scrjfarfrr ArNhczoa: Scieractinia!. Coral Reefs 2: 159 - 164. Kufiner,I.B., Ondrusek, M.E. & Lesser,M.P. 995!. Distributionof mycosporlne-like amino acids Inthe Casern ol Hawaiianscferactinia.. lrr, "Ultraviolet Radiation and Coral Reefs, D. Gulko andP.L Jokiel eds!, HIMB Technical Report ¹41. UNIHI-SEAGRANT CR-95-03. University cf Hem&. Maragos,J.E. 972!. A studyof the ecologyof Hawaiianreef corals. Ph.D. dissertation, Universityof Hawaii. Richmond,R.H. 8 Hunter,C.L. 990!. Reproductionand recruitment ofcorals: comparisons amongthe Canibean, the Tropicaf PacNc, and the RedSea. Mar.Ecol. Prog. Ser. 60: 185- 203. Shibata,K. 969!. Pigments and a UV-absorbingsubstance in corais and a blue-greenalga living inthe Great Barrier Reef, PlantCell Physrol.10: 325 - 335. Shick,J.M., Dunlap, W.C., Chaiker, B.C. ~ Banaszak,A.T. & Rosenzweig,T.K. 992!. Surveyof uftraviokrtradia5mabsarhng rmfcosporine-like amino acids in organsof coral reef holothuroids.Mar Ecol.Prog. Ser. 90: 139 - 148. Vaughan,T.W. ~ Wews,J.W. 943!. Revisionof the suborders, families, and genera of the SderactiniaGeol. Soc. Am. Spec. Pap., No 44. 134 U rsvlolatRadhtlon snd CoralRssA. 1ggs. D. Goikol P. L Jokiai ada,!,HIMB Tach. Report faf. UNIHI-SssGrant-CR-95-03 Effects of ultraviolet radiation on fertilization and production of planula larvae in the Hawaiian coral Fungia scutaris Dave Guiko Hawaf'fInstltuas of llarfne Biology P.a. B tsfs Kana'ohe,fff geyl4 ABSTRACT: SOtarirradiaticn, ~ly withinthe ulbaVictet UV! range,hae been irrylioatadin a VarietyOf effeota On Cerate, butfNe work has beendone on its effectson coralgamehe or the ~ pianula larvae. Gsrnelm and ptanulalarvae producedby theevening-spawning Mawaiisn coral Ftargiascufarr's were exposed to levelsof tttftcisl f~ comparableto daily sofarvalues, Viable~ pnxluction was comparedamong differenti~ beatrrvants Full solar UV-B,UV-A, fL PAR!, UV-A 4 PAR, PAR Only no UV!, and Dark no solar irradiation!!. Effectof UV expo' waa morepronounced on the F. scuhumsperm than it was on the eggsor planus. A strong~ of uv damagewas the signlrrcentdeCreaae Of viable ptanuteeresulting from fertilizadcn with uv-&e~ Sperm. Previousideas ~ the evolutionof nocturnal~ng in ooraismust be reevaluatedin lightof these results INTRODUCTION Ever since Jokiel 960! first broachedthe subject of ultraviolet radiationimpacting shallow tropical marineorganisms, researchers have concentratedon a variety of shallow reef corals see partial review in Falkowskiet al., 1990!. Often this work has focused on sessile adult colonies Dubinsky et al., 1984; Gleason, 1993; Jokiel 8 York, 1982; Vareschi 8 Fricke, 1986!, or the defenses that these benthic organisms utilize to amelioratethe effects of UV Chalker 8 Dunlap, 1986;Chalker et al., 1966; Dunlap 8 Chalker, 1986; Dunlap et al., 1986; Drollet ef al.~ 1993; Gleason, 1993!, The recent interest in coral bleachinghas also led to a number of studies on UV effects Gleason 8 Wellington, 1993; G!ynn, 1993; Goodman, 1991; Lesser et al., 1990!. Few studies have examined the effects of UV on coral larvae but see Jokiel, 1985a; and Gleason & Wellington,1995!. While studies on sessile corals are important,the adult coral colonies are exposed to less UV though Overa much longer and extended peried! and often have we!l~tablished protectivemechanisms mycosporine-likeamino acids MAAs!, pigments, shade adaptation,etc.!, than the garnetesor planulae in the water column above the reef, dependingon the time of gamete release. Gleason & Wellington 995! looked at the effect of UV OnAyariCia agarf'Citea pianulae and found a decreaSein SunrivalOf p!anulalarvae releasedfrom adult coionies at different depths. They related contrasts in planula survival to differences in the concentrationsof MAAs incorporatedinto the planulae releasedfrom differen depths. Agartcia broods its larvae prior to release and produces large planulaecontaining zooxanthellae. Studies done on Pocilloporadamicomis, another broodingspecies in Hawai'i that produces planulae containingzooxantheilae, have shown the ability of these larvae to remain planktonicfor over two fnonths Richmond, 1982, 1987!, Such a situationwould place the larvae under extensive exposure to high levels of UV irradiation. Presumably,zooxanthellae, whose pigments and postulatedproduction of MAAs through the Shikimate pathway Bentley, 1990! provide shielding frofn the effects of UV, are incorporatedinto the planulae prior to release fromthe adult co!ony, Similarly,lipids or pigrnentedbodies stored in the planulaecould serve to also help protect structures such as DNA from solar irradiance. The majorityof coral species so far observedappear to broadcastspawn their gametes as opposed to brooding planulae Kojis 8 Quinn, 1962; Richmond& Hunter, 1990; Harrison 8 Wallace, 1990!, Sperm and eggs releasedinto the water coiumn, and the externally-formed planula producedfrom them, may iack some or all! of the defenses against UV seen in brooded pianuia. Since eggs are more dense then sperm, often pigmented,and considering that many coral eggs have zOOxanthellaealready in them and therefOrepresumably B source of MAA compounds!,one would expect the detrimental effects of UV on eggs to be less-pronounced than for sperm which lack such possibledefenses. While not expecting large-scalemortality of sperm, surface UV might result in a decreaseof movementor ability to penetratethe egg. Likewise,UV might cause changes in the permeabilityof the egg. 135 Thepresent investigation evaluated the effectsof UV on Fungiascufaria coral sperm and the abilityof irradiatedsperm ta suixetuifuliyfertilize F. scuterr'aeggs, the effectof UV independently an Fungiaeggs, and the effectof UV on planulalarvae produced from non-irradiatedeg gs and sperm. MATERIALS 4 METHOOS SolitaryFungfa mdaiM werecollected from various patch reefs in Kane'aheBay in order to maximizegenetic diversity among individualsand minimize self-fertilizationor non-fertilizatlandue to relatedness.Corsis were carefullyand quicklytransported to the Hawai'i institute of Marine Biologywhere they were maintained separately in glassbowis set withinflow-through wet tables, Priorto a spawningevent, the wet tables were cleaned and the water changed to cartridge-filtered .22 pm ! Now-throughseawater. The waterlevel was lowered prior ta the onsetof a spawning event,melting in eachcoral's spawnedgametes being retained within that individual's glass bowl far identNcation and colkctfon. Gameteswere cafkicfedfrom individuallyspawning corals during the annual summer spawningperiods June through September,two to four nights after the full moon, between 1700and 1900hours!. Gameteswere immediatelycollected after spawning by gently siphoning the ~ watel IYlasswithin 8 spawned coral's glass bowl, Sperm from three s~iwned males were gently mixed together in a 500 rnl beaker in order to minimize individual genetic problems with sperm that might affect fertilization success. Five ml of this spermmixture was then gently poured into each of twelve, clean, fail~ered borosiiicate30 ml shellvials corNaining20 ml of 022 pm filtere seawater . Four treatments were prepared: ~ a UV transparent UVT! treatment that allowed PAR + UV-A+ UV-B radiation ta pass througha lid fittedwNh AciarfI 33c Fluaropoiymerfilm produced by Allied Signai Pottsville, PA!, 127 tun thickness gauge!!. ~ a UV-A tmiparent UVA! treatment that allowed PAR + UV-A radiatian to pass through a lidfitted with Mylar@Type D Ruoropolyrnerfilm produced by DuPont, 127 pm thiaknee mil!!. ~ aUV opaque UVO! treatmentthat blackedboth UV-A and UV-B, allowing only PAR radia5xl to pass througha lid Itted with 100 A Clear Acrylic Safety Glazing sheet produced by K-S-H, inc 2.5 mm thickness. ~a dark Dark!treatment that preventedirradiation of the sample, but that was within the same water bath as the other treatments. Data on the opticalproperties of the filters used in this experiment is contained in Guiko et al. this volume!. Eachof the twelveshell vials was randomlyassigned and Nted with a fliter lid resultingin thiee iepflcatesper treatment n = 3!. Each of the filteredshell vials were then placed randomly underneaththe center of a lightfield generated by a water~led, AlRMASS1 filteredKRATOS SolarSimulator Sofar Simulator! and exposedto solar spectral output similar to that of natural sunlightmeasured beneath the surface in Kana'oheBay. Figure 1 shows a comparisonof s~ lnadiancediiectfy adjacent ta HlMB in Kana'aheBay with that of the HIMB Solar Simulator af 'thesetting 5 arnps! used throughoutthis experiment, This setting resulted in spectral irradiancesimilar to thatseen in Kans'oheBay at a depthof 3.5 m. Undernatural conditions, spermeggs ar planulasfloating near the surfacewould be exposedto highermi44ay ieveisof UVth usedin this setof expedmerrts.Treatments were exposedto simulatedsunlight UVplus PAR! fram the Solar Simulator for 60 minutes. The dark treatments were maintained in the same waterbath M! butautside af andshielded from the irradiationfieid producedby the Solar Simulator. 'i 36 it .20 SolarS arm arm Surfma mr! L ioo 2m 23 ar 0.80 " oj 0.40 0.20 0.00 XS" OI4$PIm'itÃaglgl ggggc-g. Waar aaoer aar! Figure1. Compa risenof sfw etnairradiance sCan Of tfie H MBSOk rSknutaCH' rrrrltr ~ krett enoeeoarle taken at ~ depths1994,have+irrsneofafaly 12~45 ~pm to and f theLSD pn.Hawse inst ukof MarineBiofogy in Kans'oneBay. Takenunder dear skieson July28, Whilethe spermtreatments were beingirradiated, an unexposed10 ml sampleof diluted sperm from the 500 ml source beaker was subsampled to determine sperm densities using a hernocytometerand then scoredfor spermmotility, Also during this time, freshly spawned Ffjrrgia scutarfaeggs were gentlypipetted into clean 30 ml shellvials containing 20 mi of 0.22 pm filtered seawater. Each vial was numbered and the number of eggs contained within was recorded; within eachvial, eachegg representeda potentialplanula larva. Anyirregular or "blown-out"eggs were discarded prior to counting. Afterexposure, 1 mlof spermfrom each vial was gentlypipetted into a 30 ml shellviai containingthe Fungiaeggs between40 - 100 eggs! andfiltered seawater. Afterten minutesof gentleagitation to providefor fnaxirnurnfertilization, the egg-spermmixture was transferredto ciean250 mljars containing 150 ml of0.22 ltm filtered seawater at 2~. Thejars were then tightly cappedand placedwithin a shadedwater table supplied with flow-throughseawater overnight. This arrangementallowed gentle agitation of the fertilizedeggs at ambienttemperature. Except for exposuretreatment of spermto UV, all handlingand incubationof gameteswas conducted under low-light conditions. Sperm samples from each treatment were sampied for motility and compared with the motility of an untreatedsperm sample from the originalsperm mixture. Twelve hours after fertilization,each sample was censused for the number of planulae or pre- planulae developingeggs! present.The mean percentageof planulaeand pre-planulaewas thencompared among treatments. Results were analyzedby arcsinetransformation of percentage survival data, and single-factorANOVA tables were generated using a Microsoft Excel StatisticalAnalysis Toolpak. r ri Eggs and sperm from recently spawned Fungia scufarfa individualswere collected as described above. Eggswere pipettedinto each of nine,clean, foil~vered borosilicate30 ml shellvials containing20 ml of 0.22 l tmfiltered seawater; the shellvials were fitted with lids as described 137 abavefor the sperm experiment Uniike the sperm and planula experiments, a darktreatment was omittedforthis experiment dueto the need to minimize time between UVexposure ofeggs and fertilization,andthe need ta count eggs under a microscof e witha stronglight field. This resulted ina totalofnine shell vials, comprising threetreatments, whichwere arranged randomly underneaththecenter afa lightfield generated bythe Solar Simulator and exposed tosolar spectraloutput similar tothat of natural sunlight measured inKana'ohe Bayas described forthe spermexposure experiment. Treatments wereexposed tosimulated sunlight UV+ PAR!from the SolarWhileSimulator the sgg treatmentsfar 60 minutes.werebeing irradiated, freshly spawned Fungia scularI'a sperm were collectedand censused for density and motility. Afterexposure, 1mlof sperm was gently poured into each 30ml shell vial containing the exposedFungia eggs. After tenminutes ofgentle agitation taprovide formaximum fertilizatian, theeggs ineach egg-sperm mixture were counted andthen transferred taclean 250 ml jars containing150ml of 0,22 pm filtered seawater al2M. Thejars were then tightly capped and placedwithin a shaded watertable supplied withflow-through seawater overnight. Except far exposuretreatment ofeggs toUV, all handling andincubation ofgametes wasconducted under Iaw-lightcandNons. Aftertwelve hours, each sample was then censused andanalyzed as describedabove for the sperm exposure experiment. n Eggsand sperm were collected asdescribed abovefrom recently spawned Fungia scufaria individualson10 September 1995 and allowed tofertilize. The resulting fertilized eggs were allowedtadevelop into planula larvae wilhin 3-gallan aquaria containing 0.22 pm filtered seawater. After24 hours, planulae were counted andpipetted intoeach of twelve, ciean, foil-covered borasiiicate30ml shell vials containing 20ml of 0,22 pro filtered seawater; theshell vials were fittedwith filterdids asdescribed abave for the sperm experiment. The twelve shell vials of planufae n~ 3per treatment! werearranged randomly underneath thecenter ofa iightfield generatedbythe Sohr Simulator andexposed tosf:ectrst irradiance similar tothat af natural sunlightmeasured inKane'ahe Bayas described forthe sperm exposure experiment, Treatmentswereexposed tosimulated sunlight UVplus PAR! from the Solar Simulator for60 minutes,Afterexposure, theplanuiae were transferred toclean 250 ml jars containing 150rnl of filtered seawater at2M!. Thejars were then tightly capped and placed within a shaded water table suppliedwithfiaw-through seawater. Except forexposure treatment ofplanula larvae toUV, all handlingandincubation afplanuiae wasconducted under lowdight conditions. Aftertwelve hours,each sample was then censused and analyzed asdescribed above for the sperm exposure experiment. RESULTSIngeneral, sperm counts within any one set af experiments didnot vary notably between treatmentsasmeasured through counts using a hemocytometer pere.obs.!. SurvivalofF. ax4xria planulae twelve houis after fertiiizatian wasdramatically decreased for pianulalarvae pnx~ fromsperm irradiated underUVT canditions thanthat seen with the other threetreatments Fig.2!. Table I shows thisdNerence tobe highly significant F= 17.382, P < 0 01!-While sperm counts inthis experiment didrot noticeably differ,sperm rnotiiity didvary betweentreatments unreported data,manuscript inpreparation! andappears tobe strongly affectedby UVT. 138 p.8 p:r pp L' ps ~ p4 K p,i Figure2. Proportionof the pohrrrtirgnrenbar of Frftgta scrrtarr'aptanuta lanrae baeadon iniTialegg count! in each ~ at 12 hoursafter sperm ~. TableI. ANQVAtable Of reeutta rrf Fgsyls ecutarr'a sperm axpaeura traatinerrh. Source of Variation df SS MS F P-value Between Treatments 3 1.171 0.3905 17.382 0.0007 Within Treatments 8 0.1797 0.0225 Total 11 1.3512 Planulalarvae produced from eggs exposed to varying UV treatments did not appear to dNer significantly in their rate of survival twelve hours after fertilization Fig. 3; Tabie II!, Oifferences in percentagelarval survival betweenthis experimentand the sperm or planula larvae! experiment are not comparabledue to dNerences in spawning periods,times and experimental set-up. 139 0,1 05 00 0.1 Rgttreg.p00partlcn cfthe ~ number d satyr«ecaflfaptarltta t«nt«e baaed CnInN«i egg munt! in each ~ ai 12 hGLI««ftar egg ~ Tabteft, ANOirAtabte d a00uttaOf Rrtgfa aaAeh egg«tppaete irealmenta, Sourceof Variation df SS MS F P-value Between Treatments 2 0.0174 0.0087 0. 0996 0.9066 Within Treatments 6 0,5251 0.0875 Total 8 0,5425 ri Thenumber of planutaiarvae surviving twelve hours after exposure did not vary between UV andPAR treatments Fig. 4; Table tll!. A dNerencewas noted between UV treatments and the Darktreatiinent, although the Dark treatment did not signiTicantty differ from the UV-opaque PAR! ~nt. Thissuggests a UVeffect, but this result was not as pronounced as that seen for the sperm experiment. rr9 oe D.6 3 os 's oi tl os o2 0.1 Figusr4, pn~rrSon of the ~ number of Frstgiasafaaria ptsnda larvae baserfon Ini5alpbrnusse counri in eachtreatment at 12 hoursafler phnuhmexposure. TS5e III. ANOVAtable Of recuse Of Finger Satan@planta larvae~ treatments. Source of Variation df SS MS F P-value Between Treatments 3 0.2541 0.0847 4.522 0.039 Within Treatments 8 0.1499 0.0187 Total 11 0.4040 DISCUSSION The resultsof thisstudy clearly demonstrate the importanceof ultravioletradiation on the productionof viable larvae in broadcast spawning species whose sperm might be exposed to suchconditions. Additionally, Fungia planula larvae exposed in the currentexperiment also showedsome effects on survivaldue to UV, Thoughlacking zooxanthellae, Fungia planulae do containIlpids and MAAs,as the eggsthey were producedfrom contain measurable amounts of MAAs Krupp8 Blanck,this volume!. Presumably,these providesome protectionagainst UV exposure;though the concentrationin the eggs may be greatlydiluted in the developed,much largerplanula larvae without an influx or productionof new MAAs. If this were the case, it might accountfor the decreased survival of planula under UV conditionswhich a was not observed in the irradiatedegg experiment, ln the wild, Fvngia planulae resulting from negatively buoyant eggs would not be swimming high in the water column and exposed to such UV levels. Additionally,Fungia larvae are thought to acquire zooxanthellaeeither whiie in the plankton or shortly after settlement, and would then have both a source of pigments and MAAs to enhance their own protection. Given the presence of concentrated MAAs in the Fungia eggs, it not surprisingthatlarvae produced from UV~sed eggsin the present experiments didnot show signNcantdecreases inpercentage sutvival. A recent study by Krupp and Blanck this volume! comparedapecNc MAA content ofMontipora verrucose eggs with data on MAA content inthe adultcolony at shallow depth. They found the eggs to have seven times the amount of one type ofMAA concentrated within them, suggesting that MAAs are selectively concentrated inthe eggs toprotect these garnetes and the developing pianula larvae! while in the water column. Itshould benoted that Qcntjxm concentrates zooxantheliae intothe eggs prior to spawning and this couldaccoUnt forthe high presence ofMAAs within the eggs. Studies done in Okinawa H~ etal 1987! suggest that few other species implant zooxanthellae within their eggs prior tospawning; assuch, the presence of~ innon-zooxanthellae eggsrepresents yetanother energeticexpense on the part of theadult, Eventhough UV-B makes Up iess than 0.3% of the solar irradiance reaching the surface of theocean, there is enough ambient UV-B present to damage pooriy-shielded cellular DNA Kohenetal., 1995!. A numberofstudies have been done on the effects ofUV on the gametes ofaquatic txganisms, butthe majority ofthese have been associated with vertebrates such as frogsand fish Grunwald & Streisinger, 1992;Dey & Damkaer,1990; ijiri, 1980a, 1980b !, Mostof thestudies have shown a steriiizingeffect on the eggs given high doses immediately after fertillzagon.When sperm were irradiated atlow doses and then used to fertilize normal eggs, survivalwas very low, while highly irradiated sperm resulted ina higherproportion ofsurviving offsprin.This phenomena isreferred toas the 'Hertwig Effect' and is thought toresult from the sperms'chromatin being totally inactivated causing the larvae ta be produced bythe maternal haploidsetof chrornoeornes Ijiri,1980a, 1980b!. Aquaculture workers have used UV light to purposelyinactivate sperm toinduce gynogenesis inactive eggs in order to produce monosex generations see Gontczko etal., 1991 !. Unlikepianufae oreggs, sperm are relatively poorly protected from the effects of UV and react stronglytoUV exposure. Fungia scutena sperm contain iittie, if any,MAAs D.A. Krupp, pere. cornrn.!and shovred signNcant effects from exposure toUV-8 in this study, Given the comparabrktsizeand energetic investment differences between individual sperm and eggs, this is perhapsnot surprising. Theresults do not clearly indicate whether UV-B prevented sperm from successfully fertilizing eggs,or alkwved sperm tofertilize the eggs but not successfully develop into pianuia larvae due to mutationofthe DNA or possibly a combination ofthese two effects!. UV-B causes malformations inMonffpoia wtrrucosa planulae produced from UV-6 irradiated sperm under conditions similar to thosedescribed forthis experiment Gulko, unpublished observations!, suggesting mutation of theDNA ~ complete de~ation offertilization capability, TwospecNc types of sperm have been identNed inscleractinian corals Harrison, 1985a; Hamson& Wallace,1990!. An ovoid- or pear-shaped sperm is generally characteristic of hermephruditicspecies, while sperm with an elongated head containing a conical structure atthe apexis more characteristic ofymtchonc families including the Fungiids!. The generalized structuresofthese two types of sperm are as shownin Figure5. Otherpossible sites of action on the sperm include specific constituents ofthe motor apparatusi~ withthe flagella such as the mitochondria, which supply the energy for flagellarmovement!, receptor sites involved insperm orientation chemotaxia! towards the egg, or asyet unidenfified sitescomparable tothe enzIirme-containing cap acrosome! ' seen on the head ofother inverhkrate sperm and involved insperm penetration ofthe egg FIg.5!, Studieson greenfk@ellates afterexposure toUV-B have shown ktse of motility Kohen etal 1995!. Similar reeutiehave been reported with filamentous, ghding cyanobacteria Donkor & Hader,1991 !. Both ofthese studies suggest that UV has effects on sites other than the DNA. For coral sperm, this couldstrongly affect the ability of the sperm to reach or penetrate the egg. Coraisperm tscka vratldefined acntsrcme, butcontain smail ~ termed 'pro-rscnsarmal"veeiake!which may have a simirtartuncticn Harrison ih Waitace, 1990!. 142 ypothesisetfsites of ection: 1! Tbe DNA ttttdetrs 2! Tbe«ttterlor apex or pro-«croeomalcomplex 3! The loeomotory strttctttre pear-shaped!sperm Figrrre5. Dlegr«m dst«ting the two types of stwrrm dsersited forscfersctinisn corets After Haziest, t988b, es shown in Hariae4 W«Nece,1fxXt! efontf with hypothesized sites of ection ~ ~ toUVR ~. A,eesgtlLrm; B, cytoplasmiccoN«r, C,lamellas; D,rnitochondris; E,pro-tron:reornef vesidee; F,DNA-cont«thing nucleus; G,«pic«I iees densenucfero zone. Spermbehavior prior to fertilizationmay also be impacted.When coral sperm encounter an eggthey aggregate to form"sperm dots" adhered head-first to theegg surface Babcock & Heyward,1986!; the function of suchbehavior is stillunknown, but may play a rolein facilitating fertilization.Inactivation, or modificationof this behaviordue to UV exposure,could contribute to the decreasedfertilization observed as representedin the currentsperm experiments by decreasedsurvival of planula larvae!, Outsideof environmentalcues such as seasonaltemperature change, lunar and tidal cydes, andwith very few exceptions Fungta coricirtna, Pavona cactus Marshall& Stevenson,1933!, Furtgiascutarta Krupp, 1983!, Pogillopora verrucasa, P. eydouxf Kinzie, 1993!'!, hermatypic coralsspecifically appear to spawnprimarily at night.Among the various cues responsible for spawning,nocturnal illumination has been proposed to coordinatethe timing of release Jokiel et aI., 1985;Jokiel, 1985a; Jokiel, 1985b; Richmond & Hunter,1990!. Accordingto a numberof propaguledispersal hypotheses, many marine animals spawn duringthe late day or early evening to avoidegg predation by diurnal planktivofeus fish, Sucha strategyhas been proposed for both reef fish Robertson& Hoffman,1977; Johannes, 1978; Meyers,1989; Robertson, 1991! and corals Harrison et s!.,1984; Wallace ef al.,1986; Batcock et al., 1986;Harrison & Wallace,1990! with the ideathat by restrictingspawning to lateday/catty evening,diurnal egg predators would either be inactive or, if active,satiated from diurnal feeding. Whilecoral spawn has yet to beshown to bea majorprey item for planktivorous fish, Westneat and Resing 988! didfind that some fish switched their diet to coralspawn during mass coral spawning events. fonzfe~ these ~ spewiSng eeffyin the rnornlne inJQpg; rXrnM«reefy, Scht«d ty 4 ~ ~ ~ SrLrCOSSrefeeeed ils ~ si nighton restein theRed See. 143 Resultsof thisinvestigaton demonstrate that free-spawned gametes, being vulnerable to the effectsof UV, havea highersurvival/success rate if spawnedat night or late in the afternoon! whenUV levelswould be very low. Undersuch a UV-avoidancehypothesis, corals or any marine organismwhose gamt:-rtes contain little, if any,defensive mechanisms against UV radiation,and whosegametes raise up off the reef and into the surface layers'! would have evolved behavioral strategiessuch as night-time spawning in orderto maximizefertilization success. Night-time spawningmay have evolved, in patt, to allow gametes maxirnurn time for fertilization and developmentprior to UV exposure,and to minimizeexposure of the poorly shielded sperm from highUV in an environmentwhere diurnal broadcast spawning would expose sperm to such oxtt~ns. Eventhose species that do not spawnat nightcharacteristically spawn at timeswhen UV levelsare bw. The resultsof thiswork show a strongUV-B effecton coralsperm which affects theirability to successfully produce pianula larvae. Such results argue strongly for a UV-avoidance hypolhesisof night-time spawning in corals,Currently, very Iittkf evidence exists to supportthe alternativepredation hypothesis for night-timespawning in corais. Ongoingwork is presently looking at the effects of UV on theeggs and sperm of a hermaphroditiccoral which releases egg-sperm bundles in theevening hours on the new moon, Preliminaryresults suggest a similareffect on the sperm as seenwith Ftjngia. Other research will exploreboth the actual effects on the sperm itself, and endeavor to offsetFurfgia scufaria individual'sbiological clocks in an attemptto inducespawning during mid-day hours so that sperm can be exposedto naturalUV conditicxts, ACh'J4QtiyLEG6QiENTS: Nhohof thiswork «ea fundedthrough the Edwin W. PauteyFoundation and the ~i I~ ofMarina ateteffy, Untmnrlty OfHews, SfteoafappnretattOn goes to Dra.p. L JokialS D. A. Kruppfor all Of their aastatanoaand menkrrahtp, and Dr. M. Lesser for hia ~ce anduae oi his U~ underwaterspectroradiometer, Than' krJ, Stanokfor her ~iataee in aaonNrgSperm and ~ LITERATURE CITED Battoxk,R. C. & Heywafd,A. J, 986!. Larvaldevelopment of certain gamete-spawning sderac5nian oorals. Coral Reefs 5: 111 - 116. Babcock,R. C., Bull,G. D., Hamson,P. L., Heyward,A. J., Oliver,J. K., Wallace,C. C. & Willis,B. L. 986!, Synchronousspawnings of 105 scieractiniancoral species on the Great Barrier Reef. Ner. Hiol. 90: 379 - 394. Ben5ey,R. 990!. The Shlkimatepathway - a metabolictree withmany branches. Cri. Rev. Blocfl. IWec. Blot. 25: 305 - 384. Chalker,B. E. & Dunlap,W. C. 986!. Bathymetricadapta5ons of reef-buildingcorals at Davies Reef,Great Barrier Reef, ~ia. ffl. UV-B absorbingcompounds. J. Exp. Mar. Bio/.Eccl. 104: 239 - 248. Chalker,R. E.~ Barnes,D. J., Dunlap,W. C. & Jokiei,P. L 988!. Lightand reef-buildingcorals. lrfferdisc.Sett'. Rev. 13 !: 222 - 237 Dsmkaer,D. M.,Dey, D, B. & Heron,G. A. 981!. Dose/dose-rateresponses of shrimp larvae to UV-B radlaticrn,Oecologis 48: 178- 182. I:tsmkaer,D. M. & Dey,D. B. 983!. UV damageand photorear~ation potentials of larvalshrifnp, Pandalusplafyeeros, and adulteuphausids, Thysanoessa raschii. Osco/ogle 60: 169 - 175. Dey,D. B. & Damkaer,D. M. 990!, Effectsof spectral irradiance on the early development of chinooksalmon. Prig. F'sh-Culf.52 !: 141 - 154.. ' Presumablyfordspasg but~ aketo avoid asroentrations ofsgg pedal onIha reef 144 Donkor, V. & Hader, D, P, 991!, Effects of solar and ultravioletradiation on motility, photomovementand pigmentationin filamentous,gliding cyanobacteria, FEMS MicrobioL Ecol. 86: 159 - 168. Drollet,J. P Glaziou,P. & Martin,P, M. V, 993!, A studyof mucusfrom the solitarycoral Fungia itrngites Scleractinia: Fungiidae! in relationto photobiologicalUV adaptation.Mar. BioL115: 263 - 266. Dubinsky,Z., Falkowski,F. G., Porter,J. W. & Muscatine,L. 984!. Absorptionand utilizationof radiantenergy by light-andshade-adapted colonies of the herrnatypiccoral Stykyhora pistillate. Proc. R. Soc. Lond. B 222: 203- 214, Dunlap,W. C. & Chalker,B. E. 986!. Identificationand quantitationof near UV absorbing compounds S-320! in a hermatypic scleractinian. Coral Reach5: 155 - 159. Dunlap,W. C., Chalker,B. E. 8 Giver, J. K. 986!. Bathymetricadaptations of reef- building coralsat DaviesReef, GreatBamer Reef, Australia. ill. UV-Babsorbing compounds. J. Exp. Mar. Biol. Eco!. 104: 239 - 248. Falkowski,P, G., Jokiel,P. L. & Kinzie,R. A. ill. 990!. irradianceand corals.Irr. 'Ecogrstems of the World25: CoralReefs', Z. Dubinsky ed.!. Amsterdam,Elsevier, pp 89- 107. Gleason,D. F. 993!. Differentialeffects of ultravioletradiation on greenand brownmorphs of the Caribbean coral Pontes astreoides. Lirnnol. Oceanogr. 38: 1452 - 1463. Gleason,D. F. & Wellington,G. M. 995!. Variationin UVB sensitivityof planulaelarva of the coral Agancia agarrcitesalong a depth. Mar. Biol. 123: 693 - 703. Gleason,D. F. & Wellington,G. M. 993!, Ultravioletradiation and coral bleaching. Nature 365: 836 - 838. Glynn,P. W. 993!. Coralreef bleaching: ecological perspectives. Conrl Reefs 12: 1 - 17. Goodman,G. 991!. The effectsof UV radiationon skeletal growthand bleachingin fourspecies of Hawaiiancoral, Masters thesis, California State University,Long Beach. 80 pp. Goryczko, K., Dobosz, S., Makinen, T., & Tomasik, L. 991!. UV-irradiationof rainbow trout Sperm aS a praCticalmethod for induced gynOgenesia, J. AppL Ichthjrol.7: 136 - 146, Grunwald,D. J. & Streisinger,G. 992!. Inductionof mutationsin the zebrafishwith ultraviolet light. Genet. Res. 59 !: 93 - 101. Gulko,D., l.easer,M, P. & Ondrusek,M, 995!. Introductionto materialsand methodsused by participantsin the 1994 HIMB Summer Program on 'UV Radiationand Coral Reefs'. Irr. 'UltravioletRadiation and CoralReefs', D, Gulko& P. L, Jokiel eds.!. HIMBTech. Rep.¹ 41, UNlHI-Sea Grant-CR-9543, pp 19 - 24, Harrison,P. L. 985a!. Sexualcharecteristics of scleractiniancorsfs: systematics and evolutionaryimplications. Proc. 5th Int'I. Coral Reef Congr., Tahiti. 4: 337 - 342. Harrison,P. L. 985b!. The finestructure of Acroporaforrrxms Dana, 1846! Scleractinia: Anthozoa!,Thesis, James CookUniversity of North Queensland,129 pp. Harrison,P. L., Babcock,R. C., Bull,G, D Oliver,J. K., Wallace,C. C., & Willis,B. L 984!. Mass spawning in tropical reef corals. Science 223: 1186 - 1189, 145 Harrison, P. L & Wallace, C. C. 990!. Reproduction, dispersal and recruitment of scleractinian corais, irr, 'Ecosystemeof the World 25: Coral Reefs', Z, Dubinsky ed.!. Amsterdam, Elsevier, pp 133- 207. Hayward,A., Yamazato,K., Yeemin,T. & Minei,M. 987!. Sexualreproduction of corals in Ok'nawa. Gfsiaxea 6: 331 - 343, Hunter,J. R., Kaupp,S. E, & Taylor,J. H. 982!. Assessment of effects of UV radiation on mannafish larvae. In: The Roleof Solar UltravioletRadiation in Marine Ecosystems'~ J. Caulksns ed.!. Plenum Press, New York, pp 459 - 497, ljlri, K, 980a!. Effectsof UV on the developmentof ffsh and amphibianembryos. In: 'Radiaticn Effectson AquaticOrganisms', N. Egarni ed.!, BaINmore,Univ. Park Press, pp 223 - 236. ljiri, K, I. 980b!. Gamma-rayinadiation of the sperm of the fish OryziasIatipes and inductionof gynogsnesis,J. Radial. Res.21 p4!: 263- 270. Johannes, R. E. 978!. Reproductive strategies of coastal marine fishes in the tropics. Environ. Sol. FIshes 3: 65- 64. Jokkii, P. L 980!. Solar ultravioletradiation and coral reef epifauna. Science 207; 1069 - 1071. Jokiel, P, L 985a!. Lunar periodicity of planula refease in the reef coral Pociilopora darnicomis in relationto variousenvironinental factors. Proc. Flftfr Irrtemationaf Coral Reef Congress, Tahiti Vol. 4: 307 - 312. Jokiel, P. L. 985b!. The ph~ogy of the reef coral Pocflfoporadamicomis and symbiotic zooxanth@las. Dissertation, University of Hawaii. 221 pp, Joklel,P. L & YNk, R. H. 982!. Solar ultravioletphotobiology of the reef coral Pociflopora ahmIcomisand symbioticzooxanthellae, Buff,Nsr. Sci, 32 !: 301 - 315. Jokiel,P. L, Ito, R. Y, & IJu, P, M, 985!. Night irtadiance and synchronizationof lunar releaseof planuh ktrvae in the reef coral Rocificipora damicomis. Mar. Biol. 88: 167 - 174. Ignzie,R, A. III. 993!. Spawningin the reef corals Pocfflaporaverrvrx~ and P. eydouxi at Seseko Island, Okinawa. Gslafiea 11: 93 - 105. Kohen,E., Santus,R. & Hirschbeig,J. G. 995!. Photobiology. San Diego, Academic Press, 506 pp. Kajis,B. L. & Quinn,N. J. 982!. Reproductiveecology of two Faviid corals Coelenterate: Scleractinia!.Mar, Ecol. Prang.Ser. 8: 251 - 255. Krupp,D. A. 983!. Sexual reproductionand early developmentof the solitary coral Fungia scuteria Anthozoa:Scieractinia!. Coral Reefs2: 159 - 164. Kiupp,D. A, & Bianck,J. 995!. Preliininaryreport on the occurrenceof rnycospoiine-likeamino acidsin the eggsof the Hawaiianscieractinian corals Mon5poraverruca~ and Fungia scuferia, I+ 'UltravioletRadiation and Coral Reefs',D. Gulko & P. L. Jokiel eds,!, HIMB Tech. Rep,II 41~ UNIHI-SeaGrant&R-95-03, pp 131 - 136, Lesser,M. P SIachaj,W. R.,Tapley, D. W. 8 Schick,J. M. 990!. Bleachingin coralreef anthozoans;effects of irradiance,ultraviolet radiation, and temperature on the activities of piotecffveenzipnes against active oxygen. Cora!Reefs 8: 225 - 232. 146 Marshall,S, M, & Stephenson,T. A. 933!. The breedingof reefanimals. Part l. The corsls. Scient.Rep. Gt, BarrierReef &yed. 3: 219 - 245. Meyers,R. F, 989!. MicronesianReef Fishes.Bamgada, Guam: Coral Graphics. 298 pp, Richmond,R. H. 982!, Energeticconsiderations in the dispersalof Pociiiopmsdamicomis Linnaeus!planulae. Proc,4fii Int. CoralReef S!rrnp.,Moorea, Tahifi2: 153 - 156. Richmond,R. H, 987!. Energetics,competency, and long-distancedispersal of planulalarvae of the coral Pociiiaporadamicomia Mar. Biol. 93: 527 - 533. Richmond,R, H. 8 Hunter,C. L. 990!. Reproductionand recruitment of corats:comparison among the Carribean, the Tropical Pacific, and the Red Sea, Mar. Ecol.Prog, Ser, 60: 185- 203. Robertson,D, R, 991!. The Roleof AdultBiology in theTiming of Spawningof Tropical Reef Fishes,In: 'The Ecology of Fisheson Coral Reefs' P. Sale,ed.!, San Diego, CA: Academic Press, pp 356 - 386. Robertson,D. R. 8 Hoffrnan,S, G, 977!. Theroles of ternal mate choice and predation in the matingsystems of sometropical labroidfishes. Z. Tierpsychol.45: 298 -320. Schlesinger,Y. & Loya,Y. 985!. Coralcommunity reproductive patterns: Red Sea versusthe Great Barrier Reef. Science 228: 1333 - 1335. Vareschi,E. & Fricke,H, 986!. Lightresponses of a scieractiniancoral Piercgyra sinuose!. Mar. Biol 90: 395-402. Wallace,C. C.~ Babcock,R. C., Harrison, P, L Oliver,J. K 8 Willis,B. L. 1986!,Sex on the Reef:Mass Spawning of Corals, Oceanus29 !: 38 - 42. Westneat,M. W. & Resing,J. M. 988!, Predationon coral spawn by planktivorous fish. Coral Reefs 7: 89- 92, 147 U IrasfsfetRediatlcn Snd COral Raefa. 1figfi. D. Gulkol P. L. Jokkii sds,!,HIMB Tech. Report s41. UNIHI-SssGrsrit-CR-QRL9. Solar UV-A inhibitionof planulalarvae in the reef-building coral Pociilopora damicornis Andrew C. Baker DlviaiOnof MarineBiofegy and Flaheries, Rosenaliel School of Marineand Atm~ SCkSnt» universfiyofMiami, 4600 Fttckenbaoker Cswy, Miami, FL 3S149,U.S.A. ASSTRACT;Newly-relMrsed pisnulae from adult OOIOniss ofthe SdereCbntan caratFtmSCSicra rfierwrorris were sutjecerd far3 WeekatO one of 3 UVtreatmenla using sofar filter. Inthose teatrrSmls where UV-A and UV+ were~ out, tf» rate of larvalsetllement i~, andthe finrd number of hrvseseNed reproductive success! reached 37.3% M.7%!, compruedto20.8'll M.9%! with remove of LIVW aks», and 13.1'tk ~,1%! undertsrntiol condfiions wfih neutnd density filters.These results arffue for the ecological Skgnffioance ofUV, partlCularfy inthe UV-A region of the ~, asan impertantfaCtor determining larval reCruitrnent. AOdimstion Of parent COral cdoniea tO fiffferent UV trestn»nts for One monthprior to release of planuiae did not affect aeNement rates or final numb' ofiavae setfied. HPLC ~ ~ thatnewly~ planuscontain at leastfive different types of mlfcosporine-fike amino adds MAAs!, which are polar ~ds knowntoabsorb ~ inthe UV region. Acdimatfon ofparent cdonkrs to cNerent Liv treatn»nfs ~ ptanukrewffich variecl in theirMAA ~ This ~ wasdepeni~ onboth fi» ~ and spectnsoompcsifion of iheUv treatment,and on theabsortianc» cheractertafics ofthe ~ MAAconasmsd. ~, MAAcontent did not appeartodirecfiy affect the setlemsnt succem of the planukt krrvae. Therelicre, whflst this study dccumenls varick+ in MAASin mpcmmtO UV radisfion, it dOes not ~ the hypolhesisthat MAAS ~ly providea proesidlve bamer Sgalnatthe elfecaiOf high energy~ lNTRODUCTlON Solarultraviolet radiation in the 290 - 400 nm band has only relativelyrecently been recognizedas detrimentafto tropicalmarine shallow-water benthic organisms Jokiel, 1980; Siebeck,1981; Worrest, 1982; Siebeck, 1988; Glynn, 1992; Gleason & Wellington1993!, Until theearly 1980s, its importanceas a physicalstressor on coralreefs wss underestimated, largely dueto i!a generalbelief that only modest amounts of UVpenetrated to signNcant depth, and s! theobservation that UV-A 20 - 400nm! and UV-B 80- 320nrn! are together only a fraction -4 - 7'A! of the radiantflux emittedin the visibleregion of the spectrum. However,data demonstratingsignificant aquatic UV penetration has been available since at leastJerfov's 950, 1968!studies, and was more comprehensively expanded over 15 yearsago Smith& Celkins, 1976;Smith & Baker,1979, 1981!. Additionally,the effectsof evensmall amounts of high energyUV radiation on a varietyaf biochemicaland subcelluiar processes in marineorganisms is welldocumented Worrest, 1982; Hader & Worrest,1991!. It is somewhatsurprising, therefore, that studieshave only recentlydemonstrated the ecologicalsignificance of UV in the often cfear tropicalwaters associated with coral reefs Gleason& Wellington,1993!. Previousstudies of thebiological effects of UVhave generally emphasized UV-B as the major biocidalcomponent of UV radiation Worrest, 1982; Smith & Baker,1989; Hader& Worrest, 1991!. However,since UV-A is typicallyan order of magnitudehigher in termsof ils contribution to total radiantflux, its detrimentaleffects may have been underestimated. Hermatypiccoral tissue has been shown to possesscompounds S-320!, commonly called mycosporine-likeamino acids MAAs!,that absorbstrongly in the UV region Shibala,1969; Dunlap& Chalker,1986!. The varyingabundance of these compoundsunder different natural conditionsof UVirradiance has suggested an adaptive value for these compounds, although the evidencefor this so far is only correlative.However, reef-building corals in situ are alsoafforded protectionby their own skeletalcomponents and by shadingfrom neighboringreef material. Thereforeit wouldappear likely that detrimentaleffects of UV on coralsare more likely to be apparentwhen the coral undergoes a planktonicreproductive or dispersalphase in its Necycle, especiallyif the reproductivepropagules are positivelybuoyant or areswept up to shallower depths, thereby receiving higher doses of UV. Sofneauthors have suggested that the larvalstage may be the moreresistant phase in a coral'slife cyde Edmondson,1946; Coles, 1985; Esquivel, 1986!. Whilstthis maybe true for SOmeaspects of hrvalsurvival, particularly conSidering icng-term cornpetenCy of larvaeand lOw metabolicdemands whilst in theplankton, the same may not be true for high UV dosage and dose rate,where the potentiallygreater susceptibility of the larvae,in part dueto surfacearea to volume considerations, is apparent. Theplanula iafvae of the herrnatypicscieractinian coral Pocil/opora afamscomis are released monthiyin Australia,Hawai'i and Micronesia Mttrshall & Stephenson,1933; Harrigan,1972; Stimson,1978; Richmond8 Jokiel, 1984!. Since planulahrvae containzcoxanthellae and also 149 havea highenergy value, the potentialplanktonic lifetime of the larvaeis thoughtto be long {Richmond,1982, 1987!. This longperiod of larvalcompetency allows for considerablesubstrate selectionby the free-swimminglarva. tarvai settlement,defined for P, damicomisas the attachmentof the planula to the substratumfollowed immediately by spreadingof the thin basalplate, formationof calcareous septa,and the appearance oftentacular buds around the central mouth {Mamgan 1972!, ishighly dependent on both substrate type and local environmental conditions. These environmental conditionsmay be bioticor abiotfc,and includelight Harrigan, 1972; Lewis, 1974; Birkeland, 1977!, sediment Birkeland,1977; Rogerset a/., 1984; Rogers, 1990; Te, 1992!, wave action Bitkehndet el.,1982!, grazing Brock, 1978; Hamott 1985!, algal competition {Sammarco, 1982; Rogerset aL, 1984!and anthropogenicmarine pollutants Te, 1991!, Settlinglevee prefercryptic microhabitats, away from direct light Kawaguti1941!, whichhave been conditioned'in someway by an algalor bactenaffilm, or some chemicalstimulus MorsefI, Morse,1992!. Thechoice of cryptichabitats, however, may be influencedby a numberaf factors. Cryptichabitals with Iow ambient PAR levels may reduce competition and overgrowthby turfand macn&gae,henos increasing recruitment Birkeland, 't977!. However,higher irradiancein the PARregion of the spectrum should provide direct autotrophic benefits to earlylarval development Lewis,1974!. Fuithermore,the inaccessibilityof crypticsites to grazingby largervertebrates and invertebrates,which is only secondarilycorrelated with lower fightlevels, may also lead to an increasein initialsuccess Birksland K Randall,1982!. Giventhat high sohr radiantfiux has been shown to have an inhibitoryeffect on initial settlement Kawaguti,1941!, this study ains to separate the different regions of the solar spectrumto elucidatea broad-bandaction spectrum for the reductionin settlementunder high naturedradiation conditions. ff is hypothesizedthat high irradiancein the UV region ol the spectrumis a significantdeterminant ol larvalsubstrate selection. When energy resourcesare iirritsd,or moralityki the planktonicphases is high, variations in timespent selecting a suitable stte translatesdirectly into differentialreproductive suites. Thepotential effects of solar UV on the settlementprocess, and indeedmost other aspects ofcoral reproductive biology, have been largely ignored but see Jokiel, 1985!. Thisstudy aims to demonstratethe importance ofsuch effects, and suggests significant gaps in ourunderstanding of environmentalfactors affecting larval recruitment in scieractinlancorals. MATERIALS AND METHODS T n Adultcolonies of Poeilloporadarnicomis, 10-15cm in diameter,were collected at depthsof 1- 2 m frompatch reef no, 40, Kane'oheBay, 0'ahu, Hawai'ion 23 June 1994. Colonieswere observedfor phrtuiarelease using planulaecollectors slmlar to those describedin Richmond 985!. A largenumber of geneticaily distinct types of P. damicomisoccur in Kane'oheBay, each planulatingon differentdays of the lunarcycle Stoddart,1984; Jokiel,1985!. Fromthose collectedcolonies which planuiated in quantity over a sixday periodfollowing the fullmoon of 23 June,12 wereeHected and randomly allocated to one of three treatments coloniesper treatment!: 150 Table I: Diffarantrnatsrtals uaad tOblOck variOua wenrfantrtha Of SOlar ~. UVO blocks Uv ~ and aliOwSOnly PAA I~ nm! through. UVA NocksUV-8 ~ istd allows wesrtsngthsgrastsr the 350 nm through, UVT allows bothUV sndPAR thrcugh 90 nmand grestsr!, Sss also Figure 1 andG ko et af, thiSvalume!. TREATMENT FILTER TYPE RADIATION PASSED LEVEL SEE ALSO FIGURE f! UVO Plexiglas.G-UF-3 Acrylic sheet PAR only .0 mm! Pot!rcast! UVA Mylar@Type D Fluoropolymerfilm PAR + UV-A 27mm! DuPorrt! + PlexigiaseG-UVT Acrylic sheet .0 mm! Rohm 8 Haas! UVT Piexlglas@G-UVT Acrylic sheet PAR+ UV-A+ UV-B .0 mm! Rchm 8 Haas! Each treatmentwss carriedout in one of three 410 litertanks suppliedwith fiow-through seawaterat 8 liters per minute. Cora!awere left in these treatmentsthroughout a single lunar cycle,from the endof planulationon 29 Juneunbl the full moonof 22 July. Over the lunar cycle,treatments and tankswere rotatedtwice, at 9-day intervals,to prevent potentialconfounding tank effects. Each treatmentthus spent equal time in each tank. Planulaereleased from the coralsover the period 21-25 July 1994 were collected. Adult colonieswere removedfrom the acclimationtanks, planulaewere collectedovernight, and the colonieswere returnedto the treatmenttanks the followingmorning, On 23 July, sufficient planulaewere releasedover a single night to enable the use af planulaereleased from adult colonies at the same time. Planulae from the 4 colonies subjectedto each treatment were pooled to produce3 groupsof planulaeproduced from coloniesacdimated to dlfferentUV regimes see Figure2!. PlanulaereleaSed frOm the COralSOn Other nightS were analyzedfOr MAAS by extraction of 25 - 50 planulaein methanoland analysis by HPLC see Gulkoet al., thisvolume!. I n r T n The planulae obtained from the colonies acclimated under the three different UV treatments were then aI!ocatedto sett!ernent containers and re-subjected to different combinations of the same three UV treatments see Figure 2!. The experimentaldesign thus consisted of two differentUV treatmentperiods: an 'acdimatorytreatment' of the parentcolonies one lunar month prior to planulation!; followed by a 'settlement treatment' of the planulae two weeks post- pianulation!.Settlement containers were constructedof roundglass dishes, 9 crnin diameterand 2 cm tall, surroundedby reinforced183 mm Nitex planktonnetting to an ovenNheight of 8 cm. Priorto the experimentalperiod, each container was 'conditioned'in seawaterfor two weeks, after whichloose Narnentous a!gae was removed,leaving behind a finealgal film. Five containers, each containing 25 randomly-selected pianulae, were utilized for each combination of settlement treatment and acdimadon treatment, for a total of 45 containers containing1125 planulae. Containerswere submergedin a shallowwater table to a depth of 6 cm, suchthat free exchangeof waterwas allowedthrough the planktonnetting. The 218 litershallow water table was suppliedwith flow-through seawater at a rate of 8.5 liters per minute, with care taken to avoid strong unidirectionalcurrents that might push planulae against one side of the netting. WAVELENGTH nm! TRANSMITTANCE Rtitlt 1, Trenemlltenceafeeareertstiae af eater tNtsrs as a functionof nrstterSwemlentrth. Rlter propertiesrid natCtenye aver ae plkitt d thisStNty S weeks!, Theshalktw water table was covered at differentpoints with three filtersproviding UVO, UVA andUVT tteatmtitnts as before.Tank effects were thvs avoidedby subdivisionof a singletank into three treatments, The numberof phnuktssettled in each container,as well as the total numberof planulas remaining,were counted over a periodof 14 days. Settledplanulae were thoseobserved to have anchoredInnly to thesubstrata and spread out a basalplate. RESULTS Meankeel settlementafter 14 days, given ss a percentage of the initial25 planulas,was greatestunder the UVO treatmtatt,at 37.3'k M.7%!, comparedto 20.8% M.9%! and 13,1% M. 1%!in the UVAand UVT treatmentsrespectively. Maximum settlement after 14 days forany indiNidualcontainer was 8N6 observedin the UVA treatment!whilst fninimum settlement for any containerwas 0% observedin the UVT treatment! See Fig. 3!. 152 AccLIMATIoN TREATMENTs OF PARENT COLONIES UVO UVA PARENT COLONIES PARENT COLONIES PARENT COLONIES PLANULAE PLANULAE PLANULAE V'IN lIN SETTLEMENT DISHES SETTLEMENT DISHES SETTLEMENT DISHES 0 0 0s 0 0 0 8 0Qs 0 0 0 8 80 8 0 0 0 0 0 08 8 0ss0 I 0 8 . . 0 00 UVO UVA UVT UVO UVA UVT UVO UVA UVT SETTLEMENT TREATMENTS OF PLANI JLAK Figuns2. Experimentalprotorxx. parent ~ ansacckretxrrt todttterent Nr treslmentsforone Itxtsr month prior to xnulahOn.Settling Ifg ~ ~ trem the parentcotoniee are thenexposed to dttfersntCcmbinstiOne Ol the samethree V treatmentsfor t4 days. Mean settlement of larvaeoriginating from parent colonies acclimated to conditions of UVT wss26.7%%u M.0A! after 14 days, whiLstfor UVA and UVO it was 28,4%%u %4,0%%u! and 18.1%%u M.7%! respectively See Fig. 4!. Planulamortality, estimated as the loss of planulaefrom the containers,did not differ between treatments,and did not exceed 25%%uoafter 14 days. Whenvariantxs in settlementdue to UVsettlement treatment is removed,accttrnatory UV treatmenthas no effecton planuiasettlement rate, However,when variance due to acctimatoty UVtreatment is removed,it canbe seenthat settlement UV treatment does have a significant effect Two-wayANOVA at eachtime interval, see TableIt!. Theeffect of UV settlementtreatment appears after only two days, There is never any interaction between the two treatments, Settlementrate in the UVOis signNcanttyhigher than ratesunder both UVA and UVT, but thereis no significantdifference between UVA and UVT StudentNewman-Keuls Test, a =0.05!, this is 153 12 10 0 2 10 12 14 TIME Days! Rgures. Seaementra@ ar pienuhlarvae of Roal~ dlrnhmfs Nea fueke of uv ~ during ~rrt iuv accknaiory~e of adultcobnhe gmupadi. alsotrue from the secondday onwards. These results argue for the significanceof UV-Aradiation as an inhibitory factor in the settlement process, since when UV-B is removed there is no significantincrease in settlement similar curves for UVA and UVT!,but when UV-Ais removed, settlement increases markedly signiTicantdifference between UVO and both UVA and UVT curves!. Anobservable decline in pigmentationof the pianulaeoccurred over the 14 dayperiod of study. This decline in pigmentationwas observed in aI three treatments, and affected both free- swimming and settled planulae. This reduction in coloration made assessments of reverse metamorphosis Richmond,1985! probiematic;quantification of this phenomenonis not included here. HPLC analysis revealed newly-releasedphnulae to contain at hast five different mya~rine-like aminoacids MAAs!:rmircosporine-glycine ~ = 310 nm!, asterina ~ = 330nm!, porphyra-334 ~ = 334nm!, shinorine ~ = 334 nm!and one unknown,thought to be mycosporine-glycine:valine ~ = 335nm!. Additionallysome samples contained smal amountsof palythinai ~ = 332 nm!and palythene ~ = 360 rrn! Karentzet al 1991!. Meanconcentrations of the fiveMAAs consistently present, as a functionof the UVacclimatory treatmentof the parentcolony, are given in Tableill. 6 8 10 12 TIME Days! Rgure4.Setttemsnt rateof planta larvae offrrrcflfcprrra darrsbmh ssa ~ ofUV aodimatory traatrment ofparent colony UVstrtesment ~ts of ptanulaegrouped!. TattleII. Resultsofa ANOVA a =D.Of! at sech time ~, showingaignlttcance ofUV Sautsment X!end AcclimationT~ . Insome cases approprtrue bansfwmsfions ofthe data were made io conform tothe condHiore of Ihs anal' as shown . TIME Days! 0 1.25 2.208 3.208 4.208 5.167 6,75 9.458 13.875 DATA NQNE NONE Squttro Square Square NONE TRANSFORMATION Root Root Root Root Root SEITLEMENT NO YES YES YES YES YES YES YES TREATMENT X! ACCLIMATORY NO NQ NO NO NO NO NO NO TREAT!REMI' Y! INTERACTION NQ NO NO NO NO No NQ NQ Xs Y! 155 Tebh W. MeanMAA concentraem {In nrrVmgprotein! as a funcsonof UVacdimatory treatment of psrsntcdony. Mean vaiussare cahulatsd from n raplfcshsof 25.50pietuhe, Thoeevahss markedwith m asterisk 'i sre notsignificantly dkferent~ treatments ~y ANOVA,a=4.05!. Mean prolain conhnt { 14.3 mg per ! is caicuiatedfrom Rlchnvml t 952!, Seealso Figure 5, nobnglogrsffhmh cosh, UV ACCI IMATORY TREATMENT UVT UVA UVO re! n=7! n=5! MEAN MYCOSPORINE- CONCENTRATION 1.097 0.665 0.511 ' GLYCINE nm/mg protein! +0.188! 0,057! 0.032! XSEM! MEAN SHiNORINE CONCENTRATION 0.877* 0,724' 0.708' nm/mg protein! 0.094! R0,114! ~o.080! MEM! MEAN PORPHYRA-334 CONCENTRATION 0.484 0.395' 0.353' nm/mg protein! M.041! R0.027! X0.033! SSEMI! MEAN MYCOSPORINE- CONCENTRATION 15.926' 24.694 16.282' GLYCINE:VALINE nm/mg protein! X0,543! X1.214! +1.168! iSEM! MEAN ASTERINA CONCENTRATION 4.830 7.594 4.633' nm/mg protein! %0.186! 0.429! %0.525! j.SEM! 54 L.OG 3 Froure5. Gmenlrslonof MAAsin phnukhas s funC5onOf UV axlimefdry eafaneea of parentcofonkrs. Hole kgarithmiC scale. 156 Newiyreleased planulae left for a periodof 24 daysunder a varietyof UV treatments containedmean concentrations that,in four out of the five MAAs concerned, were significantly lessthan those analyzed immediately post-release seeFigure 6, notinglogarithmic scale!. These changesare summarized inTable IV. 5 4 log 3 CONCENTRATION [nrntug protein] +1! DAYS Dottedcolumns s! indicate DAYS no signifeantdifference in mean a.+ tz: O> tu i Rtu MAA concentration a.gj t- 0 oz ~ g cs z L ca~~ op+ p O~ 4 Vg ts J g ~< o 5y Figure6. Contxttttrabon otMAAs as a futottionoftime attar tatsasa, Nota logarithmic scale. DISCUSSION n'''n f Thisstudy demonstrates thatsettlement ofthe planula hnrae of Pocilloporactarntcamis is inhibitedbyhigh-energy solar UV radiation, particularly inthe UV-A region ofthe spectrum. Whilst someinfluence byUV-8 is alsosuggested, although not at a significantlevel see Ftg. 3!, this effectis heavily outweighed bythat of theUV-A. This is in contrast to many studies of Ianral ecologyand reproductive success that have revealed UV-B as the major detrimental orbiocidal componentinthe solar spectrum Chalker-Scott etal., 1992; Berghahn etsl., 1993!. Previousstudies of the effect of UVon larval ecology have not always investigated the fuil rangeofthe UV spectrum, andhypotheses regarding potential action spectra forUV detrimental effectsoften appear to have been assumed a priori. However, itis apparent that the effects ofUV- A havebeen underestimated andunderinvestigated. Given that the total radiant flux in theUV-A istypically atleast an order of magnitude higher than that of UV-B, it is to be expected that detrimentaieffects could be greater for this region see Peterson et al., this volume!. Whilst for manyprocesses theenergy per quantum iscktatfy the parameterofinterest, emphasizing the higherenergy UV-B region ofthe spectrum, totalradiant flux in the lower energy region ofthe UV spectrumcannot be ignored. This is doubly true when itis observedthat the distinction between UV-Aand UV-B is arbitrary and bears no direct biological orphysical relevance; they represent the division of a continuum into discrete categories. Additionally,thisstudy reveals 'sublethal' effects af UVradiation that have direct consequencesonrecruitment reproductive success! ofthe parent organism. Differences in settlementunder UV treatment not only manifest themselves asdifferences inthe final number of planulaesettled, but also as differences ininitial rates of settlement. Variation inreproductive successislikely, therefore, tobe greater than that estimated solely from the final number of planuiaesettled, since variation inthe time spent in the plankton willalso affect overall reproductivesuccess, This is especiallytrue when planktonic predation is intense, or when strongcurrents sweep planulae off the reef and into the open ocean, where chances of encounteringsuitable substrate are drarnaticaliy reduced. 157 TStttSIV. COmpanttivsmssn aveentnttians Ot MAAS immeSStoly tMst-rstssss; snd after 2% daysunder a varietyof UV trSStinontS.Mssn vaiuss are aHcvlstodflem n ~ ot 2Sea ptsnutSS,ThOSS vatuSS ~ with an aSteriSk '! are nst stSnttiosntty~ IMsnn-Whitnsy U-tsst,s 0,05!. TIME OF ANALYSIS NEWLY-RELEASED POST-REI EASE time&! timeM< days! n='f 7! rt=8! MEAN MYCOSPORINE- CONCENTRATION 0.747 M.081! GLYCINE nm/mg protein! fSEM! MEAN SHINORINE CONCENTRATION 0.764 +0,058! 0.932' %0.150! {nm/mg protein! ZSEM! MEAN PORPHYRA-334 CONCENTRATION 0.409 &.022! nrn/mg protein! XSEM! MEAN MYCOSPORINE- CONCENTRATION 19.641 1,212! 0.906 X0.290! GLYCINE:VAUNE nm/mg protein! j:SEM! MEAN ASTERINA CONCENTRATION 5.91 0 m419! 0.308 M.157! nrn/mg protein! jSEM! Thisstudy fails to revealany differencein UV toleranceof planulaeproduced by parentcolonies acclimatedfor one monthto different UV regimes Fig. 4 and Table II!. The possibilityexists that one reproductivecycle is Insufficiento producea detectable differencein tolerance,and that kxtgeraxfimatoty periods are requiredto demonstratean effect. However, from a theoretical pointof New lt wouldappear Unlikely that such an effectcould exist, since the phnula larvaeof P. darntiCOrnisare pOSitivelybuOyant On releaSe,and would therefOrebe expeCtedtO mix in the uppersurface waters, regardless of the depthof their initialrelease. A more detailedknowledge of planktoniclarval dynamicsand temporal changes in larval buoyancy associated with lipiddeCline, are requiredtO adequately resolve these iSsueS butSee Riohrnod, 1987!. Thisstudy invesbgates sahr ~ flux, withoutartie& increaseof the sohr spectrum. In contrastto muchof the literaturereganNng the biologicaleffects of UV, rnaxirnumdose rates used in thisstudy typically underestimate maximum rates likely to be encounteredin the field, since the ftitersmatanlsn transmittance never e~ QPYo aee Figure 1!. However,mean dose over a 1~y perlcdirt any regicnOf the SpeCtrummay be mOrethan the mean dOSereceived in the field. since in naturalscenantm planulse rntty be transportedto greater depths with tower UV dosage. Nevertheless,both dose and dose rais he withinthe region of values that possess potentialecological relevance. A decreasein the earth'sprotective ozone layer, and a concomitantincrease in sohr UV penetrationwl increaseboth the amountand the spectralquality of UV reachingthe earth's surface Calkins,1982; Wortestand Caldwell~ 1968!. Significantincreases in UV-A under this l58 scenarioare unlikely, Hence if reproductivesuccess of planulatingcorais is heavilyaffected by incident UV-A, but only marginallyaffected by UV-B, further reductionsin recruitmentdue to ozone depletion are unlikely. P. darnicomisis foundonly on reefflats at shallowdepths of 0 - 3 m in Kane'ohe Bay, where the specimensused in thisstudy were collected. Dominantcorals co-occurring at these depths are Poritescornpressa, and branchingMontipora spp. From its observed depth distributionit might be hypothesizedthat developingcolonies of P. damicomiscompete wall under high radiationconditions, but avoid the initialinhibitory effects of UV-Aby settlingin the manycryptic microhabitatspresent on the reef flat, Distributionat greaterdepths may be limitedby the marked reductionin PAR in the relativelyturbid waters of Kane'oheBay. The possibilityexists that UV radiationis not havingan effect on the planulaeper se, but ratheron the substratefor which they select. For example,the absenceof UV mightaffect a parbcularalgal turf, causing it to producea certainchemical stimulus that encourages settlement Morse8 Morse, 1991!. Such a hypothesis,if true,would indicate only an indirecteffect of UV on planulae. However,it neverthelesssupports the observationthat in ecologicalscenarios UV radiationis an importantfactor determining settlement site selection. in cases where either cryptic sitesare few, or settlingtime limiteddue to strongcurrents off the reef, settlementsite selection would still play a major role in recruitmentsuccess. Thisstudy reveals significant differences in the concentrationsof some MAAsin planula larvaeas a functionof the UV treatment to which the parent coral colonies were exposed. Parent colonies exposed to UV radiationproduced planulaecontaining more MAAs than colonies screenedfrom UV, However,the finerscale spectral differences produced more counterintuitive results. When parentcolonies were exposedto UVA conditions,i.e, a combinationof PAR and UV-A, they producedplanulae with higherconcentrations of both mycosporine-glycine;valineand asterinathan colonies exposed to UVTor UVOtreatments. However, when parent colonies were exposedto UVT,i.e, PAR, UV-Aand UV-B,they producedmore mycosporine-glycine than colonies exposed to UVA or UVO treatments. This differential variation in mean MAA concentrationcan be interpretedas a functionof boththe intensityand spectralcomposition of theUV treatment,as wellas the particular absorbance characteristics of the MAAconcerned see Fig. 7!. Mycosporine-glycinehas a peak absorbance~ at 310 nm, in the UV-8; whilst mycosporine-glycine:valineand aslerina have absorbancepeaks at 335 nm and 330 nm respectively,which are both in the UV-A see Rg, 7!. Hence it would appear that colonies exposedto conditionsof fuffUV UV-Aand UV-B! preferentially invest in MAAsblocking in the more damaging!UV-B, whilstcolonies exposed only to UV-A placeal their investmentin UV-A blockers. However, this is not predicted by the settlementobservations presented earlier, which showedUV-A to be the regionof the UV spectrumcausing most of the settlementinhibition. If UV-A were trulythe ecologicallymost damagingcomponent af solarradiation, then parent coloniesexposed to UV-A and UV-B, ratherthan UV-A atone,would not be expected to withdraw investmentin UV-A blockersfor placementin UV-B biockers. Additionally,despite significant variation .5 to 2-fold!in mean concentrationof some MAAs as a functionof UV treatmentlevel, no significantdifferences in settlementwere found as a functionof the sametreatment ievels Figure4 and Table II!, These resultsdo not indicatea protectivefunction of MAAs,at hast at the ecologcalresolution of this study. Care must thereforebe taken to distinguishvariation in MAA contentin responseto UV radiation,from the assumptionthat MAAsmust have a protectivefunction and, therefore,have adaptivevalue. Thesharp reduction in MAAconcentration in planulae analyzed 2-4 daysafter release raises importantque.~cons. If MAAs are indeed providing a protective function, it is unlikelythat such a markedreduction in UVblockers could occur in ecological scenarios, since planulae are knownto becompetent over time scales far exceedingthese Richmond,1987! . One alternatepossibility lsthat the reduction inMAP+ detected during this study is dueto the observedbleaching of planulaementioned earlier, since if MAAsare synthesized by the zooxanthellaeand not the coral itself,then a reductioninzooxantheila abundance or competence! associated with bleaching mightcause a concomitantdecrease in MAAa.On the otherhand, if MAAsare not directly 159 Birkeland,C,,Rowley, D.8 Randsll,R.H. 982!. Coral recruitment patterns atGuam Proc 4th Int.Coral Reef Congr. 2: 339 -344. Birkeland,C.& Randall,R.H. 982!. Facilitationofcoral recruitment byechinoid excavations, Proc.4th Int. Coral Reef Congr. 1: 695 - 898, Brcck,R.E. 979!. An experimental studyon the effects ofgrazing byparrotflsh androis of refugesinbenthic community structure, Mar. Biol. 51: 381 - 388. Cslkins,J. ed.! 962!. The role of solar ultraviolet radiation inmarine ecosystems. plenum Press, New York. Chalker-Scott,L.,Scott, J.D., Dunning, C.& Smith,K. 992!. Effect of ultraviolet-8 radiation 80- 320nm! on survivorship ofzebra mussel larvae &afissena polymorphs!: a potentiai controlstrategy. J. ShetliishRes, 11 !: 221. Colas,S.L. 985!, The effects oielevated temperature onreef coral planula settlement as relatedtopower station entrainment. Proc.5th trit. Coral Reef Corrgr. 4:171 - 176, Duniap,W.C. & Chalker,B.E. 968!. identification andquantification ofnear-UV absorbing compounds S-320! ina hermatypicscieractlnian. CoralReefis5: 155- 159. Edrnondson,C.H. 946!. Behaviourofcoral planulae under altered saline and thermal conditions.Occas. Pap. Bernice Pauahi Bishop Mus. 18: 283. Esquivel,I.F. 986!. Short term bioassay onthe planula ofthe reef coral Poclltopora rfamicornis. lrr.Coral Reef Population Bidogy', P,L. Jokiel, R.H. Richmond & R.A. Rogers eds.!. Hawaii Inst.Mar, Biol, Tech. Rep, 37; 465 - 472.Sea Grant Cooperative ReportUNIHhSea GrantCR- 86-01. Gleason,D.F.& Wellington, GM 993!. Ultraviolet radiaticn sndcaal bleaching, IVatuie 365: 838 - 838, Gulko,D.,Lesser, M.P. & Ondrusek,M.E. 995!. Introducbon tomater@is andmethods commonlyusedbyparticipants inthe 1994 H,I,M.B summer program onUV radiation andcoral reefs.Irr. 'Ultrlviolet Radiation andCoral Reefs', D.Guiko snd P.L Jokiel eds!. HlMB TechnicalReport ¹41. UNIHl-SEAGRANT CR-95-03. pp19- 24. Hftder,D-P& Womsst,R.C. 991!. Effects ofenhanced solarultravidet radiation onaquatic ecosystems.Photochem. Photobiof. 63:717 - 725. Harrigan,J.F. 972!. The planula larvaeof Pocfltopora darnlicomis.Lunar periodicity ofswarmirig andsubstratum selection behavior. Ph.D. Dissertation, University ofHawaii. 213pp. Harriott,V.J. 985!. Recruitment patternsof sderactinian corslsatlJzard island, Great Barrier Reef. Proc.5th Int. Coral Reef Congr. 4; 387- 372. Jertov,N.G. 960!. Ultraviolet radiation inthe ses. Nature 1966: 111 - 112. Jeriov,N.G. 968!. Optical oceanography. Elsevier, Amsterdam, p.194. Jokiel,P.L 980!. Solar ultraviolet radiationand coral reef epifauna, Science 207:1069 - 1071. Joklel,P.L. 985!. Lunar periodicity ofplanuis ielease inthe reef coral Pocillopora darnicornis in relationtoVariOus envirOnmental factCm.Proo. 5thInt. COraI Reef COng. 4-307 - 312. l6t Karantz,D., McEuen, F.S. ~ Land,M.C. IL Dunlap, W.C. 991!. Surveyof mycosporine-likeamino acklcompounds inAntarctic marine organisms: potential protection from ultraviolet exposure. A4ar. Bfof. 108: 157 - 166. Kawagutl,S. 941!. On the physiology ofreef corais. V.Tropisms ofcoral planulae, considered asa factorof distrlbu5onofthe reefs. paleo T opfcafBiology ical StationSturfies, Vol. II No.2, p. 319- 326. Lewis,J.B, 974!. The importance oflight and food upon the early growth ofthe reef coral Favia fragum Esper!. J, Exp.Alar. BAxl. Ecol. 15: 299- 304. Maiahali,S.M. 4 StephenSOn,T.A. 933!. Thebreeding Of reef animals. Part 1: TheCOrala. Scient.Reports Gt. Bamer Reef Expert. 3 8!:219 - 245. Morse,D. E. 8 Morse,A. N. C. 992!. Sulfatedpolysaccharide induces settlement and metamOrphcsiaOfAganicfa humNS kSVae On SpeolfiC Cruataee red algae. AbStract.7' Internet'lCoral Reef Symp. 22 - 26 June, 1992,Guam. Peterson,P J.M.,Smith, R.C., Patterson, K.W. tL Jokiel ~ P.L. 995!. A biologicalweighting func5onfor phyloplankton growth inhibition, lrr. 'Ultraviolet Radiation and Coral Reefs' ~ D. Gulkoand P.L. Jokfei eds!. HIMBTechnical Report ¹41. UNIHI-SEAGRANT CR-95-03. pp 51 -80. Richmond,R.H. 982!. Energeticconsiderations In the dispersalof PocfNoporadamicomis LInnaeua!planulae. Proc. 4h tnt.Coral Reef Congr. 2: 153 - 156. Richmond,R.H. 985!. Reversiblemetamorphosis in coral planulalarvae. Msr.Ecol. Prog. Ser. 22: 181 - 185. Richmond,R.H. 987!. Energetics,competency and longMistancedispersal of planuialarvae of the coral PoalNoloorad'ami'comls. Mar. Biol. 93: 527- 533. RIchmond,R.H. 8 Joldel, P.L. 984!, Lunar perhdicity in larva release in the reef coral Poc¹iopor»cfamfcomis at Enewetak and Hawaii. Bull. Mar. Sci. 34: 280 - 287. Rogers,C.S. 990!. Responsesof coral reefs and reef organismsto sedimentation.A%sr. Ecol. Prog. Ser. 82: 185 - 202. Rogers,C.S., Fitz,H.C. ~ Gilnack,M., Beets,J. 8 Hardin,J. 984!. Scieractiniancoral recruitment patternsat Salt Riversubmarine caelum, St Croix,U.S.V.I. CoralReefs 3: 69 - 76. Sammarco,P.W. 982!, Echinoidgrazing as a structuringforce in coralcommunities: whole reef manipulation.J. Exp.Mar. Biol.Ecol. 61. 31 -55. Shibata,K. 989!. PIgmentsand a UV-absorbingsubstance in coralsand a blue-greenalga living lnthe Great Barrier Reef. PlantCeff Phpsiol. 10: 325 - 336. Siebeck,O. 981!. Photorsactivationand depth-dependentUV toleranceh reef coral in the Great Barrier Reef/Australia. lVatunNisaenaIMfilsn68: 426 - 428. Sebeck,O. 988!. Experimentalinvestigation ofUV tolerance inhermatypic carats Scleisctinia!, Afar.Ecol. Prog. Ser. 43: 95 - 103. Smith,R.C. 8 Calkins,J. 976!. Theuse of theRobertson meter to measurethe penetration of solarmiddle-ultraviolet UV-B! into natural waters, Lirnnof. Oceanogr. 21: 746 - 749. Smith,R.C. 8 Baker.K.S, 979!. Penetrationof UV-Band biologically effective dose-rates in natural waters. Photochem. Photobiof.29: 311 - 323. I62 Smith,R.C. & Baker,K.S, 981!, Opticalproperties of the ciearest natural water 00 - 800nm!. Appl. Opf, 20: 177 - 184. Smith,R.G. & Baker,K.S. 989!. Stratosphericozone, middle ultraviolet radiation and phytoplanktonproductivity. Oceanogr. Msg. 2: 4 - 10. Stimson,J.S. 978!. Modeand timing of reproduction in some ccrmmon hermatypic corals of Hawaiiand Enewetak. h4ar.Biol. 48: 173- 184. Stoddart,J.A. 984!. Geneticai structure within populations ofthe coral po6lkyora darercome. Ii4sr. &Ial. 81: 19- 30. Te,F.T. 991!. Effects oftwo petroleum products onPocINopora datniceNrlis planuiae. Pac. Sci. 45: 290 - 298. Te,F.T. 992!. Responsetohigher sediment loads by Podliopora dsmlcomlsplanutas. Carel Reeiss 11: 131 - 134, Worrest,R.C. 982!. Review ofliterature concerning theimpact ofUV-B re5a5on upon marine organisms.In:'The role of sofar ultraviolet radiakm inmarine exeystems', CaNdns, J. ed!. Plenum, New York, pp. 429~7, Worrest,R.C. & Cakfwell,M.M. eds.! 988!, Stratospheric ozonereduction, sofaruttravfolet radiationand plant life. Springer,New York. 163 ffnsvfalaRsdlstton snd Caret RsefL tsgs, 0.GLdto 5 P. L Jokisl sds.!. HIMB Tech. Report 441. UNIHl.sss Gnwt-CR4&M Ultravioletradiation: helpful or harmfulto growthof cultured zooxanthellae? Scott R. Santos Hawal'ilinstitute of hlarine Soggy, Kana'ohe, Hl 96744 ABSTRACT:Typical invffra culturing techniques ofmlcraelgse employ theuss af huoreecent lighting, which produces ultraviolet UV!radiation. Zaasanthelkte Culturee from the Sea anemOne A5rfasfa pufafrast Were used ta tact the hypothesisthatUV prOduced byftuareeaent bulbeused inlaboratory culturing may bedstrirnsntal tatheir growlh. Aliquatsofforty millllllers ofdiluted culture were placed under a lighting system that consisted oifour 30 watt WeNnghausebrand'CaOI Whffe' fluOreecent bulbeOn a 12:12haur Hghhdark phatcPeriod. Thia~ ProvidedSn totalirradiance of15.5 W m'S' 0467 W m'S'at 300 - 320nm UV-B!, 0.495 W nr*s' at320- 400 nm UV-A! and15.0 W m' s' at400- 700 nm PAR!,nrapecSv@y!. Thehypothesis wsstested by subjecting 3 Pyrass test tubesaf zoaxanthetlae culture to each of the following regimes: fullspectrum radlattan prove bythe fluorescent bulba,fluoresCent radiatiOn lacking UVW 00 - 320nm!, and ftuaraeaem ~ lackinguv-B and UV-A 20- 400nrn!. Pyrexts was chosen fortwo reasons: 1!because ofits low reducdon &5! of biologicatly ~ chforohurocarbonradlatiOn Smith 5 Ac Baker, arts!,which1950!, tranerrftsand 2! its wavelengthscomrnOn usegreaterin milarcsrlgae than250 nm.culture. The The~ trayfirat tray waswas ffftedffftsd wffhwtths caverafpolyester film DuPantMyta4l! that blacked autwavelengths below320 nm but tramrmffted mostcf the kmgerwave engthradiattan.Thethird tray was fitted with acqver ofV V0 fxdtrcarbanate!, whichwas opaque toboth uV-Band UV-A radiation, Culture tubes were laid at a 45"angle; this cOnfiguration alkrvred Cultures tOreceive unshadedirradiance throughout the12 hour light period. Cell counts were cceducled using a hemccyrtameter arxl lightmicrowxrpy every 3 days over the t5 day sxperlmsnkd period. Eight chamber caurrfa perlast tubewera taken tc pravkfeeetfrnatsd papufat!an denaltkre ofxaoxanthellas. Datafram twO runs Were tested using ANQVA; no niftcantdifference was found ~ thethree treatments. These results ~ Ihstzoossnthellae raised ir arenat ~ byfluarascent~luced UV. INTRODUCTION interestin the ecological importance ofsolar ultraviolet UV! radiaticn has been stimulated by concernover the possibledisruption of the earth'sprotective ozone layer by anthropogenic atmosphericpollution. UV radiation measurements taken by Jerlav 950! showedthat clear, oCeaniCWaters, suCh aS thcee fOund OVer many COral reefS, tranemit considerable amounts cf UV radiation,yet the previously held view that UV is without ecological importance in all types of watersremained predominant for a considerableperiod, However,in the last 15 years, numerousobservations have given extensive support to Jerlov'sview of UV'simportance in aquaticenvironments {see Calkins, 1982 for review!.In particular,UV radiation at andabove ambientlevels has been found to be harmfulto manyforms of microalgae McMinn et al., 1994; Cullenet al,, 1992;Cullen & Lesser,1991!. Effects of seriousUV radiation damage range from reduCticnSin Carban aaeirnilatiOn Hazzard, 1990! to "bleaChing"OfChlOrcphyll and reduCtion of cell motility Gerber& Haeder,1993!. Hermatypic,or reef-building, corals are predominantly found in theeuphotic zone of warm, tropicaloceans and seas Falkowskietal., 1990!. Their success is partlydue to theirsymbiotic relationshipwith zooxanthegae {Sjrrnbiodinf'fjm sp.!, single-celled dinoflageliate algae that live withinthe endodermaltissue of the coral animal. Thesealgae requirelight for photosynthesis, andprovide the animal partner with photOSynthetic prOduots that COntribute to nutfftfon {MuSCatine & Porter,1977! and aid in the constructionof thecoral's calcium carbonate skeleton Goreau, 1961;Pearse & Muscatine,1971! Zooxanthellaehave been isolated from symbiotic partnerships andcultured in vitrosuccessfully Jokiel 8 York,1982; Lesser & Shick,1989!. Jokieland York 984! demonstratedthat in vitrocultures of zooxanthellaegrown under natural irradiance unahieldedfram UV wereSeverely impaired in wayssimilar to OtherrniCrOalgae. Typicalin vitroculturing techniques employ the use of fluorescentlighting, which produces both UV-Aand UV-B, The experimentdescribed here was designedto test the hypothesisthat fluorescentlighting may impair the growthof laboratorycultures of zooxanthellae. If the UV producedby fluorescentlighting is eliminatedby usingone, or a combination,of filters,it is hypothesizedthat zooxantheliaedivision rates will increase. MATERIALS AND METHODS Zooxantheilae Syntxbdfnium sp.!from the sea anemone Aipfeaa pulche//a was obtained fromthe Coconut island zooxantheliae collection. All cultures were maintained in Pyrex@ test tubes 5 x 150mm diameter!. Pyrex. wes chosen for two reasons: 1!because ofits low reductkw 3%! of biologically effective radiation Smith 8 Baker,1980!, and 2! ite common use Inmicroalgae culture. The culture tubes were washed with 10% Liqui-Nox., rinsed with tap water,rinsed with 10% HCi, rinsed with dlstified water, stoppered with cotton and autoclaved. Oneliter of 0.45 micronfittered seawater was filter-sterilized using 0.22 micronMiliiporeS filter paperand enriched tomake "fi2" medium. An inNal cell count was made on the Coconut Island masterculture using a hemocytometer.A dilution was made from this master culture, and aliquotsof fortymllllliters, containing a known zooxantheiiae density, were placed in the sterile PIpexOand stoppered with sterile cotton. The cotton stoppers were capped with ParafilmS beforethe tubeswere placed under their resi:ective irradiance regimes. Lb&lag The lightingsystem used consisted cf four30 wattWestinghouse "Cool White" fluorescent bulbson a 12:12 hour light:darkphotoperiod, which is typicallyused for in vitromicroalgae culture.This systemptovided an totalirradiance of 18.5 W m's' .047 W m~s' at 300 - 320 nm UV-B!, 0.498 W m~ s' at 320 - 400 nm UV-A! and 18.0 W m s' at 400 - 700 nm PAR!!, reepec5ely, when measured using a Li-Cor LI-1800UW UnderwaterSpectroradiometer at Coconut Island, Kene'ohe, Hawai'i on June 16, 1994. Nine test tubes of zooxanthellae culture weresplit among three treatments. These treatments were: full spectrum radiation provided by thefiuoreetxW bulbs, fluorescent radiation lacking UV-8 00-320 nm!,and fiuorescent radiation laoklngUV-B and UV-A 20-400 nm!. Thefiiet tray was fitted with ChlcraflurOCarbon Acier.!, whichtransmits wavelengths greater than 280 nm. The secondtray was fitted with a coverof ~eater film DuPontMyla&! that blocks out wavelengths below 320 nrnbut transmits most cf thelonger wavelength radiation. The third tray was fitted with a coverof UVO polycarbonate!, whichls opaque to both UV-B and UV-A radiation. Culture tubes were laid at a 45'angle; this configurationallowed cultures to receiveunshaded irradiance throughout the 12 hour photopehod. Cellcounts were conducted using a hemccytometerandlight microscopy approximately everythree days over the 15 day experimental period. Eight chamber counts per test tube were takento provide estimated population densities. Test tubes were agitated each time a sample dropwas extracted to ensure homogeneous mixing of eachculture. Cell count data was subjectedtoANOVA todetermine ifthere was a differencebetween cultures ofeach treatment foreach sample day. RESUI.TSAND DISCUSSION Tworuns were conducted totest the hypothesis thatfluorescent lightingUV may impair in vitrocultures ofzoxcanthellae. Cultures tended toconcentrate inareas nearest theirradiance sourcewhen left undisturbed. Cellsin both motile and non-moble phases were obse ed d cell counts.InNal cell denalty perml for each run was 7000 and 8000, respectively. IV Over urlngthe courseofthe experiment, celldensity increased exponentially forthe first twelve days ofthe experimentbeforeleveling offbetween Day12 and Day 15 Rgs.1 and2!. Th' is I evelingoff of densi ma y havebeen a resultofcultures reaching senescence. ANOVA was used to d if the difference etermine significant.s between treatments weresignificant. Differences werefooun d tot be not Culturesgrown under the full UVtreatment showed slightly higher densities for a majorityof the eXperiment.JOkiel and YOrk {1984! elec Obaerved thiS phenamenOn when Stimfriodinium miCraadristicum,PhaeOdSCtylum trfCOmufufn and TetraseIrrriSSp. Culturee Where grown under conditionsof lightlimitation '% intensityof naturalsunlight!. One explanation for this higher densityis that somespecies of algaeuse UV radiationphotosynthetically under light-limited growthconditions Jokiel 5 York,1984!. Halldal 988! demonstratedthat the action spectrum for photosynthesisin S. rnicrosdnaficumshows oxygen production in the UV-Arange. E 220 X 160 CO140 ~ 120 100 0 80 60 20 0 0 12 15 DAY Figuret. FlnrtRon. Zoecanfheilee ~ when grownunder different treefnwnte ofPAR, PAR + UV-A,end PAR + UV-A+ UV8. E 240 220 g 180 rlf 160 w 140 u 120 ~ 100 80 60 40 K 20 0 9 DAY Rgureg:.Second Run.Zoorornthefiae ~ when grownunder different treetrnentrr ofpAR, pAR + UV-A,rrndPAR + UV-A+ UV8, 167 Theseresults, although preliminary, suggestthat our stock cultures arenot being adversely affectedbythe UV radiation producedby flourescent lamps.However, thecultures usedinthis experimentwerektolated manyyears agoand given therapid division rateofthese organisms, untoldgenerations haveoccurred underthese condNons. Therefore,selection for resistant forms mlgtrthaveoccurred inthese cultures. Thktcould result inthe selection ofcultures thatare UV- tolerantandnon-representative ofnatural populations. Thenext logical experiment wouldbeto competeresponse offreshly isolated zooxanthellae ofthis type with the laboratory formsthat havebeen grown inculture under Iuorescent lightssince they were isolated fromtheir host over 15years ago. Onthe other hand, Joklel andYork 982, 1984! used thesame strain intheir worksnd found thecultured cellsto be sensNve tosolar UV-A sndUV-B. More work dealing with eubSNesolherthan growth needs tobe conducted, inciudirtg analyzes ofpigment composition andconcentration, toensure that cultured zooxanthellae arean adequate representation of natural populations. 8Obqy.UV~ On. Tttla COr»fr»»»arch Reef»waaat OOnduoted the Hawarl aaInatfhrt»part Of theOf Marineteed Edwin sfOlogy W, and pauley Wa» supp»rtedSLrnvner pl»grambythe EdWinln Marfne W. patrtey Fountfatkrn. LITERATURE CITED: Caikins,J. 982!. Therois of solar ultraviolet radiation inmsrire ecosystems. Lenurn Press, New York. Cullen,J.J., Neale, P.J. 8 Lesser,M.P. 992!. Biologicalweighting function for the inhibition of phytoplanktonphotosynthesis byultraviolet radia5on. Science 258: 848 - 650. Cullen,J.J. 8 Lesser,M.P. 991!. Inhibitionof photosynthesisby ultravioletradiation as a functionof doserate. Results from a rnaffnediatom. Mar. Blot. 111: 183 - 190. Falkowsld,P.G., Jokiel, P.L. 8 Kinzie,R.A. 990!. Irradianceand Corals. lrr, 'Ecosystems of the Woffds25: CoralReefs', Z. Dubfnsky ed.!. ElsevierScience Publishers, Amsterdam. pp. 91 -92. GerberS. 8 Header,D.P. 993!. Effectsof solar irradiationon motilityand pigmentationof threespecies of ~ankton. Etrirort.&p. Bot.33: 515- 521. Goreau,T.F. 981!. On the relationof caicNcationto primaryproductivity in reef building organisms.Irr. 'The Biologyof Hydrml,H. M. Lenhoft8 W. F. Loomis eds.!. Universityof Miami Press, Coral Gables, Fla. pp. 269- 255. Halkfai,P. 988!. PhotosyntheticcapacNes and photosyntheticaction spectra of endozoice otc alalgae e of the massive coral Fsvis, Biol. Bull. 134: 41 1 - 424. Hazzard,T. 990!. Biologicaieffects of uttrevioletradiation. Pac. Scl. 44: 188. Jerfov, N.G.. 1 950!. Ultra-violet radiation in the sea. Nature 166: 111 - 112. Jokiel, P.L 8 York, R.H. 982!. Solar ultraviolet photobiology of the reef coral Pocil Jokiel, P.L. B York , R,H, 984!. Importance of ultraviolet radiation in p hotoinotoinhlbition of i esser,M.P. & Shick,J,M. 969!. Effectsof irradianceand ultravioletradiation on photoadaptationinthe zooxanthellae ofAiptasla paliida, primary production, photoinhibition, andenzymic defenses against oxygen toxicity. hfar. Biol 102: 243 - 255. McMinn,A., Heijnis, H. 8, Hodgson, D. 994!. Minimaieffects of UV-B radiation onAntarctic diatomsover the past 20 years. Nature 370: 547 549. Muscatine,L.8 Porter,J.W, 977!. ReefCorais: Mutuahstlc symbioses adapted tonutrient-poor environments. Biascience 27: 454 - 460. Pearse,V.B. & Muscatine,L. 971!, Roleof symbiotic algae zooxanthellae! incoral calcification. Biol. Bull. 141: 350- 363. Smith,R.C. 8 Baker,K.S. 960!. Biologicaily effective dose transmitted byculture bottles in' C productivityexperiments. Limnol. Oceanogr. 25: 364 - 366. U revtoietRedtetton ent} Gml fee}L 1085 D.Gclkc a P.L, Jokiel ede.!. HIMS Tech. Report 441. UftlHI-Sea Gran}CR-f543. Surveyof mycosporine-iikeamino acids in macrophytes of Kane'ohe Bay AnastaziaT. Banaazakl'and Michael P. LesSeR DepertmentofSdegM sdenot», untventity ofCeltfomie, Sente~ GA f6106 2DeperltmentofZootaoy, ~ Mdina,ure+retty aff}ew H~, Dud»m,NH 93a24 ABSTRACT:Mtrooeportne4tce aminoedda MAAe! were ~ by highperromence ftqutd HPLC!inf9 ~ ot RhodOPhirrte redelgee!, 11 ~ of ~+le algae!,S ePeC}t» OtChtonCithyte gn»n !, end3end spadedtwOepeC}r» ofCyenaprhyte Ofpheeophytt» btueCteret~ td etN»!. ieeetSeVen I» NMAA. Ae Htwere Cf e»ideNIM MAIe ertddefeated el b«4 heveIhn»been abeerved d inmerine orgenien». Myoattfto}~ne ~ a 310nrn! end ~ ~ 334nm! were t» meet ~ MAAe, bathbetn9hundb549% 851! Of e» ePeC}t» etuctted. PtOrPhirie 334 ~ = %4nm! wee faLNd > 314% 851! d theepee}«» etudted, etylthtr» ~ = 320rvn! wt» found in19 6% 051! and eetertr»-330 ~ ~ 3Xt rvn!Wee rOund in15.7% 851! epeC}ee etuctied, The}eeet ~ MAAe deh4dWere ~, whiChweefc«ltd in 351epeotr», end r»tythene which Wee found inaniy «ee epeC}ea etu«ted. tMTRODUCTfCNOverthe lastseveral decades there has bean an increasedinterest in the potentialfor enhancedbiologicaliy effective ultraviolet UV! radiation impinging onthe earth's surface. S6ar racfiatkrnreaching the earth's surface lscomposed af both photosynthetically activeradia5on PAR,400- 700 nrn! and UV radiatktn 90 - 400nm!, Photosynthetic crgftntsms which require PARfor primary production arealso exposed tothe potentially damaging effects of UVradiation. Thehigh transparency oftropical ocean water to solarPAR and UV radiahcn suggests that shallow-waterdwelling organisms areexposed tohigh fluxes of UV radiation, posably requiring a trade-offbetween the requirements formaintaining highrates of photosynthesis whilepreventing damageBiochemicalfrom UV defensesradiation. against the damaging effects ofUV radiation indude the pret}ence of UV-absorbingcompounds known as rnyccraporine-like aminoacids MAAs!. These compounds, firstidentled as 'S-320' compounds incorals and a cyanobacterium Shibata, 1969!, have since beenfound in a widevariety of marineorganisms spanning cyanabacteria Shibata, 1969! to teleasts Dunlap etal., 1989! and ranging geographically from tropical Dunlap et ai.,1986! to Antarctic waters Karentz etal 1991!. IAVh have absorbance maximaranging from 310- 360 nm and together may provide abroad-band filter ta UV radiattcxt. ThephOtOprOtective function Of theae COmpaunds haabeen inferred from their UV abSorption porperties Price & Forrest,1969; Shibata, 1969; Dunlap etel., 1986!, the concentration ofthese compoundsin coral reef-dwelling species Shibata, 1969; Dunlap et el, 1986!,the positive correlationbetween the 325nm peak in Poritesit}thats with light intensity Maragos, 1972!, the deCreaaein COncentratian af theSe campaunda aa depth increaaea in COralspeCiea Dunlap et at., 1988;Scelfo, 1988; Shick et al., 1991!,the modulationof the concentrationof thesecompounds by exposure to UV radiatiort Shick et al., 1991!,and that corals grown in full spectrum solar radiation produce higher concentrations of 'S- 320'~! thanthose screened from UV radiation Jokiel, 1980; Jokiel & York,1962!. The ptT}duCtianaftheae CompOunds haabeen ShOwn ta be indurx}d inthe preSence of UV radiation in the dlnaffagefiateBrymbiadr'nium microedriafictum, symbiotic with the jellyfishGassiapeia !tamachanrr Etanaazak, 1994!. Althoughthe path>myfor thesecompounds is restrictedta bacteria,fungi and algae, these campounds may be passedthrough the foodchain to higher trophiclevels aa was found in hoiathurians feeding on bacteria Shick et at., 1992!. Macrophytesare important biological contributors of primary productivity and biomass in eCO}P!fatemSSuChas COral reefa and fleahy speCies afalgae serve aS the baSe Of fppd Chains whiohmay alSo SerVe aS a SOurceOfUVwbsarbing Ct3mpr3unda fOrinvertebrates andvertebrates. Calcareousalgae are also important geological conf}fbuters to coral reefs by sediment formation pteeenteddreee: Smtthe«I»rn Envtronmenbtt Reecrtuoh Cenert, p.o, B«er28, Ed rev»err,MD 21037 171 framthe breakdown of theskeleton which aids in the build up af thestructure of reefs. Thepurpose afthis study was to survey the MAAs inmacrophytes fromKarIe'ahe Bay, Hawai'i andto determinethe taxonomicdistribution of thesecompounds. MATERIALSAND METHODS Macrophyteswere collected InJune and July, 1994 during daylight hours by SCUBA diving or Snarkalllngatdeptha af One ta tWO rnetrea at patch reefS in Kane'ohe Bay, Hawai'i 1 29'N, 157o50' W! representing turbid, tropical waters, All specimens were collected from fully exposed andunshaded haNsN. Underwater measurements of UV and PAR radiation were taken at the pointof collection close to noon on sunny, cloudless days. The measurements weremade using a U~ 1800UW spectroradiometer equipped with a 180ocosine corrected sensor. Measuremetswere taken at 2 nmintervals at a rangeof 300to 850nm. Threescans were taken at each site and averaged. Thespecimens were rinsed in sea water, cleaned ta removeepiphytes and other organisms andfrozen at -50DGunI requiredfor further analysis. For analysis by high performance liquid chIamatography HPLC!, approximately onegram af wet tissue was extracted overnight in5 mlof 100%HPLC grade methanol. MAAs were separated byreverse-phase, isacratic HPLC on a BrownleeRPW column Spheri-5, 4.6 mm ID x 250mm! which was protected with an RP-8 guard column Spheri-5, 4.8 mm ID x 30mm!. The mobile phase consisted of40% methanol v:v!, 0.1% gfaaNaaNc ackf vw! in water and run at a flawrate af 0.6 ml rrin'. Detectionofthe peaks was cantedaut by UV absorbarxm at313 nm and 340 nrn using a diadearray abscxbance detector. Standardsfor seven MAAs were provided courtesy of Dr.Deneb Karentz. Identities af peaks wereconfirmed bythe raths af 313 nrn to 340 nm absorbances andby the maximal wavelength of abaxbance.The concentrations af the knawn compounds are expressed in nanomolesper mg protein.There were two unknowns found and these are expressed asa percentageofthe total peakarea. Protein measuremsnts were determined using the Lowry technique with the Hartree mcxlifica5on Hartrae, 1972! on the methanol extracts used for the determinatian of the concentration of MAAs. RESULTS Rgure1 showsthe sixtctraI irre5ance profile for Kane'ohe Bay on a sunnycloudless day for variousdepths. The amount ofUV-B, integrated from 300 - 320nm, ranged fram 1.2 to 2.9 W m.~ atthe depth of collection and UV-A, integrated from 320 - 400nm, ranged from 27.4 ta 53.0 W rn-2. TableI showsthe distributkxtaf MAAsin the fourdivisions of algaestudied. There appear to be sometrends in the taxonomicdistribution af MAAsin the variousdivisions with red algae havingwider varieties ofMAAs than green algae, brown algae ar cyanc4ecteria. The most commonMAAs found are mycoepori~lycine which absorbs maximally at 310nrn, shinorine whichabsorbs maximally at 334 nm and porphyra-334 which aha absorbs maximally at 334nm. Palythene,which absorbs at360 nrn, Is rarely present possibly because iong UV-A wavelengths aretequlred toInduce repair mechanisms. This table gives a bnmdoverview ofthe distribution af thesecompounds but does not contain the concentrationsaf these compounds. TheChloraphitta green algae! in general contain fewer MAAs than the other divisions and lowerconcentrations af the variousMAAs found in the differentdivisions Ta5e II!. Thereis also anunknOwn which COnaiStently appears. hlaNmeda, the Calumet algalgenuS, cantainS ah unknown almost exclusively. TheRhodophyta red algae! contain the highest concentrations of MAAs and in general exhibita widervariety of ccxnpaundswith few excep5ons Table III!, Theunknown 1 alsoappears in thisdivision as doesanother unkncwvn although bath make a lawcontribution relative to the totalpeak area. The calcareous md algae contain high omcentratians of MAAs although they are not as diverse as in the fleshy rhodophytes. 172 ThePhaeophyta brownalgae! contain highconcentrations ofMAAs but do not exhibit the diversityofMAAs found inthe red algae Table IV!. Again, theunknown-1 and-2 show upwith theformerThe Cyanophytaoccurring in blue-greena largenumber bacteria!of phaeophytescontain relativelysurveyed. lowconcentra5ons ofMAAs with the unknowns1 and2 appearing again Table V!. Unknown 2 appears inhigher percentage in cyanobacteriathan it didin anyother division, 1.00 x 10 1.00x 100 1.00 x 10 I 1.00x 10 1.00x 1 3 1.00 16 1.00x105300 350 400 450 500 550 600 650 Wavdcagth om! Rgurs1,~ inadlermdepth peae br Kans'Oha aayon a~day. DISCUSSIONAtleast one MAA was found in 90.2% 8/51! species examined. Seven specNc MAAs were detectedwith the most abundant MAAs being rnycosporine-glycine andshinorine. Mycosporine- giyCIne ~ = 310nm! and shlnoflne ~ = 334nm! Were found 'In55% Of the species studied.Porphyra-334 ~ = 334nm! was found in31% of the species studied, palythine ~ = 320nm! was found inf9% and asterina-330 ~ax= 330nm! was found in15% species studied.The least common MAAs detected were paiythinol, which was found in 5% species, and palythene,which was found inonly one species studied. None ofthe species examined containedallseven MAAs, however a number ofspecies contained fiveor six of the MAAs detected.InAntarctic macrophytes, themost common MAAs found were palythine and shinorine KarentzRhodophyteet al.~ 1991!. andphaeophyte species are found more commonly onthe patch reefs in Kana'oheBaythan chlorophyte andcyanophyle species, Generally, chlorophytes whenfound arepreeem inlow bicmaSS, whereas thered and brown algae are mOre abundant whiCh may be relatedtothe presence ofUV-absorbing compounds inthe tissues as protection against the damagingeffects of UV radiation. There are two unknowns which consistently show up in samplesofalgae spanning allof the divisions. These compounds need to be identified and the chemical structures elucidated. Theobservation that red and brown algae which are more abundant on reefshave more diverseMAAs and higher concentrations ofthese UV-absorbing compounds is purely 173 correlative.What is reallyneeded is to demonstratecause and effect in the effectivenesof these comprende in photoprctection. We know that MAAs absorb UV, but what happens to the eneqp/P To be photoprotective,these compounds need to convertthe lightenergy to some otherform of energy heat or fluorescence!or to dissipatethe energyby a cis.transmovement such as is found in carotenoids. The pathway s!of MAAsalso need to be determined.The probleminvolved in studyingthis pathwayIs that it lsso complexand so intricatelyirwolved in so manyother metabolic pathways thatthis weal be verydifficult. A goodstarting point, though, is to lookin speciesin whichthese compounds are induced in the presence of OV. Lang-termacclimation studies will be very importantin determiningthe abilityof speciesto acrctimateto changingUV condNonssuch as the effectof increasedUV radiationon communities. Greenalgae, which do notseem to containUV-absorbing compounds, may be a goodindicator speciesof effectsof enhancedUV radiation.Comparison with red or brownalgal species may thenbe a usefulparameter to studycornrnunity effects. In thiscontext we needexperiments whichwilt complement the measurementof UV radiationin tropicalcoastal ecosystems. Te5eI. DsttfbudonOfmlrSrvperlne-lasr Stdnoedda inmeorophress OfKsneahe Bejr, CHLOROPHYTA Neomah amulet RHODOPHYT* Adkroafalrfs Centroams drnstfsatm Gdecaaa ngeee GskSslsecwoes Gnyalfarh jretVflrpOnr Gredkrtfs eskxxBfe HjSknalfrnn Legends ~s Legendststodss Lkpote arflefpX/krte Pkxrstnftsn eandi/c~ X X AmNInon~ 8pjekN Istrenkrse Tdybde& pksnerukrkr Tifdxybee tequlen6rlr PHAKIPHYTA ~rmsnfa sfnttoes Qfcl~artfs «edsR OfctpoOracagdtsar Ofdjtofsbstfayresf DCIItdssendlk8llsfs PsdnsJspanea Srryesasn ~pgs Sarge~ pdgApkm 'I 75 Q~len effffrreeerrrrfnaefre ernfneedde fnrneCIO+jhbe Of~ Be1r CanL! TeNeIL Cms~re ef myooepe~ffreemfnrr ecfde ln fferrhy end eeferuewe preen efpaa CHLQROPHYTA4eahy 63$ OAQ 125 OM Qal~ieerfe Dci~eah &eemaphe h4ee4af~ 1.13 GXI ilgXlnlrIHI HfeNnerfeernAea 83l CHLCRI~YTA- 176 Tabbill. Gxcentredoneorrnl~&Nse errrirroaddein heehy andcrdcarerae redal9aa. RHODOPHYTA- 8eehy AorAglrora 1m% 184 18k70 12A8 4IAI4 Cerrrrocerarr 27AQ 2S? cllrrrrerrLlrr Gekjelh 8J%I 8!8! 11,79 2833 6~ 527 19MO 82220 5128 17A6 8082 4697 9725 17.77 1124 51926 - 2198! SLSl 0.10 9 Q1 24 eeee9e 5L% 5129 rn6lrrrrorrla 591 1&82 588 11AO 292 euM!rra Raoarntm 94A1 Si Redeea rNe/cdr Q2 To09riodecde gl&Mftgeer 5 33 74SR fIHIXIOPHYTA- Ard&W&h SA2 Adfnodk&fe I6.15 tnyds Galway rrr9Oea 11820 2184 Hyreallrlrerr 84,'12 2037 2LN 954 HpdrONrrrOn 554 Q87 Llepwa 'ItL% ~lleta Plptol9rrrn 418$ - 553I RVS 0,10 151 17 TebhIV,Ooh+fnerefone of coeporlne4ke Or@noaotdeIn tfeehy andoalarreoue brownalgae. PQKIPff'fTk 2eehy 14 G~~ 12 Ar5torrrerde 19! gfeffetperh 14 4 451 7t2l at4 rarusofrNa atrttsoftaM hah!real 19 Nfcer~e fletl%ere 32 478$1l8 4L42 1233 16 2.18 Serffaeeurn ~ftf rNeten ljarfrherMpm' 7MAeh~ PHAB."IPNWA. octo%.as 15 ~bftoorbe 13NR 1 AS Q.12 0.10 T~ V. 0 rneelera5OneOf mleoepOOtneake amktO «dde ln blue-greenalgae CYAfeffRhrTA Htrrrnoylerlrrt/ter 257 1,73 15 4j wrbrrelrsphofM ffngftfe meieouh 27.17 6 3 rtgbtette, 2.11 54 AdAmrrfe~e Wethank Dr. Ieebrrfa Abbott for hrepIn idenllfytng algal epeotee and Mr. MkhaN Qndrueek and Me. Nea Ku5nerIor help weh HPLC dNermkreNOn. TNe ~ ~ canduofedae part of theUV Summer Prrtgrem at theHawall etWeufeof MarineSobgy weh 4ndtng frrtm Ihe Bah W.Paukry FounchNon. tftfe thank ete hazy and~ at Hid for~ eupportend vafuebkr eeetetanoe In~g thtenreeerch, 178 LITERATURE CITED Banaszak,A. T. 994!. Theeffect of ultravioletracNa5on on the biologyof twomarine symbiotic aSSOciations.Ph.D. Diss. Univelalty Of CalifOmia, Santa Barbara. 140 pp, Duniap,W. C., Chalker, B. E. 8 Oliver,J. K. 986!. Ba+rmetrfcadaphNom of reef-building coralsat DaviesReef, Great Barrier Reef, Australia III. UV-B absorbing compounds. J. Exp. Mar. Biol. Ecol. 104: 239 - 248 Dunlap,W. C., Williams,D. McB Chalker,B. E, & Banaszak,A.T, 989!. Biochemical photoadaptationin vision. U.V.-absorbing pigments in fisheye tissues. Comp. Biochem. Physiol. 93B: 601 - 607 Hartrae,E. F. 972!. Determinationof protein: A modlfkmtionofthe Lowry method that gives a linearphotometric response. Biochemistry 48: 422 - 427 Jokiel,P, L 980!, Solarultraviolet radiation and coral reef epifauna. Science 207: 1069-1071 Jokiei,P. L. & YorkJr., R, H. 982!. Sdarultraviolet photobiology if the reef coral Po6liopora damicomisand its symbiotic zooxanthellae. Bull, Mar. Sci. 32; 301 - 315 Karentz,D., McEuen, F.S., Land, M. C. & Duniap,W. C. 991!. Surveyof mycosporlne-like aminoacid compounds in Antarctic marine organisms: Potential protection fnxn ultraviolet exposure.Mar. Biol, 108: 157 - 166 MaragOS,J,E. 972!. A studyOfthe ecology OfHaVasISs1 reefCcrals, Ph.D. Disa, Univeraity of Hawaii, Honolulu, 290 pp. Price,J.H, & Forrest,M.S. 969!, 310 nm Absorbance inPfrpsails physallis: Distribution ofthe absorbanceandisolation ofa 310nm absorbing compound. Comp. Biocibem Physrof. 30: 879 - 888. Sceifo,G. M. 986!. Relationshipbetween sdar radiation and pigmentation of the coral Monfiporaverrucose andlls zooxanthellae. lrr.Coral Reef Population Biology', Jokiel, P.L Richmond,R.H. ~ Rogers,R,A. eds.!.Sea Grant Coop. Reports: Hawaii. Shibata,K. 969!. Pigments anda UV-absorbingsubstancein corais and a blue-gteenalgaliving inthe Great Bamer Reef. Plant Cell Physroi. 19: 325 - 335 Shick,J.M., Lesser, M.P. & StochaI,W.R. 991!. Ultraviolet radiation abdphotooxidative stress inzooxanthellate Anthozoa: Thesea anemone Phyiiodiscus semoni and the octocoral Ciawluh sp. Symbiosis10: 145 - 173 Shick,J.M., Duniap, W.C., Chalker, B.E., Banaszak, A.T. & Rosermveig,T,K. 992!. Survey of theultraviolet radiation-absorbing mycosporine-like aminoaids in organsof coral reef holothuroids.Mar. Ecol. Prog. Ser. 90; 139-148 ~ ~ srrdcoal Rsefa 1905. C Gvk>t P L Jokbl ed'.!, HIMB Tech. Report «it. Uff IHI4ea GrereCR45Ck Effectsof ultravioletradiation and nitrogenenrichment on growthin thecoral reef chlorophytes Dictyosphaeria cavernosa and Dictyosphaeriaverslvysii. Scott lamed DepartmentOfZcclcgy, University OfHexad'I, HonOlulu, Hl gSS22 ABSTRACT:Deepltepoten5aily harmfullevels OfSOlar u~ ~ Uv!, meetcarel reef aoroalgrte are~ tcshallow reefhats and fsw ~ are abundantondeeper reef rdopes. Forrrecrcalgae onreef ffate, exposure to growth.tjvmay Forinhibit macroslgae growth, asongrowthdirrrtting reef rdopes, growthnutrients inhlbltxrnere directed may decret»etop~ with anddecreard repair pathwaysUV expire. ratherInthen this tostudy, Leeffects ofUV exposure andnitrogen enrichment ongrowth were examined intwo ' ' chlorcphyk» framKana'ohe Bay,HeWal'I: thereef ffat epeciee Dictycepfraerfa ver»frrraff, and the reef epeok» Dot!roepfreerfaCevemCrea Reeultefrom culture experimenta indioettedthrd ammcnlum en~ enhanCed growthrates inboth species. Atall nltrogrm levelsused, D.cavervrosa growthrates were krwer when ~ to UV-Aand UV-B than when ~ fram Uv.Growth inD. verrerutrrrfl wasnot ~ by Uvexposure atany nitrogenkrvel,sugffeslng thatUV delenees inthis specie areconst ttuth». Unlike some reef flat anthoxrm» with COnetllutiveUVdefenrsea, D.verirrlrSff isnOt an obligate reeffktt Spedee inKanebhe Bay;transplant experiments kxgcatedthatchanges indepth did not signfffcarrtly a%etgrowlh rates, Nitrogen avagebility andgrazing pressure appearedtohave stronger effects ongrowth than dkl UV exposure, butvarktblitty ingrowth rate meesuremene mey have ~ some UV effects. INTRODUOTlONMaCrOB!gaearerarely abundant OnpriStine !OW-latitude COndreef Slapes, butare Often abundantonshallow coral reef fiats Gaines &Lubchenco, 1982; Hay, 1991!. These distribution patternshave been attributed tohigher grazing intensity onreef slopes than on reef fiats, and to lowproductivity ratesrelative tograzing rates Hatcher 8 Lsrkum, 1983; Hay, 1984a; Mcrrrison, 1988!.High leva!s ofsolar ultraviolet radiation UV!make reef flats potentially stressful habitats, hcrwever Jokiel, 1980!. This raises the question ofwhether mecroaigae foundon reef fiats are obligateorfacultative reefflat dwellers i.e., whether reefflats provide optimal conditions for productivity,orserve asrefuges from herblvores butare suboptimaf habitats!. Obligate reefflat algaemay be expected tominimize thecosts afdefenses toradiation, relative toproductivity. ln facultativereef f!at algae, the relative costs of defenses are expected tobe higher. Forany macroalga, synthesis ofUV-protective agentsand the repair orreplacement ofUV- damagedcellconstituents arelikely toincur metabolic costs. lnnutrient-limited algae,nutrients maybe alloCated toUV prateCtiOn ordamage repair atthe expense OfgrOWth orreprodUCtion. Mostmetabolic pathways associated withUV exposure require nitrogen, eg.,synthesis ofUV- absorbingmanna-like aminoacids MAAs! and superoxids radical- and hydrogen peroxide- inactivatinge~imes Lesser 8 Schick, 1989; Lesser etal., 1990!, and resynthesis ofdamaged protein-pigmentcomplexes Somman, 1989!, For algae with broad depth distributions, the aliocationofnitragen toUV protecten ordamage repair may increase with increasing UV eXpOSure,i.e.,With decreasing depth. GrOwth rates Ofmaorcra!gae reetriCted tcShallow reefflats maybe independent OfUV eXpOSure, eitherbecause high MAA levels are COntinuOUsly maintained,orbecause metabolicaliy inexpensive means olUV protection areused. Growth undervaried conditions of UV exposure and nitrogen availability has not been examined in macreeopicShallow water algae. tropical and subtropical marine macroafgae from four taxonomic classes have beenshown to contain MAAs Sivalingarn etal., 1974; Wood, 1987, 1989; Post & ~m, 1993!, Increasesin MAA concentration with increased UV exposure have been obsenred in zooxanthellateanthozoans Jokiel & York,1982; Duniap et aI,, 1986; Schick ef al,, 1991; Kinzie, 1993!and macroaigae Sivalingam etal., 1974; Wood, 1987, 1989; Sherer et al., 19M; Post & Lafkum,1993!. These observations suggest that UV protection is induced and may be metabolicallyexpensive. Exceptions tothis pattern have been found among zooxanthellate anthczoanSWhiCh are reStricted to Sha!IOw water Scelfo, 1985; SchiCk et Bi., 1991; Stochaj et al. ~ 1994!;MAA levels in these organisms appear tobe constitutive rather than inducible. ln contrastto thenarrow distributions of coral reef macroalgae described above, some algal specieshave trecofne abundant oncoral reef slopes near large human populations. These ChangeahaVe been aseoCiated withreduCtiOns inherbivOfe denetties and/Or increaerkS innutrient loading Hay, 1984b; Naim, 1993; Hughes, 1994; Littler et al., 1994!. Algal species which expand 181 their distributionsdown reef slopes under such conditionsare clearly facultativereef flat dwellers. for reefflat specieswhich do not invadereef slopes under the sameconditions, the question raised above remains unanswered. On the owl slopes of Kane'oheBay, 0'ahu, Hawal'i,the chlorophyte Oictyosphaeria cavemosahas occupied large areas of reefslope for at least30 years,overgrowing and displacingcorals Hunter & Evans,1994!. The success of D. cavemosahas been linked to anthropogenicnutrient enrichment Smith et al., 1981!. Most macroalgalspecies in Kane'ohe Bay are restrictedto the shallowflats of fringing and patch reefs, but D. cavemosa has a broad depthdistrltwtlon, extending from outer reef flats downreef slopes to the muddylagoon floor at 6- 13 m depth Hunter & Evans,1994!. Thisdistribution spans a broadrange of tightpenetration, as indicatedby highextinction caefffcients approximately 4.23 m ' bay-widein 1992,L, Watarai, unpublisheddata! relative io nearbyoceanic water approximately-0.07 m ', Smithet al., 1981!. Sotldsubstrata, required for attachmentof thalli,rather than photosynthetically active radiation PAR!ap>ere to Setthe tOWerdepth limit for this alga. UkeOther Kane'ohe Bay rnacraalga, the congener Dlctycvtptuterlaversluysit appears to be restrictedto shallow reef flats and reef crests. Diicfyosphaertacavemosa growth is stronglynitrogen-limited in Kana'oheBay; sustained inorganicnitrogen ~~trations of > 1 II.Mare requiredto maintainnet growth Stimsonet al., in press!. D. cavemosagrowing under condNonsof high UV and low nitrogenmay therefore experiencecompetNon for nitrogenbetween UV protectionand damagerepair, and growth.As a result, the optimal physiologicalconditions for growth may be on reef slopes, below the depth of g~ UVexposure. D. vemiuysiigrowth is alsonitrogen-limited In Kane'oheBay Lamed,in we-!- In the preserrlstudy, I testedthe hypotlxMIsthat high levels of UV inhibit growth in Dictyosphaeria csvemoaa and D. veraiuysii,and that inorganicnitrogen enrichmentalleviates the effectsof UVexposure. The narrowdistribution of D. verstuysiisuggests that this alga maybe an obligatereef flat dweller,and an alternativehypothesis is that UV protectionin D. veraiuysiiis constitutive,making the algainsensitive to changesin UVexposure. Before starting UV experiments,a ffeldexperiment was carried out to determinewhether D. versluysiiis a facultative or an obligate reef flat alga i. e., whether its narrow distributionis associatedwith intense herblVOryOn reef skeee, OrWith phyaiOIOgiCal reStriCtiana! ~ and to meaSurepatential and net gewth in O. versluyalland O.cavarnass over a depthgradient, MATERIALS AND METHODS ThalllIn herbivoreexclusion cages and control thalli were used to measurepotential and net growlhrates over a rangeof depthson a reefslope from the reefcrest to the lagoonfloor. Dlctyosphaerlavetsluysii thalii were collected from the windwardreef flat at CoconutIsland and allowedto acclimatein the fieklat 1 mdepth for 4 daysbefore the startof eachexperimental run. D. cavemosathalll were collected at 1 m reefflat!, and 2, 4, 6 and8 mbelow the reef flat on the slopeof a patchreef near Coconut Island, transplanted within 1 hourto the samedepths on the windwardCoconut Island reef slope, and allowed to acclimatefor 4 days.Acclimation periods wereintended to minimizethe affectsof tissuedamage during collection, and to reducevariabiNy amonginNal thallus growth rates and tissue nutnent levels. ThalIIwere kept in fiat cages cm high!to preventself-shading during acclimation periods. Followingacclimation, thsili of bothsf:ecies were grown at 5 depthson 1.5cm mesh vinyl- coatedmetal trays both within and outside of 8 rnmmesh herbivore exdusion cages. The 5 depthsused were 1 outernmf flat!, 2, 4, 6 and8 mbeiow the outer reef ffat at CoconutIsland; thereef IIat represents the approximate level of loweriow tides, and the water depth over the reef fiatat hightides is 0.5- 1rn. Twothalli were attached to eachtray outside the cage, and 1 or2 thalliwere attached inside the cage. Four to five replicatesets of cagedand uncaged thalli were usedat eachdepth. SpecNcgrowth rates g g-' d'! werecalculated from initial and final fresh weights,Three runs of theexperiment ware carried out from May to August1994. acne cavemoaa runs were 7 - 8 days Iong; D. venrluyaiiruns were 12-20 days iong to fadmat growthmeasurements in this slow-growing species. Irradiance PAR! was measured at thestudy site during the field experiment on 10July 1994 a submersiblesensor and a 4 pi collector BiosphericalQSI-140!, UV intensity was 182 measuredbyM. P. Lesser inspring of1991 atapproximately 1.5m intervalsfrom0.15 to 7.6 m depthalong a transeia10m fromthe study site, using a spectroradiometer ~ I I-1800UW! and a cosine collector. Laboratoryexperiments werecarried out in an outdoor culture system toexamine theeffects ofUV, ammonium enrichment, andinteractions between UV and ammonium onthe growth of Dicfyospheeriacsvernasa andD. verstuystr'. Thalllwere maintained inopaque white 3 liter containers,each holding 3 D. csvemoss thalli range 1-3 g wetweight! orthree D. veretuysNthalli range0,75 - 1.5g wetweight!. Each container wassupplied with flowing, 100 pm filtered seawaterend aeration. Neutral density filters were used to reduce pAR to approximately 4tyY of fullsun. Results from a preliminarylaboratory experiment indicated that growth inDid!!rospheeria cavemoseislight-saturated atthis irradiance level. Three levels ofUV were provided wllh PAR- transparentfilters; UY-opaque clearacrylic filters UVO!, UY-A-transparent Mylar@filters UVA!, andUV-A and -B-transparent AdarS filters UVT! see Gulko et el., this volume!. Thieve levels of ammoniumwere provided: ambient seawater, ambient +2.5 p,M, and ambient+5.0 gM. Ambient inorganicnitrogen levels dunng the experiment were0.38 Ii.M ammimium and0.44 p.M nitrate N = 3water samples!. Each of the treatments was replicated twice for each species. Thaliiwere continuously suppiied with ambient or ammoniumenrichedseawater during the experiment.A peristaltic pump was used to pump NH4CI solutions tonutrient-seawater mixing chambersupstream from the thalli containers. Fluorescein dyewes used to check for complete mixingbefore the nutrient solutions reached thecontainers. Seawater flowrates 00 rni min-'! weremaintained with headboxes, and both seawater and nutrient concentrate flow rates were checkedfrequently. The culture system was deaned every 3 daysand the pceitions of treatmentswith respect tothe headboxes were rotated tocontrol for posibon effects. Thalli were cleanedand weighed every 3 days.Specific growth rates g g 'd '!based on fresh weights were calculatedfor each 3-day interval. Plots of growth rate vs. elapsed time confirmed that Qrowih rateswere fairly stable at theend of experimentalruns. samaj!imForthe laboratory experiments, thelast set of growth increments calculated foreach experimentalrunwas used in analyses tominimize theeffects ofearly acclimation totreatments onlater growth rates. The mean growth rate of the three thalli in each culture container was treatedassingle data point. For the fieki experiments, growth nates were calculated using initial andfinal fresh weights. Factorial ANOVAs and Tukey multiple comparisons were used to comparedifferences among treatments. Where replicate experimental runswere carried out, runs were used as blocking variables. RESULTS Spectroradiometermeasurements fromspring of 1991 indicated that UV-A and UV-B 00- 400nm! was slrongly absorbed inthe first meter of seawater atthe study site; the UV intensity at 1.6m depthwas 6.9 W m-z,or 0.9% of the level just below the water surface. UV intensity wes a decreasinglogarithmic function of depth,and the relationship Inlz = Inl0 - K z!, wherelz andf0 are the UV intensities atdepth z andjust below the surface, respectively, was usedto calculatea UVextinction coefficient linear regression coefficient of In/zon depth! of K =<.53 rn-' R2= 0.85!.PAR was measured on a singledate 200 hr, 10July 1994! at the watersurface above the study site, at thereef crest .5 m! andat the5 depthsused for the experiments.PAR was a decreasinglogarithmic function of depth, ranging from 2023 pmol quantam- - e-'at the Surface tO293 p.mOI quanta m-2 s-' 4%,Of SurfaCe! at 8 m.The attenuationcoefficient, calculated as for UV intensity,was 4.38 m ' R2 = 0.88!, The growthof cagedNctp~haerf'a csvemosathalli decreased with increasing depth while the growthof uncagedthalli showed no clearpattern; growth rates withinall treatmentswere variable Fig. t!. Therewere signNcant main effects of depthand cagingon growthrates, but the Interac5onbetween caging and depth was notsignificant Table I!. Comparisonsof growthrates amongdepths indcated that thalli at 1 rndepth grew signNcantlyfaster than thosebelow 2 m Tukeytest, 0.025 > p > 0 05!. Tablel. FaCferfalanafyaie Cf Varlanee Cf DRtyeefehssrfecevemaaa gnvwln ratea in the fieldexperlrnent. Cage levels: caped and ~. Deplh levefa:1, 2, 4, S, end 8 m, 0.025 0.020 0.015 0,010 0 e 0.005 0.000 -0.010 10 Depth et! Figure1. GrOwlhratea g g rf ! ofOfef~phaantr oarsrmeaa thafiiin the fiefd experiment. Values Shewn aremeans ofS runs,with4 -5 rspgilostedtreatmentsper run. Error bars are ~ errors, Aswith Dfcfyospfraetfa csvernoss, the variabiiity of O. versluysii growth rates within treatmentswas high Fig. 2!. Growthrates of caged D. vsrsluysir thalli were significantly higher thanthose of uncaged thalli, but there was no effect of depth on growth rate, and the interaction behveencaging and depth was not signNcant Table ll!. Depth-averagedratesof tissue loss to herbivofy mean of all uncaged treatments! were nearlyequal for both species: 0.17% d-' in Oictyosphaerf'a cstremosa and 0.11'% d' inO, versluysii.Mowever, thedepth-averaged potential growth rate mean of all caged treatments! of D.cavemosa ,19% d-1! was much higher than in D, versfuysii .02% d '!. At 1 fndepth, wherethe ranges of the two species overlap, the potential growth rate of D.cavemosa was 17 timesthat of D. frersluysiinet D. catrerrtoss gfowth rate at 1 mdepth was 7 timesthat of D. veraluysfy Rgs. 1 and2!. TableII. Fechxtal~ Of variantOf OfsiyOSphaafe vWStbytergrowth nttes inthe tteld experiment. Cagelevele: end . Ievela:t 2 4 S snd8m. 0.0010 %.0010 0-0.0030 0 10 Oepth m! Rgureruns,2. Gteatthwtth 5 nateentptioatdtreatmnte gtr tr ! ofCtteteatpfteatie perrun, Error vvvstgerthellibent are slenchttl lnthe snore.Nekl expettment Values shawn are mesne OfS OIcfyosphaeriacavemass growth rates in ammonlu~richedseawater were nearly 10 limesthose in unenriched seawater Fig. 3!, confirming that growth in this species is nitrogen- limitedin Kane'oheBay. Bothammonium enrichment and UV level had signNcant effects on growthrate Tableill!. Therewas no ammonium by UVinteraction, however, indicating that the effectsof onefactor were not inhibited or enhanced by the other factor. Gmwth rates in boththe ambient+2.5 p.M and the ambient +5 AM ammonium treatments were signNcantly higher than the ambienttreatments Tukey tests, p c 0.001for eachcomparison!; differences between the two enrichmenttreatments were not significant. Growth rates in the UVO treatments were significantlyhigherthan inthe UVT treatments .025 < p< 0.05!;noother significant differences were detected among UV treatments. TableIII, FaCtorial enalyaia olvartance Ofgrcwfh rates SII d f!Of Ofotyeaphaenlacrhemeaafhalll inthe arnmcnium enrfohmentby uv leVelexperiment. 0.05 0,04 0.02 e 0.01 ~ e 3 0 Et 0,00 -0.01 UV-T UV-A UVO UV level Rgure3.Growth rates gg d ! ofol~raerfa cavemoM lhalliin the ammonium enrichment byuv ~ry experiment.Growth rates shown are for the final 2 daysofthe experiment andare means oftwo replicated treatments.Error bars are ~ errors. Growthrates of DictyospttaenaversfuyN in ammonium-enrichedseawater were 2.&4.5 timesthose in unenrichedseawater Fig. 4!, Indicatingthat, like D. cavemosft,O. versluysii growthis nitrogen-limited.Variability ofgrowth rates within treatments was greater in O. verstuysiithan in D. csvemosa Ammonium enrichment significantly affected O.versluysrr growth 187 rates,but there was nosignNcsnt effect of UV level,artd no ammoniumby UV interaction Table IV!. Comparisonsof treatmentmeans indicated that growth rates in the 2.5 lt.Mammonium ttaatmentswere sign~ higher than the ambienttreatments pukey test, 0.001 < p c 0.005!; no other signicant differenceswere detected. Table IV. Featettalahalttal ~ OlvU4hcs OI grOWlhretee g g 4 ! of DNjeSptraenlevendoyatl thalll ilhthe ammonium ~htitthmettt by Uv lect expettment, ch ~.OOe-3 2.00+4 I O.QOe+0 -2.00e-SUV-T UV-A UV lovel Gfowm%tea4 ShOwA i + ~~"atwtaatatOr the tthal ~g thattt 4ayeOtthe tattttatmeht th the ammonium ah4~ehttohmeht ~ ~ by~uy ~~ lata~ ~te Eh' betaare ~ chore. DISCUSSION Resultsof the field experiment suggest that herbivary and, on the upper reef slope - 3m!, depthaffect the grawth ofDictyasphaerie cavsvnosa, Although netgrowth was very low between 1 ande mdepth, this is a zoneof high D. caverns ccwer on many Kana'ohe Bay reef slopes. Thestudy site has no macroaigae within about 50 rn to etther side, however, and grazing intensity wasprobably higher there than at sites with abundant rrumoalgae. The interaction between cagingand depth was not significant, butthe sharp decline ingrowth rate at 2 mdepth is probablydue to severegrazing losses Fig. 1!. GrowthinDictyasphaeria cevernasa showed a negative relation toUV level in the laboratory experiment.Atell nitrogen levels, growth rates were lower InUVT treatments thanin UVO treatments.Thehypothesis thatnltragen enrichment alleviates thegrowth-suppressing effectsof UVexposure wasnot supported. Rather, thedifference between growth rates inUVO and UVT treatmentswas similar inall ammonium treatments .5-11 10+ g g' d'!. Resultsofthe laboratory experiment withDfctyasphaeria veraluysfi suggest thatgrowth inthis speciesisunaffected bythe level of UV exposure. However, thehypothesis thatinseesitivlty to UVis associated with physiological restriction toshallow, UV-rich environments wasnot supported.Protectian fromherbivory resulted inenhanced D.veraluysii growth, butchanges In depthdid not significantly affectgrowth rates. Apparently, D.verstu~i isnot an obligate reeffiat dwellerinKane'ohe Bay, but may be restricted tareef flats by high grazing pressure onreef slopes.D.verstuysii hasbeen collected inoffshore Hawaiian waters asdeep as 110 m F. Nonis,pere. comm.!, indeating thatthe species isnot physialogically restricted toshallow waters outsideofKana'ohe Bay. The observations thatgrowth inD. verstvyetr' isnitrogen-limited, and thatnet growth inthe fieid was not sustained below 2 rn depth, independent ofcaging, suggests analternative hypothesis: D.versluysii maybe restricted tareef flats by e higheravailability of dissolvedinorganic nitrogen DIN! there. Water column DINconcentrations aregenerally higher overthe reef flat near the study site than aver the reef slope near the study site reef fiat DIN: approximately1.3pM; reef slope DIN: 0.5 pM! Snidvongs & KInzie, 1994; Stimson etat ln press!.Inaddition toelevated nutrient concentrations, ratesofadvection ofnutrients maybe moreAlthoughrapid across growthshallow isnitrogen-limited reef fiats than indeeperDiciiyasphaeria reef slopes, cavemosaas well as in D. veraluysii, D. cevemosaisnot restricted toreef flats, and thelli growing onthe reef slope may therefore have a meansofacquiring nitrogen notshared withD. versfuysii. Stimson eta/. in press! suggested thatammonium excreted byinvertebrate epifauna onD. cavemosa thalli, and regenerated nitrate andammonium fromsediment patchee isolated bythalli previde nitrogen subsidies, which allow D.cavemoes togrow and persist onreef slopes with law water column nutrient levels. Thelow level of replication inthis study ancVor thevariability inherent ingrowth rate measurementsresulted inlarge error terms, and may have precluded thedetection ofsome significantdifferences amongtreatments. Tissuegrowth Isa campaelte processwhich integrates a largenumber ofcomponent pnamees eg.,photochemical reactions,carbon and nitrogen fixation,amino acid and protein synthesis!, andchanges inrates ofcoml:enent pn:eesses may confoundexperimental treatment effects ongrowth. Ina studyofthe effects ofUV exposure on theintroduced rhodophyte ~apaphycus strietum inKane'ohe Bay,Woad 989! did not detect aneffect ofUV exposure anthallus growth, butdid measure significant reduclions inchlorophyll a andcarotenoid concentrafians, andincreases inMAA concentrations inUV~xpaeed thalli,UV-B exposurehasalso been shown toIncrease respiration andreduce photosynthetc rate oxygen evolubonrate!in the macroscopic chiarophyte Prasiatscrispa in Antarctica Past& Larkum, 1993!.Measurement ofa variable whichise componentafgrowth, and upon which bothUV leveland nitrogen aveiiabiiity havedirect effects mayprovide moreinsight intothe relationship betweenUV and nitrogen than does tissue growth rate. LITERATURE CITED Bornman,J.F. 989!. Target sites ofUV-B radiation inphotosynthesis ofhigher plants, Pfrotocllrem.Phatobiat. 4: 145 - 158 Dunlap,W.C., Chalker, B.E. 8 Giver,J,K. 988!. Bathymetric adaptations ofreef-building coraisat Devise Reef, Great Barrier Reef, Australia. Ilt.UV-B absorbing compounds. J.Exp. N¹r. Blot. Ecol. 104: 239 - 248. Gaines,S.D. & Lubchenco,J. 982!. A unifiedapproach tomarine plant-herbivore interactions, II. Biogeography.Ann. Rev, Ecot. Syst. 13: 111 - 138. Hatcher,B.G, & Larkurn,A.W. D. 983!.An experimental analysis offactors controlling the standingcrop of the epiltthic algal community ona coralreef, J. &p. Mar.Blot. Ecol. 69: 61- 84. H¹y,M. E. 984a!. Predictable spatial escapes from herbivory: how do these affect the evolu5onofherbivore resistance lntroptcat marine cornmunt5esV Oecologia 54:396 - 407. Hay,M. E. 984b!. Patterns offfsh and urchin grazing oncoral reefs: Are previous resutts typicatf Ecology85: 446 - 454. Hay,M. E. 991!.I|eh-ewwItxxf interac5ons oncoral reefs: effects of herbivorousfishes and adaptattonsoftheir prey. la 'TheEcology ofFishes onCoral Reefs", edited by P. Sale, ActxhsrntcP~, SanDiego, pp. 96 - 119. Hughes,T.P. 994!. Catastrophes, phases shifts and large-scale degradation ofa Caribbean coral reef. Scvence285: 1547 - 1551. Hunter,C.L. & Evans,C,W. 994!. Reefs inKaneohe Bay, Hawaii: two centuries ofwestern influenceandtwo decadss ofdata. Proceerfr'ngs ofthe Colloquium onGlobal Aspects of GmdReehv Health, Hazanh and History. University ofMiami, Miami, Rorida. Jokisl,P. L. 980!. Solar uttravichet radiation and coral reef epifauna. S6ence 207: 1069- 1071, Jokiel,P.L. & York,R.W. 982!. Solar uttravioiet photobiology ofthe reef coral PocNlopora dsnxtcomtsand symbio5c zooxanthellae. Bull. Mar, Sci. 32: 301 - 315. Leaser,M.P. & Schick, J.M. 989!. Effects ofIrradiance andultraviolet radiation on photoadapta5oninthe zaoxanthellae ofAlptasla pallids: primary production, photoinhibition, andenzymatic defenses against oxygen toxicity, hfar.Biol. 102: 243 - 255. Lesser,M.P., Stochaj, W.R., Tapely, D.W. & Schick,J.M. 990!. Bleaching incoral reef anthozoans'.effects of irradiance, uitravtolet radtabon, and temperature onthe activities of protectiveenzymes against acthre oxygen. Coral Reefs 8: 225 - 232. UNer,M.M., LiNer, D.S. & Lapointe,8.E. 994!, Modiffc¹5on oftropical reef community strucluredueto culturat eutrophication: thesouthwest coast ofMar5nique. Proceedings of theSeventh Intsmatfonsl Coral Reef Symposium. Morrison,D. 988!, Comparing fishand urchin grazing inshallow anddeeper coral reef algal communNes. Ecology69: 1367 - 1382. Naim,O. 993.!Seasonal responses ofa fringing reefcommunity toeutrophica5on Reunion Island,western Indian Ocean!. lM¹r. Ecol. Prog. Ser. 99; 137 - 151. Post,A.& Larkum,A.W. D. 993!. UV-absorbing pigments, photosynthesis andUV exposure in Antarctica:comparison of terrestrialand marine algae. Aq. Bot. 45: 231 - 243. Scelfo,G. 985!, The effects ofvisible and ultraviolet sofarradiation ona UV-absorbing compoundand chlorophyll e ina Hawaiianzoanthid. proceeoring of tfre Flftfr International Coral Reef Symposrum6: 107 - 112. Scherer,S.,Chen, T.W. & Boger, P. 982!. A newUV-A/B protecting pigment inthe terrestrial cyanobacteriumNostoc comrrxIne. Plant Physio/. 88: 1055 - 1057. Shick,M., Lesser, M. P. & Stochai,W.R. 991!. Ultraviolet radiation and photooxidative stress inzooxanthellate anthozoa; the sea anemone Phytlodiscus sernoni and the actocoral C/svolariasp, Symbiosis10: 145- 173. Sivalingarn,P.M., lkawa, T., Yokohama, Y.& Nisizawa, K. 974!. Distribution ofa 334 UV- absorbing-substanceinalgae, with special regard ofits possible physiological rates,Hot. Msr. 17: 23 - 29, Smith,S. V., Kimrnerer, W,J., Laws, E, A., Brock, R. E. & Walsh,T.W. 961!.Kaneohe Bay sewagediversion experiment: perspectives onecosystem responses tonutrit'onaI perturbation.Pac. Sci. 35: 279- 402. Snidvongs,A.& Kinzie,R.A., III, 994!. Effects ofnitrogen and phosphorus enrichment onin vivosymbiotic zooxantheilae ofPociiiopora dsrnicomis. Man'ne Biotogy118; 705- 711. Stimson,J,, Lamed,S.T, 8 McDermitt,K. Seasonal growlh ofthe coral reef macroalgae Oicfyosphaeriacave/nose Forskal! Bsrgesen, and the effects ofnutrient availability, temperatureand he*ivory on growth rate. J. Exp. Mar. Biol. Ecol. In press. Stochaj,W.R., Dunlap, W.C., Shick, J.M, 994!. Two new UV-absorbing nrieceponne-like aminoacids from the sea anemone Anthopl'evra e/eganf/ssims andthe effects of zooxanthellaeandspectral irradiance onchemical composition andcontent. Mar. Biol. 118: 149- 156. Wood,W. F. 987!. Effect ofsolar ultra-violet radiation onthe kelp EaU'onis radiafa. A4ar. Biol 96: 143- 150. Wood,W. F. 989!.Photoadaptive responses ofthe tropicai red alga Evcheums sfristum Schmitz Gigartlnales! to ultra-violet radiation. Aq. Bof. 33: 41 - 51. 191 UkrevksetRedtsthn snd COralReefL 1fSS. D,Gvao t P.L. Jokiel ede.!. IIIMB Tech. Report sll. UNIIII-SesGrsntCR4I543, Phototoxicityin a coralreef flat commLIrtlty RltaL Peacheyf DonaldG. crosby avtmerrtofZooogy Univaslty ofHavvel'i 2538 The Mall «152 Edmondson, Handuh Hawai1 98822 20epartmentOfEnvtmnmental Ttadacfogy, Unheasfly OfC«NOmta, Davte,Ctdtfcmia 9381Se688 ABGITfbsCT;The effectofpolyolfcNc ~ hydnasatons fPAHs! and ultraviolet ~ UVR! wasfasted on coralref It«I . Onmnhmstiom 5animal phyla C~, Ann«Nda,Mosueca, Porifere, and~! ~ ~ ln eoiukcneofate prAH« ensV«Cene orpyrlyle «IOoncentralcra belowfhe Saturattcn ofthese compounds in ~, ~ by expoauretOuVR, Cnaeaoeana, pott~ andsane cridadaa sere ~ «I PAH ccstcenlralcnsandlight Inlensi5ea that~ occur inthek mef %«t habkah. These mrs:enlnssons ofPAHs dkl not ~ men«ayinthe abeam of UVR and UVR lsd nct Cause ~ inthe abeam OfpAH. pAH4ntksM photekatCfly ol maikaorgantsrrst vmsdegstnckrtt on5e ooncentr«son ofpAH, rtanssy and waeetent+ ofINR, and fascreni: efsnky; andOCCumed atPAH COIVO«ntrslione 3 tc4 ader« Ofmagnitude belowtl1«t reported for~ by PAHakne, PAH4ndaml I:haCSaxltitycouklbe enhandno thegeneral degnatalon Ofmaine aOrrvrtunllee ~ Laban areae. Theee elf«cat may be saaetbatsdbvincr««ant inUVh due to mxee ~. ok«etmoraslty ofocta@ taaaata and aga ~ mayhave tesapectadeffSCfa Onreef communa«a. The evtNetM elfaote are ake unkncwn. INTRODUCTION Phatotoxicityrefers to the harmful effects of solar radatien an atganisms. These effects can bethe direct result of sunlighton the tissues of anarganisfn eg. sunburn! or they can be chemicallymediated. Written records of the sun's ability to enhance the toxicity af certain chemicals indirect phototoxicity! goback over 3000 years. Coal tar and anthracene were shown in the 1890sto causeblisters on humanskin, uponiater exposure to sunlight,and to promote skincancer. Reports of indirectphatotaxicity to aquatic organisms are more novel. Anthracene photaloxicitywas first observed infreshwater fish by Bowling et ai. 983!. Subsequentresearch ona varietyof fnsshwaterorganisms, using anthf3cene and other pal!~clio arafnstic hydtocartens PAHs!, have demonstrated thatphototoxiciiy affects a varietyaffreshwater afganlsITls OIis et al., 1984; Allied 8 Geisy,1985; oris 5 Giesy1965, 1986, 1987; Kagan et aL, 1987;Oris et al.,1990; McCloskey tLOris, 1991!. There have been no reportsaf phototaxic effectsof PAHson marineorganisms except for the brine shrimp, Affernia salina Morgan tt Warshawsky,1977; Kagan et el, 1987!. Results of freshwater studies indicate that phatotoxic effectsare dependent on PAHconcentration inthe aquatic environment, UVR wavelength Ortis lf Giesy,1986!, and intensity of UVR Oris tL Giesy, 1985!. Previse studies in fresfyafater also determinedthat the synergistic effecls af lightand PAHs induce acute toxicity in organismsat concentrationshundreds to thousandsof timesbelow those reported for PAHalone {Oris et at., 1984!. PAHsare chemicals composed of 2 ormore fused benzene rings and are an importantand ubiquitousclass of pallutantchemicals in theenvironment. PAHs do occurnaturally, but their concentrationsin aquatic sediments are closely tied to anthropogenicfossil fuel utilization {Hites et fit.,1980!. PAHsare produced by many processes induding incineration of industrialand domesticwastes and the generation of powerfrom fossil fuels Neff,1979!; major sources of PAHs in the marine caastal environmentare municipalwaste, urban runaff, atmospheric precipitaten,and petroleum spills NRC,1985!. Waters in close proximity to lafgehuman populationshave high PAH content but these compounds are not distributed uniformly: sedimentshave the highestconcentrations, intermediate concentrations occur in aquatic arganisms,and the ~ concentrations arefound in thewater column {Neff, 1979!. Experimentalevidence indicates that acute toxicity of PAHsto marineorganisms occurs only at concentrationsseveral orders of magnitudehigher than those found in the mostheavily polluted marinewaters Neff, 1979!. Nthoughacute toxicity to marineorganisms occurs at high concentrations,relative to those found in the environment, PAHs are carcinogenic and chronic exposureposes a riskto the healthof aquaticorganisms Neff, 1979!. Thereef flat communityin Kane'oheBay, Hawsi'i is exposedta largeamounts of urbanrunoff, especiallyin thesouth basin which supports a humanpopulation af morethan 60,000 people US 193 DeperimentofCommerce, 1993!. PAHs in urban runoff are derived from vehicle drippings and exhauetamhnaae, aaphait wear and leaching, and wear Of vehele tireS Neff, 1979!. An addl5onalsource of PAHs to the south basin is sewage eNuent. PAHs are produced by cooking andwashing, and ate contained ln urine and feces. Historically, the south basin was subjected to largeinputs of sewage eNuent from circa 1935 to 1978 Maragos, 1972!, and although sewage is nowdirected to a deep-wateroutfall ~PAHs incorporated into anoxic sediments may persist on a gfxsioig5me scale Neff, 1979! Also, during periods of heavyrain faII, sewage spills into the southbasin of Kaneche Bay, e releasecompounded byPAHs entering the bay in runofffrom channelizedslraams. Corx~ra5cea ofPAHs inwater samples increases significantly after heavy mlnfaNand reports ofPAH concentra5ons inthe pg/I ppb! range are common for marine waters nearunpen areas Neff, 1979!. TrOpioalreef flat Organieme aISO are expOeed to high dceee Of SOlar UVR due to the tranegerentnature of tropical waters Jerfov, 1966, Kufner, 1995! and the shallow depths characterie5cofthki habitat. Reef liats are generally covered by less than 2 mof water and are aelfafiyexpOeed during neap lcw 5dee. Our inveetigation waeprOmpted bythe lack Of informa5On Onthe phCANOxIC eleCte Of PAHS Cn marine OrganiSmS andthe certain expoeure to PAHs and SOlarUVR that reef flat Organiems muStexperleri We pased the following queetions: 1!Are coralreef tlat organisms seneI5ve toPAH-induced phototoxicity, 2!does the phototoxic response dependonthe intensity orwavelength ofUVR, and 3! is the intensity ofambient solar radiation adequateto Inducephotottsdclty in reef ffat organlsmsf INATERIALSAND METHODS Thephototoxlc effects ofthe PAHs, anthracene ancipyrene, were tested onrepresentative speciesfromthe coral reef flat txmmunity, Thesepreliminary experiments weresimply designed todetermine Ifreef flat organisms aresensive toPAH-induced phototoxicity. Laboratory axpedmentsusingan ar5ffckd lightsource were performed withorganisms collected from wild populalcne.Thecogtx~n «Ite was along the east side of Coconut island locatecl lnKans'ohe Bay,O'ahu, Hawal'i. During experiments, larger-sized oqymisms werecontained in50-250 ml teakersand larvae were held Indepressions onporcelain culture plates. Stocksokj5ons contained 0.2g/I anthracsne orpyrene Aldrich Chemical Co.!in methanol. A eeifeeOfpAH CcnoentraNOne Waeprepared byinjeCting aliquOte OfStOCk SalutiOn belcw the water surfacewitha mlcrtwlpfnge and0, 16, 32, 48 pg/I PAH. The solubility ofanthracene inseawater 3o/oo 25o C!, 32 pg/f Whitehcvse, 1964!.Isconsiderably lowerthan that ofpyrene 5o/oo, 250 C!90 pg/I Roeei, 1981!. Thehlgheet concentra5on ofanthracene 8 pg/I!probably wasabove It'eeatura5on eolubity lnseawater butwas Included forosmfxsnsces oftoxicity between anthraceneandpyrane; thehigheet concentra5on ofpyrene was well below itssaturation point. Tocontrol forpossible tcadceffects ofmethanol, beakerscontaining 0 pg/IPAH were injected with theamount ofmethanol usedto carry PAH into the highest concentra5on inthe series. Each experimentconsistedof three sets ofeach concentration safes,two of which were exposed to UVR Series A andSeries S!and one was shielded fromthe light source ~ Series!with aluminumfo0.Experiments warepksced Intowaterbaths tomaintain temperature atthat of ambientseawater 4 -26' C!. Organisms wereIncubated lnthe seawstter/PAH salu5onfor2 hr beforeexposure toUVR, thesource ofwhich wasa GE" F40 Siacklight poei5oned 4.5cm above theplatform. Duringexposure toUVR the organisms wereobserved everyhour forup to 8 hr. UVRintensity waemeasured witha Spectroline ModelDM-365N lightmeter andranged between 975to1000 pWcm<. Moffle animals thatdid not respond togen5e prodding wereconsidered dead.Fornon-moliie organisms othercrfferta wereused todistinguish affectedorganisms, such as,discolora5on ofsponges and bleaching ofcorals. 194 Anthraceneand pyrene, show absorption peaks in the UV portion of the electromagnetic spectrum,Light sources with peak output inregions coindding with UV absorption byanthracene GE'F40 Blacklight! and pyrene Westinghouse" Sunlamp FS20! were selected totest the hypothesisthatphototoxclty isdependent onwavelength ofradiation available. The absorption spectrumofanthracene peaks in the long wavelength UVR region 35- 375nm!, whereas that of pyrenepeaks at shorter wavelengths 1 0- 335nm!. Arfemia salirra were selected forthis experimentbecause they have been shown to be susceptible tophototoxicity inprevious studies Morgan8,Warehawsky, 1977;Kagan efal., 1987!. One day old A. seNna were ircubated for2 h withanthracene or pyrene,irradiated with the long-wavelength UVlight source, and observecl after1 h. Anotherset of A.salina was incubated in anthraceneorpyrene and then irradiated with the short-wavelengthsource for comparison. Theeffects of UVintensity on phototoxicity were investigated using the same protocol describedabove, except that exposure to UVfrom an artificial source was replaced by exposure tosolar UV. The intensity ofambient solar UVR during experiments ranged behemn 407 irW cm 2 undercloud cover to 1428irW cm< under clear skies. To test the effects of radiationintensity, oneday old Artemia salina were treated to the same concentration series of pyreneor anthracene usedin aboveexperirnenis and then exposed tc ambientsolar radia5on, The control series wes coveredwith acrylic Plexiglas~ G UF-3!that is transparent tothe visible spectrum oflight but is opaque to UVR. Experimentswith a varietyoforganisms were conducted using the same series of PAH concentrationsand afnbient solar radiation tc investigatephototoxicity in a lightregime more doselyapproximating fieldconditions. These organisms included branch tips of the corals Morrtiporavena~a, Porites compressa, and PocNropora 6'amiccrmus, andkrlvae of Fungia scufaria.Other organisms tested included the anthozoan Zoerrfhus peciNcus, the sponge Cailr'spongiadNusa, the polychaete Platynereis dbmerrlii and the gastropod BiNium parcum The crustaceanspecies included zoea larvae of the alpheid shrimp Afpheopar's sp.,and the amphipod ArrrphNocuslikeNre. Beakers with larger organisms wire wrapped with aluminum foll and larvae, heldin porcelain culture dishes, were covered with UV-opaque acrylic to shield the control series fram UVR. RESULTS Branchtips of the corals Mcvdrpcra verrucesa and Pccillopora dsmiicomis, andthe gastropod Sittiumparcurrr, showed no observable phototoxic effect with pyrene and UVR. Larvae of the unidentifiedblack sponge had 100% mortality in atl treatments exposed to UV,indicating a direct phototoxiceffect of UV on these larvae. PAH-induced phototoxicity was observed with the amphipodAmphilocus likeNke. The percent mortality increased with increasing concentrations of pyrene Frg. 1!, while no mortality was observed inthe controls. The percent mortality ofthe polrrchaete Piafyrrerels dunMrnfN increased dramatically atthe highest concentration ol pyrene 8pg lite4 andranged from 90 to 100percent Rg. 2! withno mortality in thecontrols. Series A Series B Q Control 16 32 48 PyreneConcentration p,g/I! Rffle1. 8featef ptrrerre andUVR «r ere arnphipad, Arlylrarrerre Stertee. hmphif:Oda irrthe pyre' oorrceotrettorr SertaeAend S «rpeeedtOUVR, errqhlpode llrer COrrtrotSerNre rrrere ~ frOmuVR, Series A Series B 0 Control 16 32 «rdSerlee S terre ~~UvR~e to UVR; polychm«ef ~p«e trr the Cored ~~w~t,th,~~~~~~a Sertee wereahtetdad framOVR. 196 Arfemiasalina incubated inanthracene which has peak absorption inthe lan~velengfh UV!,showed higher mortality whenexposed tothe Iong-wavelength UVlight source thanthose exposedtothe short-wavelength UVlight source Fig.3!. A. salina incubated inpyrene which haspeak absorption inthe short-wave lengthUV!, showed higher mortality when exposed tothe short-wavelengthUVsource than when exposed tothe lang-wavelength UVsource. There was somemortality afA. salina controls during these experiments, butthe overall mortality ratewas verylow. These results support thehypathesis thattoxicity iswavelength-speciwc andcoincides withthe peak in the absorption spectrum ofthe PAH utilized. Mortalityofbrine shrimp was 100 percent inthe upper concentration ofanthracene orpyrene within1 hr after exposure taambient solar UVR FIg. 4!. At the same carx~trations afPAH in the laboratorymortality ranged between 0 and 70 percent Frg.3!. These results support the hypothesisthatphototoxicity dependson light intensity which overall was greater outside ona partlycloudy daythan inthe laboratory underlamps. Asthe intensity ofoutdoor gghtvaried greatly,theactual amount oftime for mortality taoccur ata particularlightintensity isunknown. Callisparrgr'adouse,a purple tubular sponge which iscommon onthe reef flat, showed no responsetoPAH under ambient solar UV, Similar results were obtained withthe black gastropod Bittiumpar@urn andthe zoanthid Zaerrfhus pacÃcus. Branch tips of the corals Manfr'pora verruccea,Pocillapara damicamis andPorifes campmssa showed various responses topyrene in ambientsolar UV. The day following treatment, M.verrucose tipswere bkMched inthe pyrene concentrationsof16, 32 and 48 pg/I in treatments exposed toUV; the contrai series appeared normal.Tips of P. darnicomis werebleached oniyin the 48 pgll concentrations exparredto UV andP. campressa tipsappeared normal inall cases. Larvae ofthe alpheicl shrimp, A~psis sp., showed100% mortality inanthrecene orpyrene atconcentrations of32 and 48 digit within 1 h af eXpoSuretOUV. Larvae Ofthe muehraOm coral,Fungla SCufarr'a, Showed 100% mortality inthe highestpyrene concentration within 1 h. DISCUSSION Thisis the first report ofPAH-induced phototaxicity intropical marine organisms. The phatotoxicresponse depends ona combinationaffactors: 1!the concentration ofPAH in the water,2! theintensity andwavelength ofUVR, and 3! the sensitivity ofthe organism. During theseexperiments, theauthors were careful tokeep the concentrations ofchemical andthe experimentallightconditons within those that can occur inthe reef flat environment. Acute toxicitywasobserved forsome organisms atccrncentratkxrs ofPAH as low as 16 pg I' ppb!under lightcanditions thatwere considerably lessintense than freld canditons. Results ofthese experimentssuggestthat phatotoxicity maybe occurring innatural systems andthat organisms in tropicalshallow water habitats areat high-risk dueto the transparency ofthe water and the high doseaf eohr UV to whichthey are exposed. Nefl's 979! compilation afacute ~ of PAHstofreshwater andmarine animals, shows timesta achieve a 50% mortality rate to be on the order of 18 hr ta >10 days. The time scale of our experimentswasquite brief and acute toxicity ta100% ofthe organisms exposed toPAH and amL'rientader radiation took less than 1 hrfor brine shrimp, coral larvae and shrimp larvae, This demonstratesthatthese chemicals become extremely toxic when combined with UVR. As PAH alonerequires concentratiorrs anthe order of mg I' ppm!for toxicity, toxic concentratiens onthe orderof IIg I' ppb!in our tests make the probability ofharm even more compelling. Long WavelengthUVR 100 16 32 48 16 32 48 Antbnemc Concentration Pyrcne Concentration ShortWavelength UVR Pyrene Concentration RgraeS,One rtay Ctl ASsmfa aetnalaNaeInCubaetd inIrrrene Oranlhracane, theneXpceed telang-Or ehrat~attength UVR. OCntmlEachgntphSeriee repreaente wee ahtetaed 8 pAHliOm~arattcn UVR. eerlae mgtitnwhtch SeieaA andSertea B were~ tn UVRandthe 80 40 20 16 32 48 >6 32 48 Anthraceoe Coacotttrtttioo Pyrene Ctmcentratioit Rgtltt4. Oneday Oki Atttrnitt oNeltt t«wte ttttttr ~Xtbtition m ptirorWor«tthracene ~ bv expc«usio«nMerX ttotttr . EOChgraph ~I 3 pAHattxentteSXt s«ice tngitj inwhich Sert«t Aard Settee B w«6expand io sdar ~ «td theConboi cork«wt«ekedttd tmrn the UV porrtcn ofItw spectnm with acrylic. Thevariation in sensitivily to phototoxiciiyoforganisms inthe same taxonomic dase is remarkable.The coiais exposed to ambientsolar UVR are a casein point Thecoral that is most commonin theshallow waters of Kana'ohe8ay, Pcrifes aorqpressa, wae not visiblyaffected by PAHat the concentrations used, while A4onfpora verrucose, which is generally found at greater depthswas bleached and Poci lapora damicomis, whch is aimost exdueively found on the reef flatalso bleached. This difference might be associated with the natural UV blockets found in many corals Dunlap and Chalker, 1988!. Larvae of themtishroom coral, Fungia acufaria, were affected evenin the lowest concentra5on of PAH, indicating that some larvae may be highlysensitive to phototoxicity. Phototoxicitymay have a greateraffect on the developmental stages of organisms,especially thosethat spend their earfy life in shallownearshors enNronmenls. High sensNivity of larvaecould negativelyimpact coral populations ofspecies that are not suscephbie tophototoxicity asadults. Fish larvaealso appear sensitive to phototoxicity.Freshwater fish thaI found shade in outdoorflume experimentswere able to survive,while those expceed tofull radiation were kilted Bowling etal., 1983!.Habitats that provide shade, such as mangroves, may function ae a refugefor tish and larvae thatmake their way to theseareas. These relationships are fertile ground for futureresearch. Theorganisms that were not affected by photoioxicily inthese experiments include sponges, gastropods,corals and anthozoans. Their resistance tophototoxicity may be a resultof an ability to metabolizePAHs at a higherrate than olher organisms, orthe possession of UVblockers that absorbor diffuse UV energy; most resistant organistrte were dark~otwd. The ability to resist photott:eicftywill favor the survival of resist speciesin urbanizedshallow water habitats and couldchange the composition and structure ofmarine commttnitiss inthese areas. In fact, such changesmay have already taken place, Acutetoxicity to marineorganisms occurs at txxtcentratiottsof PAH vnelI beiow the saturation in seawater,Further investigation into this phenomenon may lead to regulationand treatment of tunoff andsewage effluent to reducethe PAHcontent. Toxicologists Itave been concerned about PAHs in theaquatic environment fordecades, but the subiethaI effects obtained in paststudies with PAHs did notcompel managers to restrict these chemicals. The predicted increase in solarUVR due to ozone thinningmay compound thedINculty inesfimating sate levels of PAHs in the aquatic environment. ~99 LITERATURE CITED Bowling,J.W., Leversee, G.J. ~ Landrum, P.F. 8 Geisy,J.P. 983!. Acutemortality of anthracene- contaminatedfish exposedto sunlight. Aquat. 7oxicol.3: 79 - 90. Duniap,W.C. & Chalksr,B.E. 988!. Identificatioand quantification of near-UVabsorbing compounds S-320! in a hermatypicscleractinian. Coral Reefs5; 155 - 159. HItss,R.A., LsFIamme, R.E., Windsor, J.G. Jr., Farrington,J.W. & Euser,W.G. 980!. Polycyclican:xnafic hydrocarbonsin ananoxic sediment core from the PeffaquamscuttRiver RhodeIsland, U.S.A.!. eeocfiirir. Cosmocfifrn. Acta 44: 873. Jerlov,N.G. 968!. PNpectsof lightmeasurement inthe sea. Iir.'light asan EcologicalFactor', R. Bainbridge,G.C. Evans & O.Rackham eds.!, Blackwall Scientffic, Oxford, England. pp. 91 - 98. KaganJ., Kagan,E.D., Kagan, I.A. & Kagan,P.A. 987!. Dopoiycyclic aromatic hydrocarbons, acting as photosensltizers,paificipate in thetoxic effects of acidrain'l /rr.'Photochemistry of environinental aqLNsf systems',R. G, ZIka & W.J. Cooper eds.!. The American Chemical Society, Washington, D.C. pp. 191 - 204. MaragosJ.E. 972!. A studyof the eosiogyof Hawaiianreef corals. PhD.Thesis. Department of Ocearegraphy,University of Hawaii,Honolulu, Hawaii, 290pp. McCloskey,J.T. & Oils,J.T. 991!. Effectof wastertemperature and dissolved oxygen concentration on tf photo-Irducedtoxicity of anthracenetojuvenile bluegill sunfish Lepornis macrochiius!. Aquat. Taxfopf. 21; 145 ~ 158. NationalResearch Council 985!.Oil In the Sea: Inputs, Fates and Effects. National Academy Press, Washington,D.C. 601pp. Neff,J.M. 979!. PolycyclicAromatic Hydrocarbons in the AquaticEnvironment: Sources, Fates, and BiologicafEffects. AppliedScience Publishers Ltd., London. 282 pp. Oris,J.T., Geisy, J,P., Allred, P.M., Grant, D.F. & tandem,P.F. 984!. Photoinducedtoxicity of snthreceni in equaficorganisms: an environmentalperspective. Irr. 'The Biospere:Problems and Solutions',eNed by T.N.Veziroglu. EIsevier Science Publishers, Amsterdam. pp. 839- 658 Oils,J.T. & Giesy,Jr., J.P 985!. Thephotoenhanced toxicity of anthraceneto Juvenilesunfish Leiocnifs spp.!. Aquat. ToxicoL8: 133- 146. Oris,J.T. & Geisy,Jr., J.P. 988!. Photoinducedtoxicfiy of anthraceneto Juveniiebluegill sunfish Leipcttli. mscrocibirnrRafinesque!; photoperiod effects and predictivehazard evaluation. Environ. 7axicof. Chem 5: 761 -768. US Departmentof Commerce 993!. 1990Census of Popukition.General Population Ch~rfstfcs. Hawaii.U. S. Departmentof Commerce,Economics & Statistics,Bureau of the Census.Washington D. C p. 1. Whitehouse,B.G, 984!, Theeffects of temperatureand salinity on the aqueous solubility of polynucker aromatic hydnmarbons, hfar. Chem 14: 319 - 332. 200 recognizedto be associated withfish UV vision is feeding on plankton. UV sensitivity is known to improveplanktan capture by rainbowtrout and pumpkinseedsunfish Browman et al., 1994! and juvenileyellow perch are ableto feedunder UV illumination Loew et al., 1993!. Additionally,UV sensnve stages of salmonkfsare planktivores,while the adults prey on iarger items. UV sensitivitydoes not camecheaply to fish. UV radiationmay damage the retinaaf the fish, especiallythose active during day time in shallow waters. Indeed,numerous marine teleosis have UV-absorbingpigments in their lenses Dunlap et ai., 1 989;Douglas & Thorpe, 1992; Thorpe et sL, 1993!. These pigmentsare presumedto protectagainst UV damage Dunlap et al., 1989; Thorpe ef al., 1993!, though their existence in deep sea fish suggests other roles as we8 Douglas8 Thorpe,1992; Thorpe et ai., 1993!, Someof thesecompounds have been identified as micaspxfne-likeaminO aCids MAAs! Dunlap ef a!., 1989;Thorpe et al., 1993!which are asecc4atedwith UV protectionin other organisms. These pigments,each having a narrow absorptionspectrum, when combinedtogether can absorb light over the range of 320-395 nm. INVERTEBRATE UV VISION Amongaquatic invertebrates, UV sensitivityhas beendescribed in a limitednumber of SpfeleS,rnaet af them arthrapadS.A SingfeCaSe Of UV eenSIvity iS knOwnamong the rnoliuSCS, wherethe giantdam Trkfacnahas a receptordass with maximalresponse at 360 nrn VNIkens, 1964!. Crustmxaufswhich have photopigmentswith maximalabsorbance in the UV range include the fresh water Dsiphrriamttgna- ~ 348 nm Smith& Macagno,1990!, and the common prawn Palraemanefesvuiganls- X 390 nm Wald 8 Seldin, 1966!, the spiny lobster Panulirus argus- ~ 370 nm Cummlneet ai.,1984!, several species of mantisshrimp- X 325-340 nm Cronin et af., 1994!, and several speciesof deep sea crustaceans:tanicetta spinacauda-X 370 nm, Cpfcpherussphaeus and OplqphOruSyraC@raefrfe- X 350 nrn Frank & CaSe,1988!, and S!ete/Isspisder Frank& VAdder,1994!. Thephotoreceptors in the medianeye of the xiphcsuranLfmufus ~hemvs have a ~ of 360 nm Waid 8 Krainin, 1983!, though the role of this medianeye is nat fully understood. In additionto this 'true' UV sensitMty, some organisms such ss crayfish Cummins8 Goldsmith,1981! and crabs Martin8 Mote, 1962!have visual pigmentswith maximalabsarbance in the violet-blueregion. These pigments are expected to abseb and respond to near-UV light as wali. Several marineorganisms shaw sensitMty to UV light, but do not use this sensitivity for 'true vision', i.e. using light to imagethe externalworid. The salt water bacterium,Habbacferium hsllobi'um,disphqra antayonistic responses to UV illumination-X 260 and 370 nm and to visible light-~ 360 nm,although the mechanism of this responseis not well understood reviewed by Msnzel, 1979!. The sea anemone,Andhapfeura xarrttragrammea, retracts Its tentacles in responseto UV Illuminationp 360 nm!, but bendsthem in ref4ronseto visiblelight ~ 500 nm! Clark 8 fgmeldorf, 1970; Menzel, 1979!. UV VISION AND PCNARIZATION SENSITIVITY UV sensitivityis frequentlycoupled with sensitivityto parballylinearly polarizedlight. Several fish speckxt Haraei, 1985; Hawr!rsff!rn,1992! as well as man5sshrimps Marshall et al., 1991! use their UV phaloraceptorsto sense polarized light This polarizationsensitivity may be used for navigation,spatial orientation,and for detecfingspecwc objects, The underwaterlight fieid is partiallylinearly polarized with the orientationof palarizatiandepending on the timeof day, the depth,the op5catproperties of the water, and the angle of view Tyler, 1963!. Shiny objects, such as fish, reflect tightat cyecific orientatiore of polarization C~an 8 Pugh, 1991; Hawryshyn, 1992!,and depolarizingorganisms such as planktonmay seem conspicuousagainst a partially linear polarizedbackground Loew, pere.comm,!. However,the role of UV-baeedpolarization sensitivity is as yst unknown. UTIUTY OF UV VISION A simplereason for the existenceof UV visionis to expandthe spectralrange aver which animalscan obtain visual information Jacobs, 1992!. Haver, the existence of UV-protecting compoundsin the lenses af numerousfish suggeststhat maintainingUV vision may have some disadvantages,and therefore lhe benefitsto the animalsthat preserve it shouldbe considered, The highscattering of UV tightin seawater Lythgoe,1979! may create a bright 202 homogeneousbackground, mainly atthe direction ofSnell's window. Dark objects, such as plankton,and fish will be highlighted against such a background.Cronin et al., 994! demonstratedthisphenomenon where a schooloffish appear more noticeable when viewed againststrongly scattered UV light in a horizontaldirection, using a UV-tr3nsmi5ng butvisible! light-blockingfilter. Zooplankton, which are exposed tohigh UV radiation, maintain UV-absorbing compounds,such as MAAs, that are believed to serve as 'sun screens' and protect internal tissuefrom UV damage. These animals are transparent through the visual range but wII be conspicuouswhen viewed in the range in which these compounds absorb Loew, pere. comm,!. On theother hand, the high scattering of UV lighttends to blurthe image and make a UVimage- formingsystem less useful than one based on longer wavelengths McFarand, 1986!. Theinteraction between UV receptorsand one or morereceptors in thehuman visual range 00-700nm!, may form a multichannelmechanism capable of breaking carneuffage ordetecting differencein the incomingspectrum. Such systems commonly function by looking at the differenc,or contrast,betweerl the inputs. The complex visual system of stomaiapods,with their numerousvisual pigments, may funcfion insuch a ccimI:erativeway Cronin 8,Marshall, 1989!. UVvision can be usedto detectspecific patterns which are unseen, ar appeardifferentl, in thevisible range. Harosi and Hasimoto 983! demonstrated that fresh water Japanese dace Trfbolodonhalronenals! have UV absorbing cones - 1 350- 370nrn. The UV body coioration ofthese fish display strips which do not appear when viewed through a UV-blocking green filter Harosi,1985!. It isreasonable toassume that other animals have UV~lored patterns aswell. The roleof suchpatterns in communication isyet to beestablished. SensitivitytoUV illumination bydeep-sea crustaceans presents a most intriguing problem: lightat the depths in which they live moiethan 500 m duringthe day! is very limited and is presumedtocontain very limited amounts ofUV light. Therefore, one may ask, 'What uae is there forUV sensitivity foranimals living at these depths?" UV sensitivity inay be used for discrirrination betweenbioluminescence from different organisms Frank 8 Case,1988!. As four species of deepeea crustaceans that are known to Ixaisess UV sensitivity display diurnal verbcaI migrafion, Frankand Widder 994! suggested that UV sensitivity ispart of a tunnel mechanismusecl to identifychanges in the ambient light environment, set to trigger the vertical migra5on. AN ONGOING CHALLENGE At the currentlevel of knowkidge,it seemsthat a discussionon UV visionin the marine environmentpresents more questions than answers. McFarland8 Loew 994! presenteda keydifficulty which must be answered before any seriousecological discussion about marine UV vision can occur. we need to know which animals possessUV sensitivity. Atpresent, the number of marine species examined for sensitivity toUV lightis extremely small. However, itis likely that UV vision is present among coral reef fishes McFarfand,1991! and other species exposed toUV radiation. Therefore, aneffort needs to be madeto examinemore marine animal si:xa~, bothfish and invertebrates,so thatwe can havea basis for comparison. Alongwith looking atthe animal'a se~, deteikidmeasurements andimaging ofthe underwaterUV lightfield are required so that we can start looking for specnc information which is cernedby UV light. Better understanding ofthis information willenable us to isolate tasks that can beperformed through the use of UV vision. Behavioral studies can follow and examine whether these tasks are indeed performed, The associationof UV visionand polarization ssmitivity is notclear either. What is the unique propertyofthe short-end ofthe spectrum that has such importance topolarization vision? Hawryshyn pere. comrri.! suggests that the underwater UV lightfield undergoes fewer changes duringthe day, and was more stabs on an evoiutionaryscale, than other regions of the visible range.This stability Is important when polarization sensitivity isused for navigation. Direct measurementof thepolarized light field, at differentparts of thespectrum, are required for better understandingof the informationavailable from this type of sensitivity. Thequestion of therole af UV sensitivityin deepsea organisms, living in regionsdeficient in UV iight,is anything but answered. What characteristics ofthe light fiel triggervertical migration FranklL. Widder 1994!? Does UV sensitnvity serve as a depthgauge for vertical migrating animals Weld8 Rayport,1977!? Does the bioluminesrxinf spectrum of someanimals extend to the UV- violetregion, to enablediscrimination between animals Frank8 Case, 1988!? Detailed measurements along with behavioral studies are required to answer this enigma. 203 Goldsmith 990!cautioned thatit ia anthropomorphic andnaive to ask, "What does a UV leOSptOrdO"far anlinaiSV Nonetheleaa, puraulng anSWere fOrSpecific questionS, suchaa thOSe rafsedlnthis dleimssion, willenable better understanding ofthe role UV vision plays In the life of irtetrte aninsfs. ~ dgesiippcit byN8F giant 9IR 9817927. LITERATURECITEO Bicwmsn,H.l,, Nova!as-Rarniufqus, I. 8 Ha~, C.W, 994!, Ultravioletphotcreception contributes to preysearch behavior intwo ~ of zooplanidivcrousfishes.J. &p. Biol. 188:187-198. Clark,E.D. 8 Kimeldorf,D J. 970!. Tantara response ofthe eea anemone Anthcpleura xanlfiogremina tc UV end visible iadiatkm. Nsfure 227:f58-657. Camercn,D.A. d pugh,E,N. 991!,Double cones as a basisfor new type of polarization vision in wrtsblates. Natun~ 383:t 6t-184. Cronin,T.W. 8 Marahall,NJ. 989!. MuldpleSpectnd classes Cf phctorecefnors inthe retinas of gcnodactyiokfsstornatapcsfs crusbumans, J. Comp, PhysfoL A. 166:267-275. Crcnln,T,W Marshall, NJ., Qukin,C.A. 8 King,C.A. 994!. Ultravioletphotoreception in mantisshrimp, UnionRea 34 1!:1443-1452. Cummlns,D.R. S G~, T.H, 981!. CellularIdentiikm5cvi the violet receptor in the crayfisheye. J. Cotnp.PhyslcL 142:1$9-202. Cummins.D.R., Chen,D.M. 8 Goldsmith,T.H. 984!. Spectralsensitivity of the spinylobsier, Panulirus argus. BloLBufL 168:269-276. Dougbis,R.H. 8 Thcrpe,A. 992!. Short-waveabsorbing pigments In the ocularlenses of deep~ tsieosts. J. Mar. NcL Asa UK 72:93-112. Dunlap,W.C, Wlllktms,D.M., Chalker,B.E. 8 Banaszak,A.T. 989!. Biochemicalphotoadaptaicn in vision: U.V.- absorbingpigments in fisheye tkauie. Cond. Bilocihem.Phyabl, 93B !: 601~7. Reischman,E.M. 989!. The measurementand penetrationof u~ radiatcn intotropicai marine water, LlmnoLQceangr. 34:1623-1 629. Frank,T.M. 8 Case,J.F. 988!. Visuai~ sensitMtiss of biciuininesoentdeep-sea crustaceans. Biol, Bulf. 175:281-273. Frank,T.M. 8 ~, E.A. 994!, Evidencefor ~ral silastlvltytc nearUV lightln deepsea crustacieanSyefeIfaspis debls, Mer. Nol. 118:279-284. Goldsmith.T,H. 990!. Qpdmizadcn,constraint snd history in the evolution of eyes. Quar.Rev. BicI. 65:281-322. Harosl,F I 985!. Ultterloletsnd violet absorbing vertebrate visual pigments: dichroic and bleaching picperses.ln: Faki A. ~ LevineJ.S. eds.!The visual ~. AlanR. Ussinc. N.Y. pp. 41-55, HamslF I + Hashiinotc,Y. 983!, Ultravioletvisual pigments in a vertebrate:a teirachromatic cone syAm in the Dace. ~ 222:1021-1023. Haiosi,F I 8 Fukcrotami,K. $86!, Ccrrelaticn bebveen Cone ~noe andhOrizontal cell response from300 to 700nm in fish. Irrves.Opfiffje~ NsualScL 27:192. Hawiyshyn,C.W, I992!, Polarizationvision in fish. Am Scl 80:184-175. 204 Jacobs,G.H. 992!. Ultravioletvision in vertebrates.Amer. Zooi. 32;544-554. Jerlov,N.G. 968!. OpticatoceanOgraphy, Eleevier Publ, CO, Ametetdarn. Kunz,Y.W Wttdenburg, G.. Goodrich,L & Callaghan,E. 994!. Thefate of ultraviolet reosptors Inthe retinaof theAtlantic salmon Ssirno saiat!. Vision Res. 34 1!;1S75-1S83. Loew,E.R. & McFarland,W.N. 990!, Theunderwater visual environment. irr.Douglas, R,H., Djamgoz M.B.A. eds,! The visual system of fish. Chapman and Halt. London. pp f ~, Losw,E.R., McFarland, W.N., Mills E. & Hunter,D, 993!,A chromaticacfion spectrum forptantdontc predationbyjuvenile yellow perch, Pares Ifsvssr,ens. Can. J, Zoot. 71.384-386. Lythgoe,J.N. 979!,The ecology ofvision, Clsrendon press, Oxford. Marshall,N.J., land, M.F., King, C.W, & Cronin,T,W, 991!. The ccimpound eyes of mantle shrimps C~a, Hoplocarida,Stomatcpoda!- I. Compound eysstructure: thedetection otpolarized light. Phil. 7 rane. R. Soc. London 8 334:33-56. Martin,F,G. & Mote,M,l. 982!. Colorrsosptors ln marine crustaceans: a second spectral class of retinularcell in the compound eyes of Csfttnecfes andCarcinua J. Contp,PhyeibL 145:549-554. McFarland,W.N. 966!.Light in the sea- correlations withbehaviors cffishes and Invertebrates. Amer. Zooi. 26:38~1. McFarland,W,N. 991!, The visual world of coral reef fishes, in: Sale, P,F. sd.! The ecology offishes on coralreefs. Academic press. San Diego. pp, f &38. McFarland,W,N. & Loew,E.R. 983!. Wave produced changes inuis5erwater lightand their relations to vision. Envlmn.Biol. Fhh. 6:173-184. McFartand,W.k. & Loew,E.R. 994!. Ultraviolet vtsust pigrrsmts inmarine fishes of the family Pomacenbfdae.VArlon Rss. 34 1!:1393-1396. Msnzel~ R. 979!. Spectral sensitivity andcolor vision ininvertebrates. lrt:Hutrum, H. sd.! Cornparathre physiologyandevolution ofvision ininvertebrates VIMA.Springer-Veriag, N.Y.pp, 503-580. Robinson,J.,Schrnltt, E.A., Harosi, F.I., Recce, R.J. & Dowlhg,J.E. 993!. Zebrafhh ultraviolet visuai pigment:absorption spectrum, sequence, sndlocalization, Proc.Na8. Aced. Sci. USA 90:6009-6012. Smith,K,C. & Macagno,E.R. 990!. UV photoreceptors inthe compound eysof Daphnis magna Crustacea,Branchiopoda!. A fourthspectral dasscf ommatidia. J.Comp, Physiot. A.166;597-606. S~, D.G. 984!, Blue and uttraviobrt tightineyes: primary reactions andlight-induced metabolic changes,bzH, Senger ed.! Blue light effects inbtotogicat ~. Springer-Vsrtag.Beitin.pp,60-71. Thorpe,A.,Douglas, R,H.8 Truscolt,R.J.W, 993!. Spectrat transmission andshort-wave absorbing pigmentsinthe fish lens- I.phylogenetic distribution andidentity, Msion Res. 33 !:289-300, Tyler,J.E. 963!. Estimation cfper cent potartzatiim indeep oceanic water. J tt4sr. Res.21:102-109, Tyler,J.E. & Smith,R,C. 967!. Spectronsdtornetrc characbwh5csof natural light under water. Opt. Soc. Am. J. 57 !:595-601 Weld,G.& Krainin,J.M. 983!. The msdhn eyeof Lirnufus'. anultraviolet receptor. Proc.Hst. Aced. ScL USA. 50:1011-10f 7, Weld,G. & Rayport,S. 977!, Vision inannelisd worms. Sciisnce 196:1434-1439. Weld,G.& Seldin, E.B, 968!, Spectral sensNvtty ofthe common prawn Psiaernonetes vuigsrts.J. G. Pfrysrbi.5 f:694-700. 205 Wllkene,L.A. 984!. Ultraviolet seneitMty inhyperpolarlzing photcreceptore ofthe giant clam Tiidacna. Netwe 309:448448. 206 VrsvteeetRadiation Snd Carel Reefs. 1805. D,Guiko & P.L. Joklel eda !. HIMBTech. Relet fal. LINIHI-SeaGrant-CR-IM3. Poiartzationvision as a mechanismfor c}etectionof transparent objects NadavShsshar', Loans AddessP, and Thomas W. Cronin' ' DspaenentOfBidcgfhal Scfencre, Unieratty Of~, Cetcnadlkr,trID2122S DepartmentOfOceanogrrrphy, unnarndtyOf Haves'i at~ 1fNO paperOad, HcrekAu, Hl 9ftS22 ABSTFb4CT;Pdartzetion vfskrnmay be used by aqua% orgsrv'erne toImprove ~ of targets.Inthkr study |ve domcnetnrkrthatpoferizrakn vkicnCar imprOve both~ recCgnitkrnanddeteCSrm nerge.Target ~ range l~ byup to S2% for tnaneperent targe@,|Vhfch depC&artaa Ight,whIe oray by12- 1I%, fOr tsrgele wfhan inteneky conltgg.A similar improvement in~ range isIkely to rortaf tortnrnsparant organisms suchas zooptenkton, ,wtoae Issuesdepolarize IghtBehavtcnd studireare needed toassert that ~xr vkeonisused bypkrntdlvoroue orgenianw krenhance ~ d transparentpkmktcn h the water column, INTRODUCTION Variousmarine animals, such as cnmlaceans Ritz, 't 991; Sabra 8 Glantz,1985!, cephaloprgds Moody8 Parris, 1960! and fish Carneron & Pugh, 1993; Cameron 8 Easter, 1993! are sensitive tothe orientation ofthe e-vector oflight, or have some form of polarization vision. Although the recordofsuch polarized light-sensitive animals isincreasing, thefunction ofthis form ofvision is asyet largely unknown. Asin bees Rossel, 1993!, aid in navigation hasbeen suggested tobe an importantroleof polarization sensitivity andindeed grass shrimps useit to determine thedirection ofa shelter Ritz, 1991!. Octopus areable to distinguish between targets based onpolarization vision Moody & Pams,1960!. Contrast enhancement anddetection range increase have long beensuggested aspossibie roles for polarization vision. Detection range for ~ can be increasedbyabout 20% by using various polarization techniques Briggs 8.Hatcett, 1965; Lythgoe& Hemmings, 1967!. Itis conceivable thatdetection oftransparent targets willbe enhancedto an even greaterextent. Tosome extent, the underwater lightfield is linearly polarized. Partially linearly polarized light Canbe regarded aSa CcmbinatiOnOftwo StateS; onetOtally depalariZed Withthe intenSity ld,and theother fully linearly polarized withthe intensity Ip Kliger etal 1990!. The onentatkxt orangle cf polarizationisdefined asthe onentation ofthe e-vector ofthe linearly polarized component, from thepoint ofview of an observer looking atthe source oflight orreflection Kliger etal., 1990!, The intensityofthe two states, when measured using ananalyzer oriented perpendicuhr tothe orientationofthe e-vector ofthe linearly polarized slate, isequal toIQ. Whenexamined atthe orientationofthe e-vector ofthe polarized state, the intensity isequal tothe sum of the two states IQ+ Ip . Thetotal intenaity iS,thetafcre, I,= ld+Ip. The partial polarizaflOn iSdefined aSIp, and rangesfrom 0 to1 inclusive,where0 indicates unpolarized, and1 fully-polarized light Wotff, 1 890!,Theunderwater tightfield, especially inshallow water down to50 m! shows stfortg linear poiarizationinthe horizontal plane Tyler, 1963!. This strong polarization presents a distinct backgrOundfarany Object whiCh IelieCta OrtranSmitS lightthat iSpclarized ata different Orientation orthat is depolarized, Oinoflagellates areknown toinduce circular polarization oflight passing throughthem, and numerous otherspecies depolarize thetight asit passes through theirbody Rg.I!. Many plankton species afelargely transparent, andare therefore hardtodetect byvisual predators.Itis conceivable thatpolarization visionwillenable suchpredators tcimprove detection Oftheir transparent prey.This will be eapecially truewhen the baCkgrcund wateriSpckinzed ata givenorientation whilethe plankton iseither polarized atanother angle, has smaller partial polarization,oris completely depolarized. However, ithas not yet been shown that,indeed. polarizationvision can be usedin this fashion. Inthe current experiment wechecked the hypothesis thatpolarization vision can improve detectionof transparent,yet depolarized,targets. 207 Harrish A Charrtagnath Aiand a aXelarVa Is! viewed effete 'reSLrkrr' backSrrmtnatton rfghtI,and thmUtfh Steleat freiirendlcuar orlentascn lerare en osrer, Ner ptaosd rrnderneeth andone above the serntrts Irstti. The rterteedtry rare frstarta~ ster la erainSrrieheCI bythe Other. HOWher, the tteerree Oflha ~lieth actae depcrtartrere ~ ndtrerefore cd bedeeiiy rarrer. The corn does net affect the poi~ orientationandis, therm, notseen. Under 'reetrter'NNntireare, bOih trantfralt reemehare not cearly ~. MATERIALS AND METHODS Thisresearch wasconducted ina small mangrove channel onCoconut Island Moku o Lo'e!, Ksne'oheBay, O'ahu; ata waterdepth of 2.5 m. Sixtargets were viewed through a polarization analyzktgcamera. For each of these targets the characteristics ofthe light were analyzed and the maximaldetttction range based on black and white contrast and based on polarization contrast were determined. Targetswere 18.5 x 18.5cm, and made of tranSparent Plexiglas that does not affect the polarlza5onofthe light. Each target was divided into 1.8 cm wide stripes which were then altematlrrrslycovetedby black orwhite total~que tape, or a thintransparent plastic film that eelsas a depolarizer.Coating was such that the targets had alternating stripes ofeither black and white,bktck and transparent nocoating!, black and depohrizing transparent, whiteand transparent nocoating!, white and depohrizing transpara«, ordepolarizing transparent alternatingwith no coating. ThepcktlIZatian senSOruSed iS based Onthe design OfWOlff and ManCini 992! adapted fOr portabilityandIeid work and will be fully-described elsewhere. Tosummarize, thesensor consists oitwo twisted nematic liquid crystals TNLCS! of45' and 90 rolatian, anda linearpolarizer PoiamldHN38S, which hasa highandcorrstant extinction ccrefficient ratiothroughout the VISlblelartge! both pktCed infrOnt Ofa Smalldigital Camera Electrim EDC-1000 mOnochrOmatic digitalcamera with'I92 x 165GCD array! connetded toa portable personal computer PC!.The TNLCSrotatelight by 0, 45' or 90', by applying anAC currant atdesired times controlled bythe PC!and three images areacquired. Theseimages areused foron-line anaiyssrs anddisplay ofthe poktr4atkxtchantctenstics ofeach pixel inthe image. Our analyses program presents the IntensityImage "bktck andwhile' !,the partial polarization, andthe orientation ofpolarization, ona singleelement resolution, throughout theImage. Targetsandcamera wereplaced horizontally ata waterdepth of 2 m .5 m abovethebottom! wherethecamera lineof view was perpendicular tothe direction ofthe sun. All measurements weretaken behveen 10:00and 14:00 hours, when theIrclination ofthe sun was relatively high andunder cloudy sky providing a relatively constant iightfield. EaChtarget waa~ at 1 mdistanCe flemthe Camera, andthe image charaCteristiCS l«ensity,partialpclarizaticn, andorientatiOn Ofpolarizatian! werefeCOlded frcmthis Setting, TalgetaWerethen plaCed atinCreaSing distanceSfrom thecamera, andthe dietanCe atwhich they 208 disappeared,depending onmode of vision, was recorded. For each target two modes were used;intensity black and white! only and polarization including intensity!. Ineach case, the observerofthe output display was aware of the mode used, but not of wh!ch target was used nor atwhich range it was posNoned, until after f!nal decision ofdetection range was made. RESULTS Thenatural light field in the waler of this mangrove lagoon was found to be highly polarized. Theorientation ofpolarization was horizontal par3!k!I tothe water surface! and Ihs parbal polatfzstlortwas 0.35. Measurementsofthe characterist!cs of the light coming from the diNerent targets, placed at a distanceof 1 mfrom the camera, Table I!, showeda profounddifference btstween the diferent typesof coating, especially between the transparent and the depolatfz!ng setbngs. Even at the shortdistance of 1 m,a considerableamount of polarization evolved from the scattetirtg oftight by thewater. This shortdistance polarization amounted topartia! polarization of0.07 to 0.33, ckxte tothe background polarization ofthe water. H~r, onemust consider the possibility that a smalldegree of linear polarization was introduced bythe reflection oflight off the targets themselves.This may be the explanation forthe dNerence inthe average orientation of polarizationofsome of the targets from the background orientation ofpolarlzatiort. Targetdetection range based on intensity contrast was substantially dNetent from that based onpolarization contrast for each of the targets Tabie II!. Intargets which contained a strong internal intensity contrast, polarization analyses increased detectionrange by 12 to 14%. However, when the fully transparent target no cosling and a depalarizer!wasexamirted, polariZatiOn Ccntraet ana!yeeS inCnffeeed deteCtkSn range by 82%. TsbteI. Chttntctadattce ofthe ffpht ~ g ~ ~! meastntdfortargeie ptaoed at2 mdepth. ata ~ Ot 1m. Targets vwtre trttnepttnatt ~ nct ~ the palarizaffonotffta ffgM!, tsar x taSCm, tffvtdedintO 1 gcvn vide stripes. The etttpee were than aaanaffvahjr Cevend wffh blaCk Orwhite tcteffy4patOe tapeor wffh a thintnataparem ptaaffC amthat acta as a dspolertzsr,Thiecoaffng aeahd a paaarnOfalter black endwhtte, black and traneterant nocoettng!, btack and depharlzktg Oaneparent, whffsand tn~ rw ~!, whffaand tnatepttrent,ortransptrent dvCctarizhg artemeffnff wffhno ooaffng, hNenslly ts massontdon ao-255 coats. Orientation of Psrfiat Intensity polarization pcl8ftzatlcn - 1.0! Target type - 255! - 158! Back and white Biack stripe 8,5+2.8 N/A 0 White stripe 66.31 + 7.7 168 g7 0.13 g Rat!o B/t/t/ 0.13 0.02 Blaokand tnffnaparent Sack stripe 13,80 + 3.1 N/A 0 Trans. stnpe 42.00 <8.0 172 ~3 0.34 ~0.06 Ratio B/T 0,33 Blackand depolarizing Black stripe 14.25 + 2.3 N/A 0 Depol. stripe 53.67 + 5.7 17~ 0.23+0.04 Rat!o B/D 0.27 209 Tablel orat,I.. Orientation of Partial Intensity polarization polarization Targettype - 255! - 180o! - 1.0! Whiteand transparent Whitestripe 88.31~5.3 174g10 0. 07g0. 01 Trans.stripe 71.6~2. 9 Tg10 0. 14j-0.02 Ra5o W/I l.23 0.52 Whiteand depolarizing Whitestripe 57.2~.2 3~ 0,32g0.06 Depol.stripe 37.27+.2 167g5 0.3~.04 Ra5o W/D 1.53 0.94 Transparentand depolarizing 51.91'.0 1 7~2 0,32~.01 Trans.stripe 65.3~2.5 17~3 0. 22t0. 03 Depol.stripe 0.79 1.44 Ra5o T/D TatrteS,Target detSCttcn rangebaeert on~ COntreettetrt on pclartsaSOn ~ Trrrttets were~ a peiertsattonanattare Camera ats depthOfZ m.The natrrral tight ttekl at the hortmntal dtrecttrn, which eerNSd asbaraentond % the talgata, «aa 35% petartzetien ata hertzerltaiorlenlalicn. Targettype Detection range m! Ra5o Intensity Polarization Pdarization/ contrast contrast intensity! Sack and white 3.5 4.0 1.14 Blackand transparent 3.0 3.5 1,17 Blackand depolarizing 3.0 3.5 1.17 Whiteand transparent 3.0 3.5 1.17 Whiteand depdarizfng 2.5 2.8 1.12 Transparentand 1.65 3.0 1.82 depolarizing DISCUSSION Thepartial linear polarization ofthe light Said in sea water eels the stage for a polarization vktion-basedabject's detec5on andpossible recognl5on. Thispolarization is5nearfy polarized in thehoriziontal plane down to depths ofover 50 m whenviewed atthe horizontal parallel tothe surface!orientation. However, theorientation ofpolarization is expected tochange significantly whenobserved from other Clirections. Major changes can be expected tooccur at the edge of Snail'swindow, where light which is refractedas itenters the water from the air, is indose proximliytolight which isinternally refected from the water surface. Regions ofhigh partial polatfza5cn,orof changes inthe orientation ofpolarization ofthe ambient light, such as the edge ofSne5's window, are expected tobe the preferred background forpolariza5on-based target detec5on. Lylhgoeand Hemming 967! reported an increase of20% in target detec5on range when viewedthrough cross polarizing filters. Our results sustain this observation fortargets which containinternal contrast. For transparent targets which depolarize the light, the increase in detedionrange was much more significant, vpto 82%. Such an improvement indetection range islikely to be of great significance forboth predatory and prey organisms. 210 Polarizationvision is knownto beused for various tasks such as navigation, spatial onentatlon anytarget recognltlon. Our measurements demonstrate that pokttrization vision could be used to improvedetection of transparenttargets. Severalplanktivorous animals are known to posse+, polarizationvision. We propose that this sensing ability is usedfor better detection of transp+ planktonin the watercolumn. In the currentpaper we demtmstratedthe existent' for such improveddetection; we hopethat following studies wili show the actual use of lt,through behavioral studies. Wethank Mr, G. JOhnSOn and Or. L WOtfffor their inwttusbts rois in dtwetcptngthe fOtart~ camera,Mr, F, Tefor htahelp and ~p, Or,P, Jokistfor hta ~ ~ alongthis study, and Or. E. Lowefor usefuldIscuasiona. Weare greatful forsupport pOvkkb bythe Edwin W.Pauty FanarSOn exlths US ttaastt alefartcrWt SCtencsFoundatian. Ornelopmant elthe potsrtr.allen Camera wea augpertatf by MSF grant BIR Set 7927. LITERATURE CITED Bnggs, R.O. & Hatcett, G.L. 985!. Techniquesfor improvingunderwater visibility with video equipment, Qcean Science and Qcean Engineenng 1&2:1284-1308. Cameron D.A. & Pugh, E.N. Jr. 993!. Doublecones as a basisfor new type of pokttrizationvtttion in vertebrates. Nature 353:161-1 64. Carneron,D.A. & Easter,S.S. Jr. 993!, The conephotorecaplor mosaic af the greensunfish, Lepornis cyanellus. Visual Neuroeci. 10:375-384. Kliger,D.S. ~ LeWiS, J.W, & Randall,C.E. 990!. POilanzedlightinOpfioa and Spectroaccpy. Academic press. San Diego. U.S. Lythgoe, J.N. & Hemmings,C.C. 967!. Polarizedlight and underwatervision Nafure213:893- 894. Moody,M.F. 8 Parrls,J.R. 960!, Discriminationof polarizedlight by Octopus.Nature 188:839- 840. Rossel,S. 993!. Navigationby beesusing polarizedskylight, Comp. Sioctftefn. Physiol, A. 104 !:895-708. Ritz,D.A. 991!. Polarizedlight responses ln the shrtrrtp Palaernoneftss vufyans Say!. J, Ettp Mar. Biol Ecol. 154:245-250. Sabra,R. & Glantz,R.M. 985!. Polarizationsensitivity of crayfishphotoreceptors iscorrelated with theirtermination sites in the laminaganglionaris. J. Corrtp.Phystol. A. 156:315-318 Tyler,J. E.& Smith,R. C. 967!. Spectroradkometriccharecteristics of natural light under water. Opt. Soc. Am. J. 57 !:595-601. WoN,L.B. 990!. Polatizationbased material classification fmm specuiar retie~, IEEE Tr~ion onpattern analysis and machine intelligence PAMI! 12 1!:1059-1071. WoN,LB. & Mancinl,T.A. 992!. Liquidcrystal polarization camera. IEEE worlrsttrW on applicationsof computervision. Palm Springs, CA.: pp, 120-127. Uleelolet Iledlstlonsnd Coral Iteefe. 1NNN. D,Gubic 5 P.L. Jokiel erbr.!. HIMB Tech. Repel Net. UNIHI4es GiehtCR-N5-03. Designs for submersible imaging polarirneters NadavShashar', Thomas W, Cronin',George Johnson', and Lawrence B. Wolff' 'Deptof 'ologicalSrNenrs», University ofMaryla&, Baldmcee Ccestbf, C~Ne, MD2122$, 'Computer~ Dept.. TheJohns HcpktI» Unhrendty, Bakrinora, MD21 218. ABBTFIACT:Numerous tensed andmarine organlsrr» are ~ to parhaNyNnearly ~ Nght PLPL!.The natural NghtNeld in a largeportion of the water cobrmn isparNaNy lilearly ~, endmany abjech in water raNact Nght N»t br Otaatzedat epegffhoilrenNlor». ~ hrenens i»e ~ Nght, cur ~ tO eeeit NmbaOur understreMinqcfNs dbrtrtbuNon innatura and of the ~on itcarries. By ptacbtg twotwisted neman a fberdOdartsrra Niter in Seriesin front Of a CIDarmera,we have oonetructed pcrtabte polarlmetere that the charachrirNcsiaafPLPL in a IuNImstte, on a singbrpbcel barNs. The ~ image cwt bepresented aea ccNor insge wherehue rapiiaeents ortenbtttcn OfpCNSrtzrsion, Snd~ represents theperNal palaltsaNCe. We ~ here tvrO corrNgundionsofthe potarimelar, an autcrnomousisrnsor that ueee a armcordrerfcr raoonNngimages N»t are ~ st a hier stage;and an on4nesensor, that ress a dlglhlcrsnera ~ lo a pereorar~ which consoleencl ~ theiifixmsficn. Thecurrant polartmebrrs are NrrNted toN» visibleregion of thespecba. Hawser, their baric design Nr appNCebirtoa sensoroperalng in theUV region aS well. INTROOUCTION Likewavelength and intensity,polarization is sn intrinsicproperty of everylight beam. Althoughhumans make uss of the polarizationfeatures, we are unable to sensethem directly. However,numerous animals are sensitiveto the orientationof linearlypolarized light. Theserange from invertebrates,such as crustaceans Ritz, 1991;Sabra 8 Glantz,1985!, insects Phillipsborn& Labhart,1990; Rossei, 1993; Wehner, 1976! and cephalopods Moody 8 Parris, 1960!to vertebratessuch as fish Cameron8 Pugh,1991; Hawfyshyn, 1992; Camefort 8 Easter, 1993!,amphibians Auburn & Tylor,1979! and possiblyalso birds Phillips& Waldvogsl,1988; Philiips& Moore,1992!. This polarizationsensitivity is usedfor navigation Rossei, 1993; Wehner,1976!, spatial orientation Ritz, 1991;Hawryshyn, 1992!, and for detectingof kitpge bodies of water Schwind, 1991!. The underwaterlight 8eld is stronglypolarized down to considerabledepth Tyler,1963!. SuChStreng palariZatiOn COuld be uSedby a pOiafizatianbaSed deteCtian Or identifsmtiOn visual system.ThiS baCkgrOund pelarizatiOn ariSeS mainly from the ~ng af dowrnwrptlinglight We expectthe UV portion of the spectrum,which is highlyscattered, to beconsiderably polarized. Althoughwe are wellaware of the visualsystems of animalssensitive to polarizedhght, our currentunderstanding of the informationit caniesis very limited Hawryshyn,1992!. One reason for this lackof knowledgeis our inabilityto visualizethe differentcomponents of poianzedBght, namelyorientation, partial polarization named also percentpolarization or the "amountof polarization" WDN, 1990!!, and phasedelay or circularity.The currentdisr~ssion is limitedto partiallylinearly polarized light PLPL!,and thereforethe lastcomponent circularity! will not be considered, Partiallylinearly polarized light can be describedas a mixtureof twostates, one compkftefy depolarized,with an intensity of l,, andthe other fully linearly polarized having an intensity of Ip Kligeret at.,1990!. The angle cf polarization or the orientation of polarization!istherefore the orientation of the e-vectorof the polarized state. The intensity of the two, when examined throughan analyzeroriented PerPendicular to the orientationof Polarization,equals lef2, and whenexamined atthe orientation ofpolarization it equals Ief2+Ip, Therefore, the total intensity tr! equalsle+I,, The Partial Polarization is defined as I jl and rangesfrom 0 to1, where 0 indicates unpolarized,and 1 fully polarized iight. A methodof presentingPLPL which can be readilyunderstood was developedby WoN 990!. Theangle of polarizationis multiplied by 2 toachieve a 360'range! and according 'lo this value,it is assigneda falsecolor hue!on the 360' HVS hue,value, and saturabon! scale, The partialpolarizatiOn iS aSSigned tO Saturation on a 0-1scale, and the totalintercity Q iSssgigned tO value. Therefore,depolarized light will appearon a grayscale, while completely poiarized light prossessesa saturated hue which depends on the orientation of polarization. 213 MEASUREMENT TECHNIQUES Meamrlngthe ~eristics of par5afiyfinearly polarized tight involves quantifying three parameters:intensity, angle of polarizationand polartza5on. Naturally, this involvesthree separate measurements. For instance,imaging devices such as film or video cameras could be used with linearlypolarizing filters positioned at threeseparate orientations ln front of them. Designs sugIiested include using two or three cameras, each equipped with its own filter Lythgoe & Hemming,1967; Bernard & Wehner,1977!, or rota5nga filterin frontof a singlecamera Sriggs& Hatae, 1985!. Eachaf theseapproaches has particular advantages and disadvantages. Using threeseparate cameras permits simultaneous acquisition of all threeimages, but has inherent magnIca5on and parallax limitations. Additionally,precise corrections are requiredto ccmf,'ensatefor smallverlaine betweencameras. Rotating a filterin frontaf a singlecamera solves the parallaxproblem but introdems potentialdistortion due to surface inconsistenciesand tc a5gnment Ilmita5one of the filter. Further, since the three measurements are taken in sequence,there is a limitationin the ability to measurechanging events. The Introduction of twisted nematlc liquid crystals TNLCs! Wclff 8 Mancini, 1992! solved the inconsistenciescaused bythe filterrota5cn, as the sameeffect as filterrotation is achievedwithout the useof any moving parts. Heaver, the time limita5on,resulting from the fact that three measurementsare taken one atterthe other,ie stillan intrinsiclimitation of thistype of sensor. Twistednema5c liquid crystals TNLCs! have a helicalmolecular structure, twisting gradually fernone face cf thecrystal to theother, by a numberof degrees n! thatcan be controlled during manufacture. When a voltage is appliedacross the TNI C, the mciecules are stretched and the twistis slraightened.When the voltage is 0, themolecules return tc theirtwisted stage. The orlen515cnof polariza5onof light appliedacross the TNLC follows the molecularstructure and rotate by n degreeswhen the TNLCis relaxed,or doesnot changewhen it is stretched.Some of the advantagesof TNLCsare that they do nctchange the geometrlcaiarrangements of the Incomingimage, and that they transmit light across a wide-spectrum.They are firnited, h~r, in the 5rne requiredfor change in position stretch or relaxation!. In the device described here we usedfast TNLCs with a switching5me of 18 me, By applyingtwo TNLCs in seriesin front of a linear p~ng filter,cne can obtain the same infoima5on as if thepolarizer was rotaled to asmany as fourdlfferent paeitions, without having any mechanicalmotion, Only three orientations are neededfor measurementof PLPL; we chose to use the 0', 45' and 90' orienta5ons. THE PM4RIZATION SENSORS Thedevices described here are based on thedesign of Wolffand Mancini 992! adaptedfor portabilityand field work underwater. Two models are described: a totallyautonomous device, basedon a carnamfsr,where data are collected in the field,but analysis Is conductedas a separatestage, and an on -inedigital sensor connected by cableto a personalcomputer. Theautonomous senex consistsof two TNLCsof 45' and90' rotation,and a linear polarizer Polaroid |8 HN38S,cap>Ale of pakartzinglight at wavelengthsfrom 400 to 700 nm,with a highand constant ex5nc5on coefficient across this range!both placed in frontof a YaehicaKX-V1 carrvmrder FIg. 1!. Imagesare tapedon Hl4 video fcrrntd, and the state of the TNLCs is Independen5yrecorded by piecingsmall potanzlng fiftere In the fieldof viewof the camera, ~ed to the sensor'sunderwater housing. Sequences ol individualfields of imagesfrom the threedesired orienta5ons, are tntnsferred,as 320 H! X 240 V! pixeis,2~, colorimages, througha VideoSpi~ framegrabber board, to a personalcomputer for analysis.Each pixel in the frameis analyzedseparately. This sensor has the poten5eito acquirefully-analyzed images at onethird of the videorate or 10 Hz,but in mostfield work it is usedat 5 Hzor slower,to insurethat at leastone fufi frame is availableat eachpolarizs5on position. Once transferred to the computer, eachcolor channel can be analyzedseparately, providing an eetirna5onof changesin polarization between different parts of the spectrum,or they can be recombinedto create an 8-bit black and white image, and provide informationon the over all light field. 214 rnaurhsd@rough a ppwsr«xi abolishIiw ~ be14Ny ~ala hler n this case digital camera pixekt tween resolution rdingto the unch box' type false color ra as well as The video sensor based tween the person ADJ A growingportionof the literature shows thatvision inthe ultraviolet UV!range 800 - 400 nm!isclosely related tosensitivity toPLPL Wehner, 1976;H~yn, 1992!.Furler, the ch~rfsticsof PLPL are expected tochange exerting tothe wavelength observed, and shouldbeespecially strongin the short end ofthe spectrum, Therefore, wehave a greatinterest indesigning a polarization sensorthatwill operate inthis range. Adjusting thecurrent sensors for workinthe UV range means, infact, constructing a newsensor dedicated specifically tothis task. Thechanges required arein several components ofthe sensor, theTNLCs, thesensing device, andthe housing ofthe sensor. The TNLCs should beadjusted formaximal rotation ofthe light at about350 nrn, and shouid beput between quartz plates curreritty, theyare set between glass plates!thatwilt not absorb thetight. The polarizing filterused with lhe TNLCs should have a high extinctioncoefficient inthe UV range, such as Polaroid's HNP'8 filter. The sensing device itself shouldbedesigned specifically forworking inthe UV range 00 - 400nrn!. This will require using a UV-sensitivecamera, such as a chargeinjected device CID! based camera, with lenses that 21' transmitin the UV range, and proper filters that vill blockthe longer end of thespectrum. The housingof thecamera should contain a UVtransparent window through which the light can penetrateto thesensor. However, the analysis and control software currently used can be useabtefor the UV sensor. Thoughthe adjustmentsare substantial,the informationexpected to be gatheredis wellworth the effort. Rgure2, A dktertCemtrre4erred, on4ne, poterlzslkrn ~, ThetWO TttLCe end e» ~ faer ere~ in frontof en teCttttnRDC-1000 monechromrdlc tkgkel Camera. The pateCehg ttnd ~ of theTttLCe enr controfted bya 'lunch berrtype, 488OX2, ~ oonputer Ihreughe ~ cfroufLEight-btt imtOee, 192 H!X 185 V! pixekr,are intnetrrrredto dieccetputer where Ihe pehrlztdkxt chenrckrrtrrScs ere ~ end ~ FURTHER RESEARCH AND DEVELOPMENT Numerousanimals are sensitiveto linearlypolarized light and use it for varioustasks. Dueto ourinability to visualizethe distltbution of PLPL, our understanding ofthese tasks is very limited. Ourimaging pOlalimetere ate alreadybeing ueed in the field tO meaaure the natural polarized light 5eld,and the light reflected from objects. We expectthese measurements to provide an insight to animalpolarization vision. As an add t onalstep, we hopeto adaptthe sensorsto work in the UV 00 - 400 nm!range of the spectrum. Thepresent paper describes a designof a fie d-operationasubmersible, , imaging polerimeter.We believethat ourdesign can be usedin otherfield-oriented studies. We expect thismethod of studyingand presentingPLPi to open a windowto aspectsof the visualworld, cunsntly obscured from our eyes. . We thenkthe Nelor»l Ar3uerturnIn BelImore, Ihe AuetrttlkrnNrdtonrd Museum, Uzard fektnd ~ Strrtkrn, endthe Hawei'ikteau» fortrkrrkte tkctcgy Ior ~ teCelk» end~ during drWefolmsntof theeeeenecre, TNe etutkr wrrs euppcried byNSF grant Blf8317927, enrf ARPA ~ F30IIO?-92-G4191. Reklwork witeeuppcrted bythe U.g. - krraelBinrrsor»I adence Feundelon, the Amerlcen Mueeum of Netund Hletery, the pAOI IorItrSdion,end the Edwin W, FttukryFaude&n. 216 RATURE CITED m,J.S. & Taylor,D.J. 979!. Polarized lightsensitivity andorientation inlarval bullfrogs Ranacatasbeiana. Anim. Baba v. 27: 658 - 688. ard,G.D. & WehnerR. 977!, Functional similaritiesbetween polarization visionandcolor vision.Vision Res. 17: 1019- 1028. ggs,R.O. & katcett,G.L. 965!. Techniques forimproving underwatervisiibility withvideo equipment.OceanScience anrlOcean Enginaenng 1&2:1284 - 1308. ameron,D.A.& Pugh, E.N Jr. 991!. Double cones asa basisfornew type ofpolarization sionin vertebrates. Nature 353: 181 - 164, eron,D.A, & Easter,S.S.Jr. 993!, The cane photoreceptor mosaicofthe green sunfish, Lepomiscyena/ us,Visual Naurosci 10:375 - 364. hyn,C. 992!. Polarization visioninfish, Am. Sci. 60: 164- 175. r,D.S., Lewis, J.W. & Randall,C.E. 990!. Polarized lightin optics andspectroscopy. AcademicPress, San Diego,CA. hgoe,J.N.& Hemming,C.C. 967!. Polarized lightandunderwater vision.Nature 213:893- 894.Moody, M.F,,Pams, J.R. 980!, Discrimination otpolarized light byOcfr:ipus. hafure 186: 639 - 840. lips,J.B.& Waidvogel,J.A. 988!. Celestial polarizedlight patterns asa catibrsbon reference forsun compass ofhorning pigeons. J,7heor. Biol. 131: 55-67. lips,J.B. & Moore,F.R. 992!. Calibration ofthe sun compass bysunset polarized light patternsina migratory bird.Bshav. Ecol. Scciofsoi. 3'I;169 - 193. llipsborn,A.& Labhart, T. 990!. A behavkxalstudyofpolarization visioninthe fly Musca domseflca.J. Comp. Physiol. A 167;737- 743. Ritz,D,A. 991!. Polarized lightresponses inthe shrimp Palaere~tes vuigans Say!. J. &p. Mar. Biol.Ecol. 154: 245- 250. Rossel,S. 993!. Navigation bybees using polarized skylight. Contp.Biocbem. Pliysiol.A 104 !: 895 - 708. Sabra,R.& Glantz,R.M. 965!. Polarization scnsitivity of crayfish photoreceptors iscorrelated withtheir termination sitesinthe lamina ganglionaris. J.Camp. Physio/. A 158: 315- 318. Schwind,R. 991 !. Polarization visioninwater insects andinsects livingon moist substrate. J. Comp.Physiot. A 169:531 - 540. Tyler,J.E. 963!. Estimation ofper cent polarization indeep oceanic water. J. Ntar. Res.21: 102 - 109, Wehner,R. 976!, Polarized-light navigation byinsects. Sci.Arn 238 !: 106 - 115, 217 WoN, LB. 990!. Polarizatke based material ciassNcation from specular reflection. IEEE Transaction on pattern analysis and machine intelligence PAMI! 12 i!.' 1059 - 1071. WoN, LB. & Mancini,T.A. 992!. Uquid crystal polarizationcamera. IEEE workshopon applicationsof computervLsion Palm Springs,CA pp 120- 127 0 orrtototftorti40On 4nrt COret f44444. tNNN. D. GrrlkoN P. L JaktrrlIertrr.!. Htlls Tech Report44L UNIHI-84rrGrr~xt, Ultravioiet imagery G. Losey',C. W. Ha, W.N. McFatfand',E.R. Lowe', T. W. Cronin', D. Roni' 'HrnrrrN'II of Marlr»BIOIOIBr, P.O, BOM 1348, K4rr»'ohe, Hl ft6744 'Urrivtrnrthrof ~, P.O.Box 1700, ~ BC Cer»chVsw 2Y2 ~lP K.Wrfsfrry IMarlr» Bofenrxr Canter atCatttNna, P.O.Box 394L AvatOn, CA90704 Of, CoNrraeofVetrtnery MeCNdr», COmtN Unhrtrretty, ~ NY 44BNS 4~4nt Of Sctonor»,' Of~ BsNfmcrs Caunly,5401 WB»r» AVe., tNttmOnr. Mo 2422S 'OplcrtfOr ~, P.O.BOx 82, ~, hkl 07%6 The recentdiscovery of possiblywide-spread ultraviolet-sensitive vision in marineanimals castsa newlight on the need for research on ultravioletradiation. Weiler 993! oufiinesthe resultsof ultravioletradiation workshops dating back to 1985.Throughout her summary, the centralbiological theme is Defenseagainst damage caused by UV-Aand UV-8 radiation. In the currentworkshop, Dr. David Mauzerafi cast a somewhattfifferent h~ viewon the possible importanceof UVradiation in theorigin of life. Thebiological entities that today must defend againstdamage from UV radiation may owe their very existence tothis same radialion asan energy sourcefor the originof complexorganic molecules. Recognition of the potentialimportance of UV visionin marineanimals indicates a presently beneficialinfiuence of UVradiation; various madneanimals have been selected for visualsensitirvity to the sameUV radiation that has led to defensivemechanisms in othersystems, This section of the workshopexplores our current knowledgeregarding the occurrenceand functionof UV vision,the physicalparameters of UV radiationthat mustbe exploredand the engineeringspecifications for the equipmentthat is required to study these systems. PRESENCEOF ULTRAVIOLETVISUAL SENSITIVITYIN CORALREEF FISHES Dr, WilliamMcFarland reviewed the biophysicsof mcrnochromeand color vision in animahand gavespecial attention to themarine environrnerrL Signal detection theory is especially important in consideringthe degree to whichthe absoq4on spectra of visualpigments match the spectra thatare available in thebackground of the visual field and in therefiection from the object to be detected. Given this background,the potential imptxtanceof UV vision is seen as far more than justan abilityto detectthe coloration of an object.We must consider the UV absorption patterns of objectsin themarine environment along with the nature of thespectral distribution of lightin the environmentand the sensorysensitivity to afiportions of the spectrum, OVerthe Iaat deCade the preeence Of uIIraVIOkNt Viaual SenSitivity invariOue vertebrateS haS beendemonstrated by behavioraltests. The basis of UVse silfvityhas been corrhrfned by spectrophotometricanalysis, which has revealeda classof single-conephotoreceptor cells that containvisual pigment that absorb light maximally between 350 and 400 nm. UVvision in birdsis usedin navigationand, in hummingbirdsfor example,as a uniquevisual means of guidinga pollinatorto a flower'snectar nectarguides!. In lizardsUV vision is usedin courtshipdisplays Fleishman,Loew 8 Leal,1994!. In somefresh water fishes UV vision enhances the contrastof zooplankton I oewef al., 1993!,and in goldfish Hawryshyn8 McFaffand,1987! and salmonids Hawryshyn,1992! it providesinformation about polarizafion fields that ts usefulin orientationand navigation. It was recentlyshown that somecoral reeffishes also possess this uniquevisual capacity McFarland8 Loew,1994!. Damsel fishes, which are highly territorial, display distinct courtship rituals,and lay demersal eggs, have visual pigments that absorb near 360 nm. Thebehavioral functionof thesevisual pigments, h~r, is unclear.possibilities include foraging, mate recognitiondue to 'unseen'UV markers, agonistic display signals, UV-polarization detecfion for contrastenhancement and, perhaps, for orientationand navigationalmovements within the reef communIIy.It is likelythat this unusual visual abiiity is widespreadamong reef fishes, and especlafiyso, becauseUV-A light penetrates in coralenvironments to considerabledepths 0 or mOremeters! and at a higheneugh intenaity to proVfdereaders OpbCgSIgnaIS MCFatfand, 1985' Loew& MCFariand,1990!. It is important,therefore, to Visugize'how coral ecosystems apPear In near-UVlight. This can be achieved mastefffcienfiy bythe use of a UV-sensitive,video-based imagingsystem. DrCraig Hawr'lrshI~ continued thereview ofthe broad spectrum ofevidence forUV vision in aquadcandterrestrial vertebrates eeeVisiari Research voiume 34¹11, 1994 for special issue on UVreception lnanimals!, Fewstudies have emphasized thecharacteristics anddynamic features ofUV phab~ceptlon, butthere issame limited information onthe performance charecterisfics of UVCone phatoreceptors lnrelation ta the other cone photareceptors ofvertebrates. For instance,Hawryshyn 992! examined thelight adaptation ofcone photoreceptors overa broad rangeafambient intensity. From this data, we can extrapolate tathe photic regimes under which thesecone photoreceptors wouldnormally operate and hence understand theconditions suitablefor visual behaviors, Our liinited knowledge suggests that UV cones have more in cornmoriwith the rods than the other cone photoreceptars interms of the intensities ofambient lightunder which the cones operate dynamic range tuned totwilight condNons!. Theadvantage ofhaving such data is that it helps Inanswering questions related tahow the receptors may be employedbythe organism togather informatjan vIIalto guiding their behavior. Another such studybyH~yn andMcFariand 9B7! examined theresponse offish to plane-polarized light. Thisstudy aswell as others see Hawryshyn, 1992for review! have brought tolight the complex natureaf how vertebrates detect and pracess plane-polarized iight. Various studies have shown thatthe cones respond differen5ally tathe plane qf palarization andthat this may be usedto enharcei:xxitrast of the images,but directevidence to supportthis contentionis not forthcoming.The role af UV-polarizedsan~ in orientationand navigation has been establishedinthe laboratory, but open acean studies are still in the planning stages, Havrrifshyn'sgrouphas ident%el two main research areas ta pursue: ! Optical signaling: We haveliterally no knowledge ofintraspecwc andinterspecific modes of visual communication inthe ultnwrialetspectrum, For instance, current studies of opticalsignaling in the deanerwrawl addresssignals that are displayed bythe cleaner wrasse that evoke a posingbehavior inthe host spades.In recent UV video photography during a post-workshopsession Dr, E.R. Loew!, it was dearthat juvenile and adult @caner viissees had marked differences in the pattern of UV reflectance. ! UV polarizatkxi sensNvtty may pisy a raisin guiding orientation movements of fishesan and off the reef especkiily during dusk and dawn, a periodof greatest polarization inthe lightflekl. Whole-field Imaging ofIx~tlan patterns,ora "fish'sview' of the polarizatio field in the UVand 'visible" spectrum as plannedfor the UV imagingsystem, would enable a much deeperunderstanding ofthe passible Importance ofthis capability. SPECIFICATIONSFOR A VIDEO-BASEDUNDERWATER IMAGING SYSTEM WhatIs there to seeIn the near-UV visual world of marine animals7 Dr. Ellis Laew summarized thecorclusians of theparticipants as to the type of equipmentthat is needed to answerthis quei0on.There are three visual mechanisms thatmust be considered. Rrst is simple luminosity; thatIs, are UV images coded as gray-scalewithout the potential for using the UV informationas partaf a colorvision scheme. Oh~sly, colorvision is thesecond potential use for UV information.Here, one must consider whether there is enoughdifferentia reflection of UV for hue discrimlnationin thisspectral region ta be useful,Lastly, there is piWrizationprocessing of imagesinthe UV, Anycamera system ta be designed shauld be capable af pravidinginformation in all three of these areas. Zhalen; Thisis tie mastImportant part of any UV imaging system and aLso the one item that is notcurrently available'off-the-shelf. The ideallens would be almostiderriical to the zoomsystems on personal camcorders 30 to 80 rnmmotorized zoom, manual- and ~ris, and macroring. Unfortunately, to obtainsuch a lenswith transparencyinto the near-UVis riotredly feasibleand would be a researchpraIect in itself.Rather, a numberofC-mount, quartz achnxTlatlc leilses of differeilt focal lengthand corrected for the 300 to 400 Arrl region can be turret mounted with same kind of 220 remoteswitching mechanism. The300 to 400 nm correction region for optimizatlori ofthe lenses isspecNed forvisual system reseaich. However, ifcoatings areto be used they should bevery broad-bandsothat images couLd still be obtained inthe shorter UV regkxis for use in other researchareas. The faster the lenses, the better, as the amount ofUV Light isnot great given the sensNivityofmost unintensified cameias. A lower limN Lsprobably about f/4. Theeasiest way to focus the system would beto adjust the detector-to-lens distancewNh a linearmotor or stepper. Many cameras used for remote sensing already have this capabiwty. Trialswith Dr. Loew's current underwater UV-sensitive camera demonstrated theneed for a highly-corrected,multi-Lenasystem. Hispresent camera, spedalizad forvtsualizafion of zooplanktoninthe UV, uses an'off-the-shell' quartzmeniscus lens.This camera iswell-suited for itsintended task, but could not provide images ofthe quality required forfield study ofreef animals or pelagic fishes. IbaEilfefhNextto the Lens, these are the most criteal elements. Theideal fINer isa rectangularfunction having100% transmission averitsdesignated bandpassand zero transnission outofband. Of course,suchideal fiiters donot exist and there are many tradeoffs thatmust bemade inchoosing a filter.Forimaging, thebest fiiters areabsorptive, likethe common HoyaU filters orthe Kodak 1SA.These have excellent optical cianty, lowscatter, nopinholes andare apectrally independent ofangle-of-incidence. Suchisnot the case forthin-film interference fiiteis,Unfortunately, all absorptiveUVfilters have a red-window whichleads tocontamination ofthe UV image. For underwaterwork,this may not be that significant a problem aswater filters outmuch ofthe ied light inthis window anyway. H~r, thisprobiem mustnot be ignored sincethe camera coukl be Theusedbest ln suifacedesign of'wouid on-ahore useabsorptive imaging. filtersfor band isi~on with thin-fil IRbiockera suchas thosefrom Cimelga Optical. Narrow-band absorptivefilters arehard tocome byand forthis kind of filter,thin-film edgefilters along withthe IR blocking element canbe sandwiched withtheUV isolator.Forluminosity imaging, thebandwidth shouldcover the300 to 400 nm range. For chromaticityimaging,a series of filters ofnarrower bandwidth wouldbeneeded. Fivefilters with 20nm half-band widthwould beideal. These would bemounted ina filter wheel behind thelens turretThepolarizationand could befilteV chosen isplaced via an infrontindexing ofthemotor. lens and should bedetachable. Electrically activatedpoiarization filtersforuse in the UV are currerrfiy beingdeveloped andspeicifications for thisfilter will be available from Dr. Tom Cionin. It!9 This QQEKL'isreafiy theeasiest partasmany commercial camerasystems areout fhere. Obviously, onewants a robust camerawith remote operation potential. Afterthiscomes thechoice of detector.Whiletube-based camerasoffer a number ofadvantages interms ofspectral sensitivity asthe phosphors canbespecNed forthe UV, a solid-statecameraispreferred duetoinherent ruggedness.Thebest camera atthis time CIDTEC ofNew York! usesa Charge InjectionDevice CID!detector withhighinherent UVsensitivity anda quartz window. Another advantage ofthe CIDTECcamera isthe ability tocontrol integration timeofthe cainera forelectronic shuttering whilewatching theintegrated signaiappear onthe screen. Thismakes shutter timingeasy to deduce.Various hand-held andremote controlled pan8 tilt! housings arecommercially avafiable oralready on-hand atHIMB. UTERATURECITED; Fleishman,L.J.,Loew, E.R. 8 Leal, M. 998!. Ultraviolet visionanddewlap coloration inanoline lizards, Nafure365: 397. Hawryshyn,C W. 8. McFarland, W.N. 987!. Conephotoreceptor mechanisms and the detection of polarizedlight in Ish. J. Camp.Physiol, A 180:4$~. Ha~ishyn, C.W. 'f992!. Polarizationvision in fishes,Amencan Scientist 80: 164-175. Lcew,E.R. 8 McFarland,W.N. 990!. Theunderwater visual environment, in:Visual System of Rsh.Edited by R. Douglas and M. Gjarngoz.Chapman and Hall, London. Chapter 1, pages1- Loew,E.R., McFarland, W.N., Mills, E. 4 Hunter,D. 993!. A chromaticaction spectrum for planktonicpredation by juvenileyellow perch, Perca Navascens. Canadian Jour. Zool. 71:384-366. McFarland,W.N. 966!. Lightin the sea:correlations with behaviorsof fishesand invertebrates, AmerIcanZoof'oyht 26: 389 - 401. McFarland,W.N, 8 Loew,E.R. 994!. Ultravioletvisual pigments inmarine fishes of the family PomiKentrfdae.Ãsfon Reseatcfi 34 1!: 1393- 1396. Weiter,C, S. 993!. UV-Beffects on aquatic organisms and ecosystems: a summary of recommendationsfrom a seiectimof UVwoikshop reports. Report prepared for partici pants inthe woikahcp,' 'The impact of UV-Bradiation on pelagic freshwater ecosystems, September13-18, 1993, Lake Lacawac, Pennsylvania. 222 u reerteletddtetterr end COraliteeL tN5. D. Gulkoft P. L, Joklel «trr.!.HlMS Tech. Report f41. utsHI-SeaGrarrtCft-t5Ct, DISCUSSION AT WORKING SESSIONS Moderatedby Dr. Zvy Dubinskyand Dr, paul Joldel Edttertalreee: Exteoekre dte««elorar ofrace«rCh rreode OaCvmd thmutttarut theelrerrer reeeaaCfr poer«rm Oe well ea duretS ttreworketecp, T48aocttoo below le eurrellartaed trerrl the leurrd robtll" werkthtt eeeeteh etttre WCNkehep thWhkft parttciperrteItedthe ~ty tOstele their rrrftkrr COrceo«ahd ~, Ttrkrdtekre «ererrartzee rttuotr oftfNr trder«CSOh tlirttook ptarar ttmrughout thoatrrrrrV, artd derhortetrraea thefarce etlitrttt Of Wkfety riser kfeae.Srterry oftfreee toptoa are betrrg~ pereued by the parttctpertle. DubIneky:If wewant to progress,I think we should try to distillthe knowledge. We needto compartmentalizethedifferent areas of interestand different heels of resolutionand understandingof the problem. Maybe we should try to dividequestions into biophysical, physiologicaland ecosystem levels and what ws would like to see evotvlng in termsof instrumentationand what research questions we would like to developin each of thesethree fields. Anotherthought is that we should look at controls. For example, everybody hss been doingthe same kind of experiment. PAR versus PAR plus UV, This ls the classical experimentforeverybody here. Is PAR alone the proper control for PAR plus UV7 Maybe the controlshould be an equal number of totalquanta. For example, if we subtract UV should we addmore PAR so that the system gets the same amount ol photonsdelivered. But it maybe evenmore complcatedl Maybe we want to sss the same number ofphotons absorbed. So thenws needto takeinto account differences inabsorbance coefficients for the systemfor UVand the visible. Some of ouraction spectra are not action spectra In the same way they are definedin biophysicaI research. This was brought upin the discussion oftime scales. But it is nOtjuSt a questionOftime SCakfs. For example, InSatOlu'S talkhe mentIOned thefOrmation of a chemicalinduced by UV. But that chemical isalso induced byhigh light. Maybe if we increasedthe PAR by that extra PAR equivalent tothe dose of delivered UV, we would have ended up with the same results. I thinkalso that our definitions and our conceptual tools, at leastin the same mechanistic compartment,arestill very fuzzy. I wouldtike to see a lotof development onthis side, If we try tomix the tools that are needed to understandthebasic mechanisfns ofabsorption and inibal actionof UV, and then try to moveto physiologicalandsubsequent ecosystem levels and relevantquestions, wemust move in an orderly way. We fnust start ffcrn the foundations whichare basically themOlecuiar procefMMMt anddefinions which have been ~ Out. Thereis aLsoa concernthat we standardize ourapproaches andmethodology, atleast in theinitial stages. We need to dearly define terms and concepts andexpenrnentaI protocols, measurement,etc. Isthere a needfor a workshoponthis? Chachttilck-Furman:Weshould IoOk at the enhanCed effects of UV on coral. There is a defnonstratedneed to look at Iong-term versus short-term effects, atleast over a scaleof severalmonths, and to include long-term effects on coral growth, especially interms of populationsandcommunities, andthe diffsrsntial effects ondifferent species. 8tambtertWe should look at symbiosis, therelationship between zooxsnthellae andanimals, and how this relatesto coral bleaching,etc. Sanios:Weshouldsolve themysterybehindMAAs. Whatisthe purposeof 4IAAs? Are they reallyUV blockers? There has been no validaten ofthisi Taeuchl:I would liketo see somebody workon the vertical mixing ofthe phytoptankton. Whatis theoptimum environment, thephysical environment, inrelation tothe physiology ofthe cell? Halhbauah:I thinkwe should get moreof an overallpicture of an area,Including UV measuremsnts,analyze for MAAs,stc. Then,manipulate those corals by transplantingthem to a new environment. Reepanse: Paffsreon!We needto iook at the integrationof scalesand the integrationof resourceswhen we look at remote sensing and monitoring. Satellites give us different scales; dffferentOne scalesand differentspatial scales. What varies on what scale? We need to tie everythingtogether. How do you integratesatellite data with site specific studies? What kind af overallexperimental scheme? We need to know what is appropriatefor what is being studied and what is approprtate for certain situations. Different scales require different Instruments,There is a problemwith communcationand integration. Is there a placefor satellitesand what can we learnfrom remote sensingabout coral reefs? Nausaralf: We shouldtry to Identifythe receptorsand pro@maesfor these UV-A and UV-B effects. Letsfind out what is causingthisl IIaNo: I wantto knowhow much fluctuation of UV radiationwithin the earnsday andat the same hstltuchmight affect experiments. John Morrow showed us yesterdayhow UV radiation changedwith ozone concentration. So howdace this effectthe opticalcomponents of an experiment?Biologkrts need to havemore refined data on the variationand intensity of UV. SuNe:There is anassumption that UV-B is havingan effect, yet we have no way to measure does.We need to beable to geta handleon doss. Dose equals the multiplication of incldersmtimes response the bioiogicaI or physiologicalresponse!, That is theaxis on all our graphs.We need to quantifythe action spectra on ourgraphs. Dosimetry, irradiance fiekl, and biologicalresponses all need dartfkmtke. Fisher.We need better information onthe impact of ultraviolet on the basic pmcmees involved inphotosynthesis e.g.,a betterunderstanding ofthe molecular biology of primarypnxlucers such as phytoplankton. Yaakobl:The connections between vertical and horizontal water motion and phytoplankton are varybasic and we need to know, on a mediumtemporal scale of a weekortwo, how they effectthe community. NeOtka-Kudl»:Oneof the most important problems wetace is the application ofexperimental resultsto broader scales in the field to understand some of the mechanisms involved in experimentalbiology. How can we apply our experimental resultson a scalelarge enough in thefield to predict what will happen tothose communities e.g.,on the scale of a reef!if environmentalcondltlorls chaflgs? 0Neean:Neean:There lsa misunderstanding thatremote sensing isuseful only from satellites andon a globalscale. Coral systems arevery complicated anditis nary todarlfy how togo about remotesensing and how we can best use it asa tool.We need to work on local scales and thenmake predictions. Weneed tofirst understand underwater lightfields, diffusio, and the continuouscoefficient fordownweliing andupwelllng irradiance. Andwe need to be able to measuresub-surface Irrad4me, Only then can we predict what should be determined from spaceor airbornsystems, sensitivity, etc. iilorrow:I thinkthat we need tolook atUV-A and UV-B asterms, The generalized expression of thehypothesis isthat speciTic spectral regions willimpact the typical rel hot differentl,y, We e needneedtoidentify where the relevant spectral regions are and try to focus on them independent of buzzwords. 224 Crceby:It is extremelyimportant to havea cheap,simple way to measureUV intensity and It integral,especially intropical locations where instrumentatian maynat be aviiiable. Wheth i it is a chemicalactinameter or a physicalmeasure doesn't maesr. I alSothink there ShOuld be a CentrallOCatian fOr tropiCal uttraVldet retearah, Samepiace withcoral reefs, that is readily accemible, and already equipped todirectly measure the actk of enhanced UV an a coral community. Joldet:The criticalfactor has to do withthe energeticcost and material coat and Iimttaffon of adaptationof eachof thesecases, Whatis thelevel of costat the organismlevel? Blanck:It wouldbe interestingto determinewhat the pathways or mechanismsaf action of UV an reefolganisirls aN, particularlythe Immaturestages af coiais,sa thatwe candetermine their levels of sensitivity. Amrami: I wauldtike to get an overallpicture of the energybudget of, for instance,coral and whatthe influencesare ai UV-Bon thissystem. What does UV actually do to the coral? I am aisointerested in thesynergistic effect of temperatureand UV. With the concern for global warmingwe shouldput bothtogether. What can we do? Whatshould we do? Scientistshave a responsibilityto mitigate and educate. Shaahar:I wouldhke to measureUV property, in crevices etc., the microhabitat. Is it really affectingthe coral? Thereis a needfor instrumentsthat are ixirtable and easy ta carryaround, that measurefull spectrum,that are fast, that connect to fiberoptics pl and4 pi!,and that can measure through an angle or a full 2 pi. And to do it at the coral, at the interface,to developa bettersense of UVmeasurement, It couldeither enhance UV by a prismor allow you to block out specific wavelengths. Bldwell: I wouldlike to see a betterunderstanding of the evolutionof metabolicpathways, especiallyMAA pathways and genetic repair mechanism pathways, in orderto betterpredict the effects of increasedUV radiation on marineorganisms. Peachey:We shouldknow the communitylevel effects of UV witha moredetailed community analysis that woukl go along with whateverexpenrnent we decideto do in the field. KuNner:If you cauldfind a primerof a geneinvolved in the biochemicalpathway of the MAAs, thenyau couldattach a labeledprobe and dose natural camp4ss ta find outwhat tissues are producing the MAAs. Baker: I think that we have underestimatedthe planktonic dispersal phases af benthic organisms,and UV couldbe a signNcantfactor in theseand other stages af the life cycle- I think we need to do experimentsthat boost UV rather than efiminateit, and to think more about our controls. BanaazalaI thinkwe shouldwork on eaton spectra,especially in termsaf timecosts of action specters, and relate that to dose and dose rates. Ondrueelai am eisainterested in actionspectra. I thinkthere is a problemwith people using narrowbands or differentfilteis otherthan mono- or patychromatemethods! that cut off everythingshorter than that wavelength Weneed to use narrowerbands of wavelengths thatare enhanced by prisms or that are used to blockout specific wavelengths. Byusing UV blockers,chemical fiffers in liquidsolutions!, it shouldbe possibleto filterout narrow wavelengths.We needto ffgureout actionspectra either enhanced by pnsms,or ff yau to blackout specNc waveiengths, keep eveiything and say yau are cutting off or omitting things that might react. 225 tutow: I thinkwe shouldbe lookingat biochemicalselection, "survival of the fittest,"on a blochemicaisade, includingrepair mechanisms, and we needto lookat the effectsof UV on the microbialextiogy of conti reefs. For example,lots of workhas beendone on the effects af UV on bacteriaand viruses. There must be a nutritionalaspect to that somewhere. We havebeen rerneAng UV in ourexperiments and I thinkenhancement is whatwe shouldbe doing in the future. Why not do bothin the same system? Also, we should look at boththe negativeresults and posNveresults and integrate all of thatInto an understandingof what is going an. Lewis:As a biologist,I wouldlike to havebetter information on the UV fieldand potential Inoeasesand time scales involved. We shouldbe lookingat the effectsof enhancementof UV,especially long-term, on coralgrowth. What is ecologicaliyrelevant or mostlikely to occur in termsof whatthese organisms are likely to ses? Whatare the realisticlevels of increased radiationthat we might see? Reeponee: Patterson! As far as ozone,there is annualcycle of increaseof ozoneof about 2NM10 Dobsonunits. There is an 11-yearcyckt of the output of UV radiationand a 2.5-year cycleof ozonewith many different scales and it dependson howthey get put on top of one another.This Is for the tropics. Theysay thatwith the ozonedepletion over the top of these cyclesthey are expectinga 2 - 3%differenc averthe nextcouple of decades.The satellite Imageryhas beendone polared at 30 - 60degrees and over the marineareas the satellitehas a problemof clouds. QrelellMverett: No one has addressedthe potential of a posNve Impact of any increase of UV mighthave. Hcw,for example,under differen environmental conditions, viruses in algae couldpotentially have the hostinfect the algaand get rid of themduring a bleachingevent? Thereis a poten5alfor thereto bs posNveeffects, Also, not all coraLsare effectedby bleachingor enhancedUV andthere Is a lotto be learnedfrom them. Thereare a lotof answersto our questionsthat are to be found by using those organisms. Oulko: We should have workshopson other emvprtems. We should look at other systems Includingmangroves, seagrasses, estuaries arxf freshwater streams; places where you have recruitmentand that are importantfor lots of animals. But no one is looking at themi Also, we have people who use different methods. It would be helpful if we could corns up with a chartcomparing radkxnetsrs, chemical and viral actincmeters,and include costs, nanameterranges, their pluses and minuses, accuracy, weights, etc. thatare available for experimentaldesign, including their manufacturers, by the peoplewho actually have used this equipment. Krupp:I aminterested in understarxflngthe connectionbetween biochemical effects and the organism'sresponse and what adaptive signiffcsnce, if any, may be attributedto a particular organism'sresponse. For example, I aminterested in Paul'sobservation of planulaerelease when corals are exposedto normal UV as apposedto when UV has been removed. And that thereis a reductionin growthunder UV compared to no UV. Hasthere been a biochemical shift in energy budget? Is this an adaptive responseon the part of the coral? What is the underlyingbiochemical mechanism? Lowe: I tike the ides of Gulko's that we should be looking at aspects of the tropicalmarine ecosystemother than zooplankton or phytopianktonand includefreshwater systems. I would like to look at UV ss a selectivepressure that animalsmust adaptto and how, over the longrun, animals are going to respondto eitherincreases or decreaMxin UV. Are there thingsthat are akinto heatshock proteins? This cannot be thefiIst timethat organisms on earthhave faced changes in the UVenvironment. We knowthat animals moving to new 226 environmentsaregoing to experience tanning responses. Weare acquainted withwhat terrestrialplants do with increases inUV. Is there something akin to tanning inchlorophyll. containingmarine organisms? MCFarland:CaiibratiOn isa veryCOmpieX tOpiC and nat eaeyi I WOuldlike ta ieaommend that therecould be a fewstandards orsugges«ons thatall of us could fallow within the limits of cur ability.I think we could compare ourdata bases a lot more readily. A couple ofsources or calibrationinstruments ordifferent sources would be worthwhile. Some standard guidelines wouldbe worthwhile. UV-A works in radiance and vision, but UV-B may be important inlethal and sub-lethal effects. ls thereresearch, directed research, on developing new kinds of actinometers? There is somework being done with caged compounds, compounds thatcan be tuned to 340 nanometersand used for i~lular measurementsofthings. With a biosensorand an el~ readoutit is essentially anactinometer and can be tuned very specifically, for example,the 340 line. Other possibilities mayexist for these compounds. Hawryahyn:I think we need to address some afthe more subtle effects ofUV that are important,if not obvious, onthe surface. For e~, youmay be interested in photoreceptorsandthe degeneration ofphatoreceptors infishes because ofa highphoton capture.This can, over time, lead to a dlsruptkrninthe interactions ~ween snr'mals. Coral reefsystems provide optimum environments forlooking atoptical signaling, Ifcolor vision systemsofcoral reef fishes are impaired, there could be reasonably prafaund irrrplics«ons on thebiadiversity ofcoral reefs including fishes, What strategies should be used to look at these questions? Cronin:I think it ls important tohave a betterunderstanding ofthe light field within the specific arganismsbeingexamined. Thediecussian onaction spectra isvery important andthe action spectrawill change with the induc«on ofwhatever protective pigments orwhatever respanse therels. Thereis notan actionfor everything in general. I don'tthink there is even an action spectrum fora particularorganism unless you specify whatlevel of inductionthere is. So,understanding the prrxxrs!res that an organism goes throughtoprotect itself from UV is critically important. Partof that is understarrding whatthe pigmentsarethat itwiil protect itself with and how the light Md within the coral exists. And thatincludes penetration framthe top, or perhaps thesides, and how much light there is, Chemicalprobes are par«cularfy useful. Based ontheir polarity, youknow where they are goingtobe. it would be helpful todevelop lipid-based, semi~id chemical probes. Cox."The emphasis shauld be put back an the tropics as being areas that have many diverse typesofUV environments, characterize thoseenvironments, andlook at how organisms have adapted to the environments. Te:Coral larvae settkr incracks and crerrN~. Hcw do larvae find a placetoset5e? There are compoundingfactors that affect the environment including haw UV impinges onthe organkrm,Itis important toknaw how all of these complicating factorssuch assalinity, rainfall andsuspended and particulate matter impact the organisms ahng wilh UV. Open Discussion4 Encf of Session. 227 Neeelohthewesen end Caal Reera 1%$. D,Geko BP. L Joklel ede.!, HNB Tech. Repat e41. UNIHI4ea C~~. APPENDiX I: 1994Edwin W. Pauley Summer Program inIIiarine Biology UV RADlATlONON CORALREEFS Course Syllabus Zoology715, Topics in InvertebrateZoology, 4 credit hours Instructors:Dr, PaulL Joidel HIMB!, Prof.Robert A.Kinzie III UHM Zoology!, Dr,Michael Lesser UNH Zoology!, Prof.Donald Crosby UCDavis, Dept. Environmental Toxicology!, Dr.David Krupp NtCC!. 7/5 Tue 0815 Jokiel: introI - GeneralIntroduction to Physical,Chemical and BiologicalProperties ofSolar Radiation with Emphasis on UV. IntroII - HistoricalOverview of UV on Coral Reefs 7/6 Wed 0815 Crosby: Intro- MarinePhotochemistry, Anthropogenic Changes in the OzoneLayer and PossibleConsequeces Gutko/Jokiei/Lesser: 7n Th OSiS UVReaching the Surface ofthe Earth, With Emphasis on Kaneohe Bay. Temporal, Seasonal Variation, RelationshiptoOther Factors. UV Optical Charecteristicsof Natural Water, With Emphasis on Kane ohe Bay 7/7 Th 1300Lesser: Demo. and Discussion ofUse of Spectroradiometers on Reefs DlscatssionSEMINAR Joklel!: 7/8 Fri 081 5 1!Whatisthe Overall Impact ofUV on Coral Reefs? 2!What isthe Potential Effect ofPredicted UVIncrease? 3!OoWe Know Enough toSay Anything About the Possible Dangers? 7/11 Mon 0815Crosby: UV Photochemistry - Actinometers, Dosiometers 7/12 Tue 0815 Kinzie: UVand Coral Metaboiism 7/13 Wed 0815 Crosby:UV Photochemistry - Phototoxicity and Photodegredation 7/14 Th 0815Lesser: Action Spectra -What isit andhow dowe apply it Appendix I: Course Syllabus cont,! 7/l 5 Fri 081 5 Discussion SEMINAR Crosby!: Can we demonstrate any relationship between UV photochemical change and UV coral reef biology? 7/l 8 Mon 0815 Lesser: Simulation of enhanced UV resulting from ozone depletion 7/19 Tue 0815 Ondrusek/Lesser: UV Blockers Mycosporine-likeAmino Acids! 7/20 Wed 0815 Kinzie/Lesser:Part I - UV and plant response,uniceliular algae 7/21 Th 0815 Lesser/Kinzie:Part II - UV and plant response,unicellular algae 7/22 Fri 0815 Discussion SEMINAR Lesser!: 1! Wowis an action spectrum determined? 2!What does it tell us. 7/25 Mon 0815 Discussion of research projects 7/26 Tue 0815 Krupp/Gulko: UV and Coral Reproduction 7/27 Wed 0815 Shashar: UV and visual response: an overview 7/28 Th 081 5 Student presentations of current research 7/29 Fri 0815 Discussion SEMINAR Kindle!: 1!Does UV impact primary production on coral reefs? 2!What is the evidence? 8/3 8/4 8/5 All students participate in the UV workshop at East-West Center UNraHdk~ and Ccw3IReef@ 1$8i. D.Gula S P, L Johill eda,!. HIMB Tech, Report 841. iPilHI4ee GranK84548, APPENDIX II: ULTRAVIOLETRADIATION INTROPICAI COASTAL ECOSYSTEINS WorkshopSchedule 3-5August 1994 East-WestCenter, Jefferson Hall, PacNa Roam Honolulu,Hawaii, USA Wednesday,August 3, 1994 Wednesday,August 3, 1994 8:00-10:00OPENING SESSION. Dr.Paul L. Jokiel tnoderator!. IntroductoryRemarks. DavidMauzerall - Ultraviolet Radiation and the Origin of Life. MichaelLesser - Summaryof PreviousUV workshops. Discussion. 10:00- 10:15 BREAK 10:15-12:00.PHOTOCHEMISTRY OFUV. Dr. Donald Crosby {moderator!. Don Crosby - UV Actinometry. CurtisSuttle BacteriophageDosimeter. RitaPeachey - UV Phototoxicity inCoral Reef Biota. GlennMiller - PrinciplesofUV photoreaction onSurfaces: ImplicationsforCoral Reef Biologists. Discussion 12:00-13:0013:00-15:00 LunchUV INSTRUMENTATION ANDHARDWARE: Dr.Michael Lessel moderator!. MichaelLesser - GeneralOverview of Instrumentation, ExperimentalMethods, Penetration ofUV Into Natural Waters AlanTeramura - Considerations When Using Artificial imps to SupplementUV-B Radiation. JohnMorrow - ScanningSpectroradiometers. Discussion. 15:00 - 15:15 BREAK 15:15- 17:00 REMOTE SENSING, GROUND TRUTH, MONITORING AnatoliGitelson and Yossi Yacobi - Remote Assessment of ChlorophyllConcentration in Productive Waters. KarenPatterson - Possibilities ofUsing Satellite Remote Sensing to CalcuiateUVTransparency ofWaters Over Coral Reefs and Other UV-Related Processes Discussion 17:00 Pau Hana End work day! ~~ g: WoaahopSchedule ~ufschy, August 4, 3 994. 8:00 - 10:00 BIOLOGICAL RESPONSE TO UV. Dr. Robert Kinzie moderator! RobertKinzie - UV anda HawaiianHigh Altitude Aquatic Ecosystem. MichaelOndrusek, Ania Banaszak,Ilsa Kuffner - UV Blockers Or.Dave Krupp, Andrew Baker, Dave Gulko - CoralReproduction and UV. Discussion 10:00 - 10:15 BREAK Petersonet al. presentedby KarenPatterson! - A Biological WeighingFunction for PhytoplanktonGrowth Inhibition by UV Based on Growth Responsesof ZooxanthellaeCultured Under Various UV conditions. SatoruTaguchi - UVDamage and Repair in Phytoplankton. Hiroaki Saito -Effectof UV-B on the Reproductionof Marine Copepods:Hatching Rate of F'aracalanussp. MarjorieReaka-Kudla - The Relative Effects of Temperatureand UVB on DifferentComponents of the Caribbean Reef Community. Discussion 12:00-1 3:00 Lunch. WORKINGSESSIONS moderated byDr. Zvy Dubinsky and Dr. Paul Jokiel!. Previoussessions have describedthe "state of the art". The WorkingSessions served as a processto describea UV research programfor thefuture and the roleof HIMB and others!in implementingthis program. The purposeof these sessionswas: 1. Defineand prioritize researchquestions. 2. Developa plan to answer questions. 3. Identifymeasurement probiems, means of resolving problems. 13:00- 15:00 WORKING SESSION I GroupDevelopment of Conclusions,Recommendations In Each Area. 15:00-16:15 Break 15:15- 17:00 WORKING SESSION II Group Developmentof Conclusions,Recommendations In Each Area. 17:00 Pau Hana. 232 S s] ~ ~ S p 8~ Subject Index C cent.! Acclimation 149, 182 Contrastrecognftfon... Acctimitization response...... ...,...... 14 Copeprxls Acrosome, '142 Coral Bleaching...... ...90, 107. 110, 112, 115, Acropora . .77, 121, 132 Action spectra ,...... ,.... ,. 15, 16, 51, 54-58, 121, 223, 226 127 223, 225, 227 Mucus. ...107, 121, 129, 135 Acute toxicity. 193, 197 Reproduction...... Agancia agarfcites, .....135 Spawning. ...... 110, 129, 135 . , .135, 'l42-144 Aiptasiapallida ...... 89, 94, 165 Sperm. .. ,..... 107-109, 115 Albedo. 25, 30 Transplantation.... Aipheopsissp ...195, 197 Culturegrowth . 142, 171 Arnpfrikxzrslikeliks ....195-197 Cyanobacteria. ...171, 173, 176, 178 Angleof polanzation, ...,.....,207 Gyanophyla.... ..... Antarctic . . 13-14 AnfhopleuraxanfhogrammrL . ,...... 202 Anthracene. 19M99 Dailysolar values. 29-30 Aromatic amino acids. ...79 Darnselfish. 202,219 Artemis salina,. ,193-198 Decalcification....,,109 Atmospheric gasses 27 Deepsea crustaceans ,.....,...... ,...... ,203 Atmosphericparticles ...... ,...,., 27 DePthProsies.. ..17-18, 32, 3940, ~, 137 Attenuation .. ,....,...... ,... 32-33, 37, 77, 184 Dictumpheerfa Azotobacterium . D, cevemosa. ,181 O.vsrsiuysil...... ,...,...... , .181 B DNuseattenuation coefficient.....,...... ,. .. 32 Digitalcameras. 215-216 Bacteria .1 .207 Dinoflagelfates...... , .208 Bees. D~ inorganicnitrogen DIN!. ..... ,.189 Biologicaldosimeters. ,15 D~ organicmaterial ...... ,...... ,.... 30 Biologicalweighting function ...... ,...... 51 Diurnalegg ~r.... ...... 143 Bioluminesca nce. DNA...... 16,51, 53-54,58-59, 135, 'l42 Blffiurn parcum. . .... .195, 197 Repairmechanisms,, 14 Bleaching,...... 90, 107,110, 112, 115, 121, 165, 223, 226 Dosimetry. 224 Eggs... ,...,.....,.....,.... 131, 135, 137, 139 14Q Energybudgets . 225 Cage effect. 18, 53, 53-55, 57-58 ....47 Erythema....,... ...... Csianus spp. Extinction coefficient 32, 184 Callspongiadlusa ...... ,...... ,. 95, 97 &-vector of light. ...207 Camcorder 213, 216 1 165 Evolution, Carbon assimilation of photosynthesis ...... 10-11 C~ropeia xamaciriena...... ,...... ,.. ... 1 72 Cell moNlty 165 ....., ... 213 215 221 Chargelntected device. ,132 ...... 15 Favr'apaffida. Chemical doeimeters.. ,135 Chernotaxis, .142 Fertilization, Chlorophyll,...... ,...... ,,.92-100, 107, 115-119, Filters 121-126, 165 alsosee plastics! 23, 116 . 171-173,'l75-176, 181 Neutral density Chlorophyta...... , .. 199 .89 Fish larvae, Chioroplasts. 142 .126 Cfavuiafia. Regalia. .142 25, 27-31, 226 Ragellates. Clouds 16 .1, 27 Fluorescence . CO, ,...,165-168 220, 227 pluorsecentfighting . ". Color vision. ... ..13, 16 Cixerast enhancement ...... 219 Freshwater systems --- 237 F cont.! L cont.! Fungis Larval settlement ..... . .... 149-150, 153 .143 Light F, ~. 121, 126 W ,201 F. scuferfs... 12f, 129, 135, 195, 197 The underwater light field , ....207 Ught meters. .91 Lightning, 1 107, 117-119 Gametes... 135 Lipid LcwrsyProtein Method.....,... .92, 130, 172 Gelbstoff. 30, 33 220 Gonfssfres ftrvufus 132 Luminosity ,110 Gr~rlsm,. 129 Lunarcycle. Gynogsnesls.. 142 H Macrophytes...... ,.171 . 174 181 ds sp. 172 Metabolicability. ...... ,.....,....,...., 90, 95-100 Hammerskrybanded ion formation ...... 4 Microafgae....., .165 Hammersleybasin...... ,...... .,7, 10 Mitochondrla , .142 Hatching rates. Monochromstera. ,15 Hennaphrodism. 129 Monosex. ....,... ,...... ...... 142 Hertwlgeffect. 142 Mcnftpcra,, , . 159 Haterotrophy 110 M. pafula . .77-79, 82 HOBO data recorders...... 106, 117, 124 M. verrucose...... 7740, S9, 107, 115, 131, HPLC...... . 77-79, 92, 130, 151, '154, 172 142, 195, 197, 199 Humkllty. Moon phases.. 129-130 Mortality. ,193, 197 +rcoaporlns-like aminoackts MAAs!...... 14, 23, 77, 79-83, Intercallbratlon 67, S9-94, 102, 121, 129, 137, Instrurnentatlon 141, 149 - 160, 171 - 174, 182, Air messuremerrs ...... 15, 19-20 203, 223-225 Comparison.... , ... , ...,226 8lological.. 15 Chemical. 15, 225 Nauplih Intercallbratlon . 15, 227 NemaNciiquid crystals....,. ,. ...213-214 Polarlmeters, ..213 Nitrate ion, ,.4 Pyranometers...... 19 NitrogenenrichmenL .181 Quantum sensors. 19 Nocturnalspawning 137 Radiometers... ...... 15, 1 9,2S, 37, 183 Preda5m Avoidance Spectroradiometers...... ,15, 20. 32, 37, 79, Hypothesis ,144 11S, 168, 172, 183 UV Avoidance Hypothesis . .... .144 Standardization...... 15, 223-224 Watermeasurements ,. . .....,. .13. 224 Nutrlents. ... 25,33 Wavelength~Nic,. . ... . ... .. 15 irradiance Compensationirradiance . ... ...... ,. 93 Octopus, ,207 Saturation irradiance ...... "."... "-,"-. 93 ...,~,47, 49 J Originof life. 1 9-10,219 K 0, ...... 27 Kans'oheBay ,...... ,. 2535, 3741, 77, 121, 0, meters ... ...... . ,122 ...... 13, 27, S1,89, 121 158, 136, 159, 171-173, 182 165, 224, 226 P:R ratios ....,....,...... , ...89, 125-127 Pslyffroscsrfbaeorurn... Urfrrciocerarnsdurae .. .,4344, 47, 49 Peraclsnus spp. ,49 Larvae. . ,....,.43, 149 238 P cant,! R cant! ...89-91 Pevonecactus. 143 Respirometry., Partiallylinearly polarized light PLPL!...... 213 Retina. .202 Pheeodacfyfumspp...... 54, 56-57, 167 Rhodcphyls ...... 171 ~ 173, 175, 177 Phaeophyta...,...... ...... 171,173, 175, 178 Photochemistry. 2,4, 7 S Photoinhibition...... ...... ,. 53-55,90, 95 S-320 compunds...... ,...,...... ...... ,89, 149 Photolysis,..., 2,3 Ssfmcnfds...... 201 Photoncapture. 227 Saturation points...... 89 Photoxidation, 95 Sediment...... ,...... 25, 33, 39, 108, 115, 121 Photoreceptors... .....,...... ,.... 202, 21 9, 227 Senescence, .167 Photosynthesis..... 16,19, 84, 89-91. 121-126 ....,...... ,.194 224 Shlfdmatepatfnrmy .... ,...... ,...... 79, 1 35, 172 Photosyntheticefficienc...... ,...... 89 Signal detectiontheo ry..... ...... . ...,...219 PhotosyntheticPhoton Flux...... ,....,..., 19 Snail's window . 201, 203 Phototoxiclty.. ,193 Solar slmukrtor. .... ...... 138 Phytoplankton..... ,...... ,.....33, 53, 223-226 Solar zenithangle.. 27, 89 Pigmentation,109-110 Spawning Plane-poladzedlight . 220 Broadcast. 135 P Brooding. Mass spawning events...... 143 Planuia larvae... ,...... ,...... , 135, 137, 149 Spectrorsdiorneters Plastics filters! 15 Lumiiarsheet., , .. 44 Camprrriscns UV-Atransparent .....,... 21-22,44, 116, 136, Sperm. 135, 142-144 . i43 151-152, 186 Behavior. Clots, .143 UVopaque,. ...... ,...22-23, 116, 121, 138, .199 151-152, 166 Sublethal effecia. Submersibleimaging polarlmeter ...... 21 3 UVtransparent ...... ,20-21, 116, 121, 136, 193 151-152, 166 Sunburn . Plastoquinones.. ...... , ...81 Syrnbirrr8nr'urnm/croadneficum...,...... ,... 53, Plsfynereisdumerf0i...... ...... ,...... .195-196 56, 95, 1~168, 172 PnNopors Synergism P. dsrnicornls...... 77, 87, 89, 110, 137, 195 197, 199 P. eydouxi .143 Temperaturestrew ...... 107, 115, 121 P. meendNm ...... ...... ,....77-79, 83 Terresrisl systems...... ...... ,. 1 4, 89 P. verrucose ..143 Tefresefrnis sp.. .167 Polarirneters, 213 .183 Polarization...,202-203, 207-211,213-214, 220 Tre~, Polycycticaromatic hydrocarbons PAMs!..193 Trtdscns sp. Porites Tropical atmosphere...... ,...,....27-28 P, compnrsse . ,77-79, 85, 159,195, 197 Tufresfrsee coccinee. P. lobate ...... ....., ... 171 U PrlmNve stmcsrphere...... ,...... , 24, 7 Ultraviolet radiation Primitive ocean. ,3-5, 10 Absorbing cones ...... , .203 Proteins . ., 77-78, 92, 189 Avoidance 193-199 Pyrene Coloration. Oefensss against... . 89, 121, 181, 219 imagery. .. 219 Q R Monitoring.. ,14 . 203,220 Radiom ate re Receptora., ,15 Seasons! data . Comparisons, 201, 203 Reflecting bodkrs, .... 28, 31-33 Sensitivity 219 Remote sensing. 224 V>sual sens4vrty .201-204 Reproductive success .. ...... ,...... 149 Vision, 194 Respiration .. . ...... 89. 95, 103, 121 Urban runoff. 239 Ver5cal rnlgratton . Veracal mixing. -"- 33. 223 ...,...... 79 Vtalon ....201-203 ... ...... 201 invertebrate... . Pohrtzation.....,....,...... 207-210, 21 7 Vleual plgrnent syetem...... 201 Vog. Water velocity . . ... 87 Wave action...... 33, ~1, 108 XZ Y Zooplanlrton,...... , 43, 203, 220, 228 ZooxantheNae ...... 77, 79, 88, 95, 107, 115-119, 135, 142, 165-168, 182, 223 240