UV radiation，especially the ultraviolet radiation B(UV-B)b and (280–315 nm)，has signifi cant effects onliving organisms(Seckmeyer and McKenzie， 1992;Bulter et al., 1999; Krizek，2004). Although UV-Bradiation accounts for only 0.8% of the solar energythat reaches the Earth’s surface，more than 50% ofthis radiation contributes to the photochemicalprocess in marine and aquatic environments(Neale，2000). Over the past decade，the depletion ofstratospheric ozone has slowed down， and it has evenslightly recovered. However，the reparation and reconstruction of stratospheric ozone will require aprolonged period as the organisms on earth arecontinuously exposed to high doses of UV-B radiation(Hu，2007). The UV-B intensity of Jiaozhou Bay inwinter is about 8.5–11 W/cm² in 2008(Ling，2010).And the UV-B intensity of Qingdao area in summer(June to August)is about 30–35 W/cm² in 2004(Wang and Hou， 2006).

Macroalgae are distributed mainly in the intertidalzone， and their physiological behavior exposes themto the detrimental effects of solar UV-B radiation.Previously，several studies focused on the effects ofUV-B radiation on spore size and germination(Han et al., 2004)， and the growth conditions of differentmacroalgae，such as Sargassum thunbergii，Ulvapertusa and Delesseria sanguinea(Pang et al., 2001;Cai et al., 2005; Roleda et al., 2006; Li et al., 2009;Schmidt et al., 2010). UV-B radiation can also causedamage to DNA，proteins and pigments(chlorophylls，phycobilins， and carotenoids) and inhibit importantmetabolic processes in macroalgae(Teramura，1983;Franklin and Forster， 1997; Sinha and Hader， 1998;Xu et al., 2003; Roleda et al., 2004; Schmidt et al., 2012; Pescheck et al., 2014; Simioni et al., 2014).DNA is particularly sensitive to UV-B radiation，because phototransformation due to the absorption ofUV-B leads to the production of cyclobutanepyrimidine dimers(CPDs) and pyrimidine(6-4)pyrimidinone dimers(6-4 PPs)(Ellison and Childs， 1981)，consequently inhibiting the replication and transcription of DNA.

Macroalgae have developed mechanisms tocounteract the damaging effects of UV-B throughouttheir evolutionary history. The mechanisms of DNArepair in algae post UV-B radiation damage includelight repair(photoreactivation)，dark repair(nucleotideexcision repair)， and recombination repair(Britt，1999; Pakker et al., 2000a，b). Mycosporine-likeamino acid(MAAs)，which have absorption maximaranging from 310 nm tO₃60 nm，are a class ofapproximately 20 compounds that are UV-B protective(B and aranayake， 1998). These compounds play animportant role in protecting marine organisms fromUV-B radiation(Banaszak and Trench， 1995).

Chondrus ocellatus Holm is abundant species ofred alga along the coast of Qingdao， and inhabits theintertidal and upper sublittoral zones of rockyshorelines. C . ocellatus is a kind of commercial alga，which is widely used as a thickener and stabilizer infood，cosmetic industry and medical. C . ocellatus iswell investigated scientifi cally and often as a modelspecies used in scientifi c research. UV radiation exertsmany negative biological effects on C . ocellatusdevelopment，including lethal DNA and RNA damage，enzyme inhibition，photoinhibition of photosynthesis and a decreased growth rate， and induces UV-screeningMAA compounds(Bischof et al., 2000; Franklin et al., 2001; Van De Poll et al., 2001; Yakovleva and Titlyanov， 2001; Kr?bs et al., 2002; Roleda et al., 2012). However，the sensitivity and defensivemechanisms of C . ocellatus to UV-B radiation duringits early development have received little attention.

Light quality is considered an essential factor forthe growth and other metabolic processes of marinemacroalgae(Senger and Bauer， 1987; López，1991).Several studies have focused on the interactive effectsof UV-B radiation with red and blue light. Han(2004)demonstrated that the green alga Ulva pertusaKjellman presented a different germination ratiowhen exposed to moderate levels of UV-B radiation followed by exposure to visible light， and asigni ficantly higher germination rate was found in theexperimental group that was given blue light，whereasthe germination rates were lower under white and redlight. Furthermore，some UV-B responses，such aschalcone synthase(CHS)expression are regulated byred and blue light， and there are some complex websamong the phytochrome-，cryptochrome-， and UV-Bsignalingchains in plants(Ohl et al., 1989; Boccal and ro et al., 2001; Wade et al., 2001). CHS is akey enzyme in the fl avonoids synthesis pathwaywhich can prevent UV damages(Zhang et al., 2012).

Previously，the diameters，CPDs，chlorophyll-a(Chl-a)，phycoerythrin and MAAs contents oftetraspores exposed to UV-B radiation then put intophotosynthetically active radiation(PAR) and darkness repair condition were determined by Ju et al.(2011)， and the data were used to determined theeffects of red and blue light conditions on the repair ofdamage induced by UV-B radiation. In this study，weevaluated the effects of UV-B radiation on thedevelopment of tetraspores of C . ocellatus . Our aimwas to examine the effects of red and blue light on therepair of UV-B-induced damage， and to evaluate theadaptability of C . ocellatus to increased UV-Bradiation at the early stages of its development. UVB-induced damage was evaluated by determining theCPD，phycoerythrin， and Chl-a contents in thetetraspores of C . ocellatus . The level of protectionwas evaluated by determining the content of MAAs，which are UV-B absorbing compounds. 2 MATERIAL AND METHOD 2.1 Algal material

The C . ocellatus with mature sporogenic tissuesused in this study was collected from rocks in theintertidal zones at Taipingjiao(a public beach)，Qingdao，Sh and ong Province，China. All experimentswere carried out in the laboratory. The immaturesporophytes of C . ocellatus have the samemorphological characteristics as the gametophytes.2.2 Release of tetraspores

Sporangia with tetraspores were washed withsterilized seawater and cleaned using a banister brush.The washed sporophytes were dried in the shade for4 h at 18°C to promote spore spread. The sterilizedseawater was natural seawater fi ltered through a 0.45-μm micropore fi ltering fi lm and then autoclaved. Thesporophytes were placed in sterile Petri dishes containing glass slides and 300 mL sterilized seawater.The dishes were kept in the dark until the tetrasporesattached to the glass slides(20/3 mm²)，then theattached tetraspores were used for UV-B radiationexperiments. All cultures were maintained in culturesolution consisting of sterilized seawater enrichedwith nitrogen(8.24×10^-4 mol/L NaNO₃) and phosphorus(3.26×10^-5 mol/L NaO₂ PO₄ ·O₂ O)(Starr and Zeikus， 1993). The cultures were kept at 18±1°C，under 12:12(L:D)photoperiod about 40 μmolphotons/(m ² ?s)of PAR，provided by 40-W daylightfl uorescent tubes((Philips，Ningbo，China). Eachtreatment consisted of fi ve replicates. The mediumwas replaced every 2 days until day 48 of theexperiments，when a vertical branch had formed in alltreatments. The tetraspores were then analyzed todetermine their physiological and biochemicalindices. 2.3 UV-B treatments

All experimental treatments were applied underlaboratory conditions. A bank of four fl uorescentUV-B lamps(Q-Lab，Clevel and ，OH，USA)providedsupplemental UV-B radiation. A UV-B type UVdetector(Beijing Normal University PhotoelectricInstrument Factory，Beijing，China)was used tomeasure the intensity of UV-B radiation，which wasfound to be 7.2 μW/cm² . The light was fi ltered withcellulose diacetate foil to achieve 0% transmissionbelow 286 nm. The UV-B lamps were burned for100 h before starting the experiment and steadied for10 min before the start of the exposure every day toachieve stable radiation. The levels of UV-B radiationwere setting according to the preliminary analysis.The tetraspores and germlings could not developmentwell as the dose of UV-B radiation higher than 216 J/m ² . For these experiments，fi ve levels of UV-Bradiation(36，72，108，144， and 180 J/m ²)wereemployed by adjusting the exposure time(5，10，15，20 and 25 min，respectively)(Table 1). The PAR group was used to assess the effects of UV-B radiation;the control was kept under PAR with no UV-Bradiation(0 J/m ²). The UV-B radiation treatmentswere applied each day at 9:00 am. To test the effectsof light quality on the repair of UV-B-induceddamage，the tetraspores were subjected to a 2-hrecovery period under PAR，darkness，red light，orblue light after the daily UV-B treatment，as shown inTable 1. Red and blue lights were supplied at 40 μmolphotons/(m ² ·s)using 40-W red and blue Philipsfl uorescent lamps. After the 2-h recovery period，thetetraspores were returned to their culture conditions.

Table 1 UV-B radiation and light treatments during the repair period of tetra spores released from C . ocellatus blades

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To determine the UV-B radiation effects，oneexperimental group(PAR 36–180 J/m ²)was placedback in PAR after exposure to UV-B radiation and thesamples named PAR 0 J/m ²(control)were notexposed to UV-B radiation at all(Table 1，PARgroup). To determine the effects of light quality onUV-B induced repair，experimental groups weresubjected to four different light treatments afterexposure to UV-B radiation. All treatments wereapplied immediately after the UV-B treatment.2.4 Assessment of UV-B-induced damage to C . ocellatus 2.4.1 Tetraspores diameter

The diameters of the tetraspores were determinedusing a fl uorescence microscope(CX31，Olympus，Tokyo，Japan)every 4 d from day 0 until day 48. Thetetraspore diameters were measured from imagesusing the optical microscopy software packageImagePro-Plus ver. 6(Media Cybernetics，SilverSpring，Maryl and ，USA). Normal tetraspores weredisc-shaped.2.4.2 DNA extraction and ELISA of CPDs on day 48

Total DNA was extracted from tetraspores ofC . ocellatus according to the method of Mayes et al.(2004). To avoid light repair during analysis，the entire process of DNA extraction was carried outunder dim red light. The DNA concentrations weredetermined with a nucleic acid spectrophotometer(Ultrospec 4300 Pro，GE Healthcare，Little Chalfont，UK).

CPDs were quantifi ed by enzyme-linkedimmunosorbent assay(ELISA)according to themethod of Mori et al.(1991). The monoclonalantibody，ab10347，for ELISA was suppliedcommercially by Abcam Company(Abcam，Cambridge，UK). The absorbance of the reactionmixture at 490 nm was measured using a microplatereader(Multiskan MK3，Thermo Fisher Scientifi c，Massachusetts，USA).2.4.3 Concentrations of chlorophyll a and phycoerythrin on day 48

Chondrus ocellatus was ground in 90% acetonewith quartz s and on ice，then kept at 4°C for 24 hbefore centrifuging at 8 944 × g at 4°C for 10 min. Theabsorbance of the supernatant was measured at647 nm and 664 nm according to Ritchie(2006). TheChl-a content was calculated as:

Click Browse formula

Chondrus ocellatus was washed with distilledwater homogenized and extracted in 0.1 mol/Lphosphate buffer(pH=6.5)on ice，then centrifuged at8 944 × g at 4°C for 10 min. The concentration ofphycoerythrin in the supernatant was determinedusing the method of Beer and Eshel(1985). 2.4.4 Extraction and analysis of MAAs

The algae were oven-dried at 100°C for 15 min，theMAAs were extracted for 2 h in 25% HPLC-grademethanol(v/v)at 40°C. The samples were passedthrough a 0.22-μm membrane fi lter. Tyraminehydrochloride(THC)was used as an internal st and ard，according to the method of Whitehead and Hedges(2002). THC has an absorption maximum at 280 nm，far lower than that of the MAAs， and a relatively lowmolecular weight，so it is easily distinguishable fromthe common MAAs. Prior to liquid chromatograph/mass spectroscopy(LC/MS)analysis，100 μL of asolution containing 173 mg THC dissolved in 1 mL ofMeOH(1 mol/L)was added tO 400 μL of MAAst and ard.

The concentration of MAAs was analyzed using HPLC(1120 Compact LC; Agilent，Santa Clara，CA，USA) and calculated from the peak area. The mobilephase was 2.5% aqueous methanol(v/v)plus 0.1%acetic acid(v/v)in water，run isocratically at 1.0 mL/min. Sample volumes of 10 μL were injected into theSphereclone C column with a precolumn(5 mpacking; 250 mm×4 mm I.D.). Detection of peakswas determined by the absorbance at 320 nm and 340 nm; however，for samples with the internalst and ard added，280 nm and 320 nm were monitored.

The types of MAAs were analyzed using an Agilent1100 LC/MS system with a diode array detector(DAD)interfaced to a quadrupole mass spectrometerat the First Institute of Oceanography，SOA，China.The UV wavelengths monitored were 320 nm and 340 nm. We used a C-8 column(250 mm×4.6 mmI.D.)with isocratic elution over a 15-min period. Themobile phase was 65:35 methanol/water acidifi ed topH 3.8 with acetic acid. The fl ow rate was 0.8 mL/min. The acquisition methods for LC/MS were basedon those of Whitehead and Hedges(2002)， and theconditions were optimized to produce positivemolecular ions(MH⁺)via electrospray ionization.Other LC/MS settings were as follows: gastemperature，350°C; sheath gas fl ow，11 mL/min;scan mode 50–600 amu. Each kind of MAA wasidentifi ed according to molecular weight from theextracted ion current(EIC). The structural formulaewere determined by comparing the examined massspectrum with the st and ard mass spectrum， and wereused to confi rm the classes of the MAAs detected inC . ocellatus .2.5 Data analysis

Each treatment consisted of fi ve replicates forstatistical analysis. We used one-way ANOVA and GLM-univariate analysis to test the signifi cance ofdifferences. Percentage data were square root(arcsine)-transformed to achieve a normal distribution.We used SPSS 13.0 for statistical analyses. Levene’stest was used to assess the homogeneity of variance.When the P value for the homogeneity of variancewas greater than 0.05，LSD was used for multiplecomparisons，otherwise Dunnett’s T3 was used(P< 0.05).3 RESULT

The diameters of tetraspores exposed to differentUV-B radiation and light repair treatments during the48 days are shown in Fig. 1. In the PAR group(Fig. 1a)，the diameters of the carpospores appeared differentafter day 20. The results indicated that the diametersof tetraspores increased with exposure tO₃6 and 72 J/m ² UV-B radiation on day 48. However，increasedUV-B radiation intensity(108–180 J/m ²)resulted in asignifi cant decrease in tetraspore diameter. Thediameter of tetraspores exposed to a dose of 72 J/m ²UV-B radiation was signifi cantly higher than in theother treatments. In the dark group(Fig. 1b)，thediameters of carpospores appeared different after day12. The diameter of tetraspores exposed to 72 J/m ²UV-B radiation was the highest for the tetraspores inthis group on day 48. Thereafter，the diameterdecreased with increased levels of UV-B radiation and the tetraspore size(in terms of diameter)was thesmallest at 108–180 J/m ² in all experimental treatments . The diameter of tetraspores in the 72 J/m ²treatment was signifi cantly greater than in the 0 J/m ² and other treatments. In the red light group(Fig. 1c)，the trend in tetraspore diameter was the same as thatobserved in the PAR group on day 48. The diametersof carpospores appeared different after day 8. Thediameter of tetraspores at 0，36， and 72 J/m ² UV-Bintensities was signifi cantly greater than in the PAR，dark， and blue light groups at 0，36， and 72 J/m ² onday 48. The average diameter value across the sixUV-B treatments in the red light group(189.14 μm)was greater than that in the groups kept in PAR，darkness or blue light for the 2-h repair period(P< 0.001). In the blue light group(Fig. 1d)，thediameters of carpospores appeared different after day4. The diameters of the tetraspores increased from 0 to 108 J/m ² UV-B radiation， and a peak appeared at108 J/m ² UV-B radiation on day 48. With increasedUV-B radiation levels，the diameter of the tetrasporesdecreased signifi cantly. In the blue light group，thetetraspore diameter in the sample treated with 0 J/m ²was found to be signifi cantly lower than that in theother samples. On day 48，the average diameter in theblue group was signifi cant lower than that in the otherthree groups.

Fig. 1 Variations in tetraspore diameter under different UV-B radiation and light conditions The diameters of tetraspores kept under PAR(a)，darkness(b)， and red(c) and blue(d)light after exposure to different doses of UV-B radiation. Diameterswere measured every 4 days from day 0 to day 48 after release. The letters(A–F)refer to different UV-B radiation doses(0–180 J/m2，Table 1). Data aremean values ±SD(n =3).

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Images of tetraspores treated with different UV-B radiation dosages on day 48 are shown in Fig. 2.Normal tetraspores appeared approximately discshaped，with a smooth edge and a dark color(Fig. 2a).Under low doses of UV-B radiation(36 and 72 J/m ²)(Fig. 2b，c)，the tetraspores grew more rapidlycompared with the control; however，the edges of thealgae appeared rougher. Furthermore，the tetrasporestreated with UV-B levels greater than 108 J/m ²showed blurry edges，irregular shapes，thicklyarranged cells， and a bleached color(180 J/m ²，Fig. 2d). The images show that the growth oftetraspores in PAR and red light(Fig. 2a，c)subjectedto 0 J/m ² UV-B radiation(control)was better than thatin the other two groups(Fig. 2b，d). When the UV-Bradiation level was enhanced to 180 J/m ²(Figs.3，4)，the tetraspores were seriously affected by UV-Bradiation in the four light treatments. The tetrasporesappeared to have blurry edges，irregular shapes，thickly arranged cells， and a bleached color.

Fig. 2 Tetraspores after different UV-B radiation dosetreatments on day 48 Tetraspores from different UV-B radiation dose treatments inthe PAR group were observed under an inverted microscope. a.Treatment without UV-B radiation，that is，C . ocellatus in normalculture conditions; b. 36 J/m2 UV-B radiation treatment; c. 72 J/m2UV-B radiation treatment; d. 180 J/m2 UV-B radiation treatment.Scale bar: 100 μm.

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Fig. 3 Tetraspores exposed to 0 J/m2 UV-B radiation in thePAR，dark，red light and blue light groups On day 48，tetraspores exposed to 0 J/m2 UV-B radiation dosetreatments in the PAR，dark，red light and blue light groups wereobserved under an inverted microscope. a. 0 J/m2 UV-B radiation inthe PAR group; b. 0 J/m2 UV-B radiation in the dark group; c. 0 J/m2 UV-B radiation in the red light group; d. 0 J/m2 UV-B radiationin the blue light group. Scale bar: 100 μm.

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Fig. 4 Tetraspores exposed to 180 J/m2 UV-B radiation inthe PAR，dark，red light and blue light groups On day 48 of the experiments，tetraspores exposed to 180 J/m2UV-B radiation dose treatments in the PAR，dark，red light and bluelight groups were observed under an inverted microscope. a. 180 J/m2 UV-B radiation treatment in the PAR group; b. 180 J/m2 UV-Bradiation treatment in the dark group; c. 180 J/m2 UV-B radiationtreatment in the red light group; d. 180 J/m2 UV-B radiationtreatment in the blue light group. Scale bar: 100 μm.

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The concentrations of CPDs induced by UV-Bradiation were measured on the 48^th day of culture byELISA(Fig. 5). The results shown in Fig. 5 indicatethat UV-B signifi cantly affected the DNA and CPDaccumulation in the PAR group(P<0.001)(Fig. 5a).The degree of damage to the DNA in algal cellsincreased gradually with increasing UV-B radiationfrom 0 to 108 J/m ² . Nevertheless，the CPD contentdropped sharply at a dose of 144 J/m ² . In the groupskept in darkness(P<0.05，P =0.017)(Fig. 5b) and bluelight(P<0.05，P =0.03)(Fig. 5d)，the average values for CPD absorbance were 0.35 and 0.38，respectively， and were signifi cantly lower than those in the PAR and red light groups. Moreover，enhanced UV-Bradiation did not signifi cantly affect the spores in bluelight compared with 0 J/m ² UV-B radiation treatment(P =0.117). In the red light group(P<0.001)(Fig. 5c)，the CPD content increased with increasing levels ofUV-B radiation(36 to 144 J/m ²); however，it decreasedat 180 J/m ² UV-B radiation. The CPD levels weresignifi cantly different from the control only at UV-Bradiation doses of 36，72， and 144 J/m ² .

Fig. 5 CPD concentrations of tetraspores under different UV-B radiation and light condition The CPD concentrations of tetraspores under PAR(a)，darkness(b)， and red(c) and blue(d)light after exposure to different doses of UV-B radiation. TheCPD contents were determined on day 48 of the experiments. Data are mean values±SD(n =3). Different letters above the columns indicate a signifi cantdifference among mean values(LSD test，P<0.05); similar letters denote no signifi cant difference.

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According to Fig. 6，UV-B radiation had asignifi cant effect on some pigments in the tetrasporesin the PAR(P<0.001)(Fig. 6a)，darkness(P<0.001)(Fig. 6b)，red light(P<0.001)(Fig. 6c) and blue light(P<0.001)(Fig. 6d)groups. The Chl-a content oftetraspores in the four repair-treated groups showedthe same trends. The Chl-a content decreasedsignifi cantly at a low level of UV-B radiation(36 J/m ²)to PAR group value 0.63，darkness group value0.65，red light group value 0.53 and blue light group value 0.44 mg/L，respectively. As the level of UV-Bradiation increased，the Chl-a content decreasedsignifi cantly. The Chl-a content in the control for thePAR group was higher than in all of the other controlgroups.

Fig. 6 Chl-a concentrations of tetraspores under different UV-B radiation and light conditions Chl-a concentrations of tetraspores under PAR(a)，darkness(b)， and red(c) and blue(d)light after exposure to different doses of UV-B radiation. Measurementswere carried out on day 48 of the experiments. Data are mean values±SD(n=3). Different letters above the columns indicate a signifi cant difference amongmean values(LSD test，P <0.05); similar letters denote no signifi cant difference.

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UV-B radiation signifi cantly damaged somepigments in the tetraspores in the PAR(P<0.001)(Fig. 7a)，darkness(P<0.001)(Fig. 7b)，red light(P<0.001)(Fig. 7c) and blue light groups(P<0.001)(Fig. 7d). In the PAR group there was a sharp decreasein the phycoerythrin content of the tetraspores whenthe UV-B dose was increased tO 36 J/m ² . Thereafter，the phycoerythrin content decreased with increasingUV-B dosage; however，the decrease was notsignifi cant after a dose of 108 J/m ² in this group. Inthe group kept in darkness，the phycoerythrin contentof the tetraspores signifi cantly decreased at 36 J/m ²，followed by an increase at 72 J/m ²， and decreasedthereafter with increasing UV dosage(Fig. 7b). Thephycoerythrin contents in the red and blue lightgroups showed the same trends as that observed in the PAR group. The samples not subjected to UV-Bradiation in the red and blue light groups hadsignifi cantly lower values than those in the PAR group(P=0.017，P=0.007).

Fig. 7 Phycoerythrin concentrations of tetraspores under different UV-B radiation and light conditions Phycoerythrin concentrations of tetraspores under PAR(a)，darkness(b)， and red(c) and blue(d)light after exposure to different doses of UV-B radiation.Measurements were carried out on day 48 of the experiments. Data are mean values±SD(n=3). Different letters above the columns indicate a signifi cantdifference among mean values(LSD test，P<0.05); similar letters denote no signifi cant difference.

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The MAAs in the tetraspores were analyzed byLC/MS(Fig. 8). Our results showed the presence oftwo kinds of MAA(palythine，245.113 7 and asterina-330，289.138 1)in the tetraspores ofC . ocellatus . These MAAs were quantifi ed by HPLC.The retention times of the extracted compoundssubjected to different UV-B radiation treatments inthe four groups were found to be similar，indicatingthat the chemical composition of the extractedcompounds was identical in different samples. TheMAA contents in the carpospores from the four lightrepair groups are shown in Fig. 9. In the PAR group(P<0.001)(Fig. 9a)，low levels of UV-B radiation(36 and 72 J/m ²)resulted in increased MAA synthesis， and the MAA contents increased to 0.94 and 0.98 mg/g. With increased UV-B radiation levels，theMAA content decreased signifi cantly. In the group kept in darkness(P<0.001)，the MAAs increasedwith increased UV-B radiation levels(Fig. 9b). In thered light group(P<0.001)(Fig. 9c)，the MAA contentin the 0 J/m ²)UV-B radiation treatment(0.085 mg/g)was higher than that in all of the other groups. Whenthe level of UV-B radiation was increased from 36 J/m ² to 180 J/m ²，the MAA content showed the sametrend as that in the blue light group(P<0.001)(Fig. 9d). The MAA content increased as the UV-Bradiation level was increased from 36 J/m ² to 108 J/m ²， and began to decrease at a UV-B dose of 144 J/m ² . The average MAA content in the PAR group wasabout 0.069 mg/g，which was higher than in thedarkness，red and blue light groups.

Fig. 8 Extracted ion currents of MAAs in the tetraspores of C . ocellatus Extracted ion currents of MAAs(palythine，245.113 7 and asterina-330，289.138 1)in the tetraspores of C . ocellatus . The charts show molecular weights asdetermined by LC/MS.

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Fig. 9 MAA concentrations of tetraspores under different UV-B radiation and light conditions MAA concentrations of tetraspores under PAR(a)，darkness(b)， and red(c) and blue(d)light after exposure to different doses of UV-B radiation. MAAcontents were determined on day 48 of the experiments. Data are mean values±SD(n=3). Different letters above the columns indicate a signifi cant differenceamong mean values(LSD test，P<0.05); similar letters denote no signifi cant difference.

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UV-B radiation is considered to be detrimental tomarine algal growth and development. It can cause serious damage to the photosynthetic apparatus，DNAstructure， and other cellular structures of algae(Britt，1995; Kolb et al., 2001; Xiong and Day， 2001; Apprill and Michael et al., 2003; Liu et al., 2008). Ourprevious experimental results showed the negative effects of high UV-B radiation doses on thedevelopment of C . ocellatus under laboratoryconditions. Low dosage UV-B radiation(36 and 72 J/m ²)strongly promoted the growth of tetraspores(Figs.1a，2b). However，tetraspore growth clearly decreased with increasing UV-B radiation levels(Figs.1a，2c，2d). In addition，signifi cantmorphological changes in the carpospores wereinduced by UV-B radiation; the discoidal body ofC . ocellatus showed blurry edges，irregular shapes，thickly arranged cells， and a light color. It is evidentfrom the present investigation that UV-B radiationcan induce CPD formation in the DNA， and the CPDcontent increases with enhanced UV-B radiation.Thus，the replication and transcription of DNA isinhibited by increased CPDs. However，as the dose ofUV-B radiation continued to increase，the CPDcontent was found to decrease signifi cantly. It ispossible that there are several repair mechanismsclassifi ed into dark repair and photoreactivation intetraspores that are induced by serious DNA damage.UV-B radiation was also found to adversely affect thepigments in C . ocellatus，such as the concentrationsof phycoerythrin and Chl-a(Ju et al., 2011).

MAAs absorb harmful UV radiation as effective UV screening compounds and emit fl uorescence toprevent UV-induced photodamage in many marinealgae(Dunlap and Shick， 1998; Karsten et al., 1998).Several investigations have demonstrated that MAAsperform not only as UV-B absorbing compounds，butalso protect against UV-B radiation(Warwick and Roy， 1993; Castenholz，1997; Oren，1997). It hasbeen reported that MAAs may act as antioxidants toprevent cellular damage caused by UV-inducedproduction of active oxygen(Dunlap and Yamamoto， 1995)，especially mycosporine-glycine(M-gly) and the MAA precursor deoxygadusol，which inhibit lipidperoxidation(Dunlap and Yamamoto， 1995; Dunlap and Shick， 1998) and scavenge 1 O 2 generated fromcertain endogenous photosensitizers(Suh et al., 2003). We identifi ed palythine and asterina-330 inC . ocellatus according to their molecular weights and structures by LC/MS following Whitehead and Hedges(2002) and Yuan et al.(2008). Our previousresults showed that samples not induced with UV-B also contained MAAs. Therefore，stimulation by lowUV-B radiation(36 J/m ² and 72 J/m ²)，induces C .ocellatus to produce more MAAs to protect itselffrom UV-B damage. However，exceedingly highdoses of UV-B radiation resulted in a gradual decreaseof MAAs. We hypothesize that MAAs are spent toprovide antioxidant functions as well as UV-Bprotection. MAA consumption exceeded synthesis，which led to lower levels of MAA(Ju et al., 2011).4.2 Effects of red and blue light on the repair ofUV-B-induced damage

The repair function of red light after exposure toUV-B radiation was evaluated in the present study.Erect branches formed earliest under red light and thediameters were higher under 0?72 J/m ² UV-Bradiation compared with the other C . ocellatus groups(Figs.1c，3c). We speculate that red light can promotethe growth of algae exposed to damaging UV-Bradiation at an appropriate dosage under normalculture conditions. Red light also stimulates proteinsynthesis to some extent(López，1991). Our resultsdemonstrated that the growth rate of the C . ocellatusin red light without UV-B radiation was higher than inthe other three treatments，suggesting that red light issuperior for promoting growth of tetraspores，followed by PAR，darkness and blue light in thatorder. In the red light group，the CPD content in thesamples not exposed to UV-B radiation was found tobe higher than that in the PAR group(Fig. 5c). Aprevious study indicated that dim red light has aninhibitory effect on the photorepair of DNA(Freeman et al., 1989). In our study，the CPD content in the 0 J/m ² UV-B radiation treatment in the red light groupwas higher than that in the PAR group. Thus，theCPDs in the treatment without UV-B radiation in thered light group could not be repaired completelyunder 2 h of red light. Furthermore，CPD accumulationstill decreased signifi cantlyin red light repairconditions at 36 J/m ² UV-B radiation. We speculatethat since photorepair is inhibited by red light，there isanother DNA repair mechanism that is activated，suchas dark repair. According to our results，thephycoerythrin and Chl-a contents of the samplewithout UV-B radiation in the red light group weresignifi cantly lower than that in the PAR group(Figs.6c，7c). We speculate that a dose of red light cancause some damage to the pigments in the tetraspores.Then phycoerythrin and Chl-a contents in carposporeswere signifi cantly lower at high UV-B doses(>36 J/m ²). Red light conditions possibly reduce the capacity of C . ocellatus to produce MAAs(Fig. 9c). In the redlight group，the maximum MAA content was found ata UV-B radiation level of 108 J/m ² .

In this study，the average diameter of tetrasporestreated with 0 J/m ² UV-B radiation was signifi cantlower than that in the other three groups(Figs.1d，5d，6d). Additionally，the average diameter in the bluelight group was signifi cant lower compared with thatin the PAR，dark and red light groups. This indicatesthat blue light inhibits the growth of tetraspores. Otherstudies have reported that an array of photoreceptorsin Arabidopsis can absorb different wavelengths oflight; therefore，when phytochrome absorbs onlytrace amounts of blue light，the growth of Arabidopsisis inhibited by blue light(Shao，2001). Some studiesindicated that the growth of red algae was lowestunder blue light compare that subject with white and red light. A possible explanation is that the blue lightcould decrease the ability of inorganic carbonassimilation and photo-absorption effi ciency，thenactual photosynthetic effi ciency was decreased(Aguilera et al., 2000; Korbee et al., 2005). The CPDconcentration was always lower in the blue lightrepair group compared with the PAR，darkness and red light groups(Fig. 5d). Blue light improves theactivity of photolyase，which is one of the mostimportant enzymes in DNA photoreactivation(Britt，1995; Zhong et al., 2009). As an energy source，near-UV/blue light(300?500 nm)not only promotes aphotoreactivation process，which is activated byphotolyase enzymes，but also rapidly converts CPDsto undamaged bases(Takahashi et al., 2006).Moreover，blue light initiates a number of cellularresponses，such as photolyase DNA repair，accordingto Thompson(2002). The average contents ofphycoerythrin and Chl-a were signifi cantly lowercompare with average phycoerythrin and Chl-acontents in PAR group. The blue light may exacerbatethe photosynthetic pigments damaged by UV-Bradiation. Some studies indicated that the chlorophyllcontents in the leaves of leaf lettuce，cucumber and strawberries were decreased when conducted withblue light(Chu et al., 1999; Xu et al., 2005，2007).

The MAA level was highest when the UV-Bradiation level was 108 J/m ² in the blue light group(Fig. 9d)， and was relatively low compared with thatin the PAR group. Blue light possibly reduces thecapacity of C . ocellatus to form MAAs. Thetetraspores in the blue light group had the smallestdiameter on day 48. This indicated that there issynergy between blue light and UV-B radiation.Although，blue light induces photolyase in algae，C . ocellatus showed poor growth. This may bebecause blue light repair conditions are not benefi cialfor the growth of C . ocellatus .

In observations of the growth of tetraspores，anappropriate amount of red light was found to be betterfor recovery from UV-B radiation(0.72 J/m ²)damage and the growth and development ofC . ocellatus tetraspores. Our results suggest that amoderate amount of red light could induce the growthof this alga in aquaculture. Conversely，blue lightinhibits the growth of tetraspores. Although blue lightcan promote the activity of DNA photolyase and greatly improve remediation effi ciency，it was foundto have a negative effect on the repair of UV-Binduced damage. Red and blue light may reduce thecapacity of C . ocellatus to form MAA. Our resultssuggest that red and blue light play different butimportant roles during UV-B induced damage repairprocesses.5 ACKNOWLEDGMENT

The authors would like to acknowledge the staff ofthe First Institute of Oceanography(SOA)(Qingdao，Sh and ong，China)for the use of their LC/MS.