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Chinese Journal of Oceanology and Limnology

Vol. 28 No. 4, P. 738-748, 2010

DOI: 10.1007/s00343-010-9908-2

Optimization of conditions for tetraspore release and

assessment of photosynthetic activities for different

generation branches ofGracilaria lemaneiformis Bory*

WANG Zhiyuan () , WANG Guangce () ,**, NIU Jianfeng () ,WANG Wenjun () , PENG Guang ()

College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

Received Jun. 23, 2009; revision accepted Aug. 17, 2009

Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2010

Abstract Gracilaria lemaneiformis Bory is an economically important alga that is primarily used for

agar production. Although tetraspores are ideal seeds for the cultivation of G. lemaneiformis, the most

popular culture method is currently based on vegetative fragments, which is labor-intensive and

time-consuming. In this study, we optimized the conditions for tetraspore release and evaluated the

photosynthetic activities of different colonies formed from the branches ofG. lemaneiformis using a PAM

(pulse-amplitude-modulated) measuring system. The results showed that variations in temperature and

salinityhad significant effects on tetraspore yield. However, variations in the photon flux density (from

15 mol m-2 s-1 to 480 mol m-2 s-1) had no apparent effect on tetraspore yield. Moreover, the

PAM-parameters Y(I), Y(II), ETR(I), ETR(II) and Fv/Fm of colonies formed from different branches

showed the same trend: parameter values of first generation branches>second generation branches>third

generation branches. These results suggest that the photosynthetic activities of different colonies of

branches changed with the same trend. Furthermore, photosynthesis in G. lemaneiformis was found to beinvolved in vegetative reproduction and tetraspore formation. Finally, the first generation branches grew

slowly, but accumulated organic compounds to form large numbers of tetraspores. Taken together, these

results showed that the first generation branches are ideal materials for the release of tetraspores.

Keyword: Gracilaria lemaneiformis Bory; tetraspore; pulse-amplitude-modulated; photosynthesis

Abbreviation:ETR (I): Relative rates of photosynthetic electron transport

of PSIETR(II): Relative rates of photosynthetic electron transport

of PSIIF0: Minimum fluorescence yield

Fm: Maximum fluorescence yieldFm': Maximum fluorescence yield in illuminated samplesFv/Fm: Optimum quantum yieldOD: Optical densityPE: Phycoerythrobilin

P700: The reaction center of PSIPAR: Photosynthetic active radiationPSI: Photosystem IPSII: Photosystem IIY(I): Effective PSI quantum yieldY(II): Effective PSII quantum yieldY(NA): Nonphotochemical quantum yield of PSI caused by

acceptor side limitationY(ND): Nonphotochemical quantum yield of PSI caused by

donor side limitation

1 INTRODUCTION

The genus Gracilaria is found worldwide and hasbeen reported in temperate, tropical and arcticregions. The genus Gracilaria, was first described byGreville in 1830. Originally, the genus only includedfour species; however, Agardh reexamined the genus

in 1852 and the number of species increased to 23.

Currently, the genus Gracilaria contains more than100 species that covered worldwide (Bird et al.,

Supported by the National Natural Science Foundation of China (No.

30830015), the National Key Technology Research and Development

Program of the 11th Five-Year Plan (No. 2006BAD09A04), and the

National High Technology Research and Development Program of China

(863 Program, Nos. 2006AA10A402, 2007AA09Z406, 2006AA05Z112,

2006AA10A413)

Corresponding author: [email protected]

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No.4 WANG et al.: Optimized tetraspore release conditions ofG. lemaneiformis 739

1984; Liu, 1987; Fei et al., 1998), of which 30 havebeen recorded in China (Wu, 1998).

Gracilaria is characterized by a typicalpolysiphonia-type life history with an alternation ofisomorphic generations. Specifically, mature

tetrasporophytes (2n) produce tetraspores (n) throughmeioses. Some of the tetraspores develop into malegametophytes (n), while others grow to femalegametophytes (n), which then form spermatangi andcarpogonia, respectively. After fertilization,cystocarps are formed on the female plant, whichsubsequently release carpospores (2n) that developinto tetrasporophytes (Ogata et al., 1972; de Oliveiraet al., 1984; Yamamoto et al., 1988; Kain et al., 1995;Engel et al., 2001).

Most species belonging to the Gracilaria genus

are economically important algae that are used foragar extraction (Craigie et al., 1984; Glickman, 1987;Santelices et al., 1989; Marinho-Soriano, 2001;Marinho-Soriano et al., 2003; Freile-Pelegrn et al.,2005) or to obtain natural products with importantbioactivities, such as phycoerythrin (PE) andhemagglutinin (Mazumder et al., 2002; Melo et al.,2002). Gracilaria also serve as efficient bio-filtrationsystems (Neori et al., 1996; Troell et al., 1997; Yanget al., 2000; Yang et al., 2005). In the last fewdecades, most of the Gracilaria biomass has beenobtained from wild stocks, while marine aquaculture

has only contributed a small amount to the totalharvest. However, the need for Gracilaria hasincreased greatly with the development of the agarindustry. Thus, greater attention has been given to thedevelopment of artificial Gracilaria cultures in manycountries, especially in Southeast Asia (Glenn et al.,1996).

By 1991, approximately one-third of theGracilaria harvested worldwide was obtained fromcultured sources (McHugh, 1991). Cultivation ofGracilaria in China began in the 1950s (Tseng, 2001;

Zou et al., 2004), and the main species cultivatedwere Gracilaria asiatica Zhang et Xia, Gracilarialemaneiformis Bory and Gracilaria tenuistipitataChang et Xia. Among these species, G.lemaneiformis was regarded as the most importantcommercial species due to its rapid growth and thehigh-quality gel produced when compared with otherspecies (Craigie et al., 1984). G. lemaneiformisnormally grows in gravel in the lower intertidal zone.G. lemaneiformis growing in these areas arecharacterized by multiple branches, while the bases

of the thallus are covered by sand. The thallus ofG. lemaneiformis is reddish purple. The thallus come

out from a large holdfast. The rate of biomassaccumulation peaks in spring and autumn (Li et al.,1984).

Most propagation methods of G. lemaneiformisare dependent on vegetative fragments rather than

tetraspores or carpospores (Hurtado-Ponce, 1990;Santelices et al., 1995; Nelson et al., 2001; Ryder etal., 2004; You et al., 2004). However, it has beenfound that thallus farming is not economical becauseit requires large amounts of propagating material tobegin a plantation (Glenn et al., 1996). For example,approximately 20%30% of the harvest may be usedas seed material in small-scale culture(Hurtado-Ponce et al., 1992). Furthermore, thispractice is labor- and time-consuming. Conversely,the spore-cultured method is economical and highly

efficient. Thus, development of a method ofcollecting spores and culturing sporelings in the seawould be very useful and has therefore attracted agreat deal of attention (Levy et al., 1990; Glenn et al.,1996; 1998).

People rarely use tetraspores to cultureG. lemaneiformis because there is still littleinformation available regarding tetraspores (Albertoet al., 1999). Therefore, the development oftetraspore cultivation methods forG. lemaneiformisrequires more details regarding the release oftetraspores and the selection of culture conditions.Here, we reported the optimization of tetrasporesreleased from different branchlet colonies and thephotosynthetic activities of these different branchletswas studied.

2 MATERIALS AND METHODS

Mature tetrasporohytes ofG. lemaneiformis werecollected in November 2007 from the intertidal zoneof Zhanshan Bay, Qingdao (360N, 12003E),China. After being washed in seawater to remove theepiphytes and sand, rinsed with sterilized seawatertwice and soaked in 1% sodium hypochlorite for2 min, the organisms were acclimated in sterilizedseawater (temperature, 20C; photon flux density,30 mol m-2s-1; salinity, 30) for one week and thealgae were then used for the following experiments.

2.1 Branches ofG. lemaneiformis

G. lemaneiformis has multiple branches that leadto the production of different colonies. Specifically,G. lemaneiformis consist of first, second and thirdgeneration branches. First generation branches are

caulis that grow directly on the holdfast, whilesecond generation branches are produced by the first

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740 CHIN. J. OCEANOL. LIMNOL., 28(4), 2010 Vol.28

generation branches and third generation branchesare produced by the second generation branches(Fig.1).

Fig.1 Mature Gracilaria lemaneiformis collected in the field

The differences in colony development of the branches are shown by the

beeline

2.2 Treatment of thalli used for the release of

tetraspores and measurement of the fluorescence

and P700 levels

The thalli used for the release of tetrasporesrelease were soaked in seawater containing 0.1 g L-1kanamycin, 0.3 g L-1 penicillin G, 1 g L-1 streptomycinsulphate, 0.02 g L-1 cefotaxine and 0.1 g ml-1 GeO2for about 6 h. Next, the first, second and thirdgeneration branches were detached and cut into smallsegments (about 5 cm). All of the branches werestored in sterilized seawater prior to use. The firstgeneration branches were prepared for optimizationof the release of tetraspores. The fluorescence and

P700 levels of the three generation branches weremeasured using the Dual-PAM-100 system.

2.3 Optimization of tetraspore release conditions

Twenty small segments of first generationbranches detached from the same individual wereplaced into individual petri dishes and the weights ofthe thalli were then recorded. Next, the temperaturesof those small segments were measured once a dayusing a temperature indicator (Midwest Group,M307477, China). The photon flux densities weremeasured using an illuminometer (PhotoelectricInstrument Factory of Beijing Normal University,ST-80C, China). The salinities of the seawater weremeasured using a hand-held refractometer (ATAGO,S-10E, Japan).

The effects of five different temperatures (10C,15C, 20C, 25C, 30C) were evaluated at a photonflux density of 30 mol m-2 s-1 while the salinity of

the seawater was 30. The effects of photon fluxwas evaluated by culturing sets of thalli at six photonflux densities (15, 30, 60, 120, 240 and 480 mol m-2s-1) at 20C while the salinity of the seawater was 30.Finally, the effects of five salinities (10, 20, 30, 35,40) were evaluated at 20C and a photon flux densityof 30 mol m-2 s-1.

All branchlets were placed in petri dishes (9 cm indiameter) and cultured in PES culture medium incultivation chambers. Each petri dish contained2 grams of the first generation branches, and each

treatment was replicated in a parallel Petri dish. Allmaterials were incubated under a photoperiod of12:12 h light:dark scheme and the culture mediumwas replaced once a week throughout the studyperiod. After the tetraspores were released, the petridishes were shaken gently so that they would beevenly distributed, after which five fields of view ineach petri dish were selected at random and thetetraspore number in each field was counted. Thenumber of released tetraspores in each petri dish wasthen calculated.

2.4 PSI and PSII measurements

The first, second and third generation brancheswere all detached from the same individual and thencut into small segments. Next, the fluorescence andP700 parameters of the branchlets of differentcolonies were examined using a Dual-PAM-100Measuring System (Heinz Walz GmbH, Eichenring 6,91090 Effeltrich, Germany) that had been optimizedfor simultaneous assessment of P700 and chlorophyllfluorescence using the pulse-amplitude modulated(PAM) method and the Saturation Pulse (SP) method.

The PAM and SP method can be used to obtain P700and fluorescence signals simultaneously.

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A PAM fluorescence measurement that wasnondestructive and could be conducted in-vivo wasused in this study. For measurement, the samples(different generation branches) were placed directlybetween the ends of two 10 10 mm perspex rodsand then measured to generate an automatedinduction and recovery curve as described by themanufacturer. All data were recorded using theDual-PAM windows software.

The Fv/Fm ratio (maximum photochemicalquantum yield of PSII) was calculated according tothe following equation (van Kooten et al., 1990):

Fv/Fm = (FmF0)/Fm (1)

where F0 represents the dark fluorescence yield,

which was determined after keeping the tissue indarkness for 15 min. Saturating actinic light pulses(SP) were applied to obtain maximum fluorescence(Fm) in the dark-adapted samples. The maximumfluorescence yields in the illuminated samples wererecorded as Fm'. The effective PS II quantum yieldwas calculated using the following formula:

Y(II) = (Fm'F)/Fm' (2)

The relative rates of the photosynthetic electrontransport of PSII (ETRII) or PSI (ETRI) were

calculated as the effective quantum yield (Y)multiplied by the photosynthetic active radiation(PAR) received by PSII or PSI:

rETR = YPAR0.5 (3)

The P700 signals ranged from a minimum level(P700 fully reduced) to a maximum level (P700 fullyoxidized). The maximum level, which was denotedas Pm, was determined by application of SP in thepresence of far-red light (FR). ThePm is analogous tothe Fm, Pm was determined after Far-Red

preillumination through application of a saturationpulse. Similarly, Pm' was analogous toFm'.

P700 red, which was determined using a saturationpulse, represents the fraction of the overall P700 thatis reduced in a given state. The nonphotochemicalquantum yields of PSI caused by donor site limitationand acceptor side limitation, Y(ND) and Y(NA),were calculated as follows:

Y(ND)=1P700 red (4)

Y(NA)=PmPm'/Pm (5)

Y(I) is calculated from the complementary PS I

quantum yields of nonphotochemical energydissipation Y(ND) and Y(NA):

Y(I) = 1Y(ND) Y(NA) (6)

The light curves (LCs) of different generationbranches were measured. The LC consists of thefluorescence responses to 13 different actinicirradiances (PAR 0, 14, 21, 30, 61, 103, 134, 224,347, 539, 833, 1 295 and 1 960 mol m-2 s-1). Togenerate these curves, PAR was applied in increasingorder, with duration of 30 s at each level. Tenrepeated measurements were conducted for each typeof branch.

2.5 Growth measurements

Different colonies of branches were placed in petridishes. Specifically, each petri dish contained threegrams of thalli and each branch was replicated in twoparallel petri dishes. All branchlets were cultured inPES culture medium in cultivation chambers at 20C,with a salinity of 30 and a temperature of 20C. Allthalli were kept under a photoperiod of 12:12 h LD.The culture medium was replaced once a week andall thalli were weighed after 30 days.

2.6 Pigment content

The branches of each colony were finely cut andthe pigments were then extracted using the followingtwo methods.

2.6.1 Extraction of water-soluble pigment

A total of 0.2 g thalli were placed into a mortar,after which 0.5 ml of distilled water was added andthe thalli were ground with a pestle. The mortar wasthen placed at -20C for about ten minutes, afterwhich the samples were ground again. The abovesteps were repeated four times and the slurry was

then centrifuged at 8 800g for 5 min. Next, thepigment was then scanned using a spectrophotometer(Du650, BECKMEN, USA) at 400 to 800 nm inincrements of 2 nm with a 1-cm light path. Thephycoerythrin content was calculated according tothe following formula (Kursar et al., 1983):

PE=155.8OD498.540.0OD61410.5OD651 (7)

2.6.2 Extraction of lipid-soluble pigment

The pigment was extracted and calculated

according to the method described by Jensen (1978),with some modifications. Specifically, 1.0 ml of 80%

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acetone was added to a mortar containing 0.5 g offresh thalli. The G. lemaneiformis tissue was thenground with a pestle until the acetone turned bright,deep green. The liquid was then transferred to acentrifuge tube, after which the mortar and pestle was

rinsed with another 1.0 ml acetone. All of the acetoneextracts were then combined and filtered into avolumetric flask using a glass funnel. The finalvolume of the extract was diluted to 25 ml withacetone and the content of the chlorophyll a wascalculated using the following formula:

C= (D666-D730) V10/890 (8)

2.7 Data statistics and analysis

The results were analyzed according to one-wayANOVA (analysis of variance) followed byTukeys-test when appropriate. Statistical analyseswere performed using SPSS13.0 with a P

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overall, with the yields of the first generationbranches being the highest and those of the thirdgeneration branches being the lowest. As the PARincreased, the parameters changed in different ways.Specifically, the maximum values of Y(I) and Y(II)

were observed when the mean photon flux densitywas 22 mol m-2 s-1 (first generation branches: Y(I) =0.58, Y(II) = 0.505; second generation branches: Y(I)= 0.48, Y(II) = 0.379; third generation branches Y(I)= 0.4, Y(II) = 0.366), but these values decreasedsharply as the photon flux density increased. Theminimum values of Y(I) and Y(II) were observed at aphoton flux density of 1 362 mol m-2 s-1 (firstgeneration branches: Y(I)=0.23, Y(II)=0.016; secondgeneration branches: Y(I) = 0.02, Y(II)= 0.0127;third generation branches Y(I) = 0.012, Y(II) = 0.01)(Figs.5, 6). However, as the PAR increased, the

ETR(I) values increased, with the maximum valuesbeing observed when the mean photon flux densitywas 99 mol m-2 s-1. Specifically, the followingvalues were observed: ETR(I)=14.51; secondgeneration branches: ETR(I) = 10.4; third generationbranches ETR(I) = 9.5. These values then decreasedas the PAR increased from 99 mol m-2 s-1 (Fig.7).Similarly, as the PAR increased, the ETR(II) valuesincreased until the photon flux density reached370 mol m-2 s-1 (first generation branches: ETR(II) =8.99; second generation branches: ETR(II) = 8.32;third generation branches: ETR(II)=5.68), after

which they decreased (Fig.8). The Fv/Fm (MaximalPS II quantum yield) did not change as the photonflux density increased, as indicated by mean valuesof 0.527 4 for first generation branches, 0.420 5 forsecond generation branches and 0.401 4 for thirdgeneration branches (P

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Fig.10 Increase in biomass of different colonies of branches

after 30 days of culture (P

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Fig.11 Absorption spectra of water-soluble pigments ofGracilaria lemaneiformis

A. 1st generation branches; B. 2nd generation branches; C. 3rd generation branches

been examined using pulse-amplitude-modulated(PAM) fluorescence techniques (Aline et al., 2007).Chlorophyll (Chl) a fluorescence measurements arewidely used to assess the physiological state ofhigher plants and algae due to their being rapid,

simple and non-invasive methods (Juneau et al., 2005).

Information regarding the photosynthet icperformance can be obtained with the aid of LightCurves (LC). Specifically, the parameters Y(I), Y(II),ETR(I) and ETR(II) obtained from LC reflected thephotosynthetic capacity of different colonies of

branches, and the Fv/Fm (Maximal PS II quantum

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Fig.12 Absorption spectra of lipid-soluble pigments ofGracilaria lemaneiformis

A. 1st generation branches; B. 2nd generation branches; C. 3rd generation branches

yield) represents the potential maximumphotosynthetic capacity of different colonies ofbranches. In this study, it was found that differentcolonies of branches had different parameter values,and that these parameters changed in a similarfashion, namely first generation branches>second

generation branches>third generation branches.These findings indicate that the sequence ofphotosynthetic activity and the potential maximumphotosynthetic capacity was as follows: firstgeneration branches>second generation branches>thirdgeneration branches. These findings completely

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contradict our original presumption that thephotosynthetic activity of third generation branchesis stronger than that of first generation branches.

Based on our findings, photosynthesis is involvedin two important steps in the cultivation of G.

lemaneiformis, vegetative reproduction and theaccumulation of organic compounds to formtetraspores. Additionally, we found that firstgeneration branches grew slowly and accumulatedlarge numbers of tetraspores, while third generationbranches grew rapidly and accumulated fewtetraspores, indicating that first generation brancheshave a stronger photosynthetic activity than thirdgeneration branches. The pigment content ofdifferent colonies of branches also reflected thisphenomenon, with first generation branches showing

the highest content of phycoerythrin and chlorophylland third generation branches having the lowestlevels of these compounds.

Normally, when spores are collected fromGracilaria they are collected from third generationbranches rather than the first generation branches.However, according to the results of this experimentand those of our previous studies, first generationbranches of tetrasporohytes possessed more than80% of the total tetraspores, while second and thirdgeneration branches contained only 20% of the total

tetraspores (Ye et al., 2006). Therefore, firstgeneration branches are ideal materials for sporerelease. Accordingly, future studies to evaluate thecultivation ofG. lemaneiformis should focus on firstgeneration branches.

G. lemaneiformis is a valuable alga that has notbeen extensively exploited. With the development ofthe agar industry, the demand forGracilaria and itsprice have increased sharply, which has resulted inserious damage to natural stocks (Hanisak, 1998).Indeed, the price of dried G. lemaneiformis was1 500 US dollars per ton in China in 2007.

Additionally, Gracilaria has become scarce alongthe coast of China. Thus, artificial cultivation ofGracilaria should be widely developed to complementthe available natural resources. In addition, furtherstudies should be conducted to facilitate the cultivationand improve the available strains ofGracilaria.

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