PRIMARY RESEARCH PAPER

Effects of UVB radiation on the carragenophyteKappaphycus alvarezii (Rhodophyta, Gigartinales): changesin ultrastructure, growth, and photosynthetic pigments

Eder C. Schmidt • Marcelo Maraschin •

Zenilda L. Bouzon

Received: 21 September 2009 / Revised: 11 March 2010 / Accepted: 22 March 2010 / Published online: 7 April 2010

� Springer Science+Business Media B.V. 2010

Abstract Damage to the ozone layer has led to

increased levels of ultraviolet radiation at the earth’s

surface. Increased ultraviolet radiation can affect

macroalgae in many important ways, including

reduced growth rate, changes in cell biology and

ultrastructure. Kappaphycus alvarezii is a red macro-

alga of economic interest due to its production of kappa

carrageenan. In this study, we examined two strains of

K. alvarezii (green and red) exposed to ultraviolet B

radiation (UVBR) for 3 h per day during 28 days of

cultivation in vitro. UVBR caused changes in the

ultrastructure of cortical and subcortical cells, which

included increased thickness of the cell wall and

plastoglobuli, reduced intracellular spaces, changes in

the cell contour, and destruction of chloroplast internal

organization. While the green strain exposed to

photosynthetically active radiation (PAR) showed

growth rates of 6.75% day-1, the red strain grew only

6.35% day-1. Upon exposure to PAR ? UV-B, a

decreasing trend in growth rates was observed for both

strains, with the green strain growing 3.0% day-1 and

the red strain growing 2.77% day-1. Significant

differences in growth rates between control and UV-

B-exposed algae were also found in both strains.

Furthermore, compared with control algae, phycobili-

protein contents (phycoerythrin, phycocyanin, and

allophycocyanin) were observed to decrease in both

strains after PAR ? UV-B exposure. However, while

the chlorophyll a levels increased in both strains, the

green strain showed no significant differences in

chlorophyll a levels. Taken together, these findings

strongly suggested that UVBR negatively affects the

ultrastructure, growth rates, and photosynthetic pig-

ments of intertidal macroalgae and, in the long term,

their economic viability.

Keywords Ultraviolet radiation � Kappaphycus

alvarezii � Cell wall � Chloroplast � Growth rates �Photosynthetic pigments � Red algae � Ultraviolet

radiation B � Ultrastructure � Culture

Introduction

The stratospheric ozone layer provides natural pro-

tection against ultraviolet radiation (UVR) exposure

Handling editor: T. P. Crowe

E. C. Schmidt (&) � Z. L. Bouzon

Department of Cell Biology, Embryology and Genetics,

Federal University of Santa Catarina, CP 476,

Florianopolis, SC 88049-900, Brazil

e-mail: edcash@ccb.ufsc.com

M. Maraschin

Plant Morphogenesis and Biochemistry Laboratory,

Federal University of Santa Catarina, CP 476,

Florianopolis, SC 88049-900, Brazil

Z. L. Bouzon

Central Laboratory of Electron Microscopy, Federal

University of Santa Catarina, CP 476, Florianopolis,

SC 88049-900, Brazil

123

Hydrobiologia (2010) 649:171–182

DOI 10.1007/s10750-010-0243-6

for all biological organisms (Madronich, 1992). It has

been nearly three decades since the first reports about

man-made changes in the stratospheric ozone layer,

which resulted from atmospheric pollutants, such as

chlorofluorocarbons (CFC), halocarbons, carbon

dioxide (CO2), and methyl chloroform (MCF) (Kerr

& McElroy, 1993). Increasingly, ultraviolet B radi-

ation (UVBR) (280–320 nm) reaches the earth’s

surface as a result of this ozone layer depletion

(Mitchell et al., 1992; Lubin & Jensen, 1995). UV

energy induces photodamage in proteins, nucleic

acids, and other compounds in biological tissues

(Mitchell et al., 1992), as well as physiological

processes and ultrastructure (Bischof et al., 2006).

Similar to other regions in its latitude, southern

Brazil has been exposed to a gradual increase in the

levels of UVR. According to the Brazilian Institute

for Space Research (INPE), this region receives

ultraviolet radiation from 2.2 to 3.5 W m-2, based on

daily UV-A and UV-B irradiances that vary from 9 to

14 during a typical summer (Global Solar UV

Index—UVI). As a consequence, the effects of

ultraviolet radiation (UV-A and UV-B) on biological

matter have become an increasingly important issue

(Holzinger & Lutz, 2006). Ultraviolet radiation

affects all biological organisms, especially those in

the aquatic ecosystem, in many important ways.

Accumulated DNA damage in diverse macroalgae

has been studied in brown macroalgae, including

Laminaria digitata (Hudson) J.V. Lamouroux,

L. saccharina (Linnaeus) J.V. Lamouroux, and

L. solidungula J. Agardh (Roleda et al., 2006b), and

in red macroalgae, such as Mastocarpus stellatus

(Stackhouse) Guiry and Chondrus crispus Stack-

house (Roleda et al., 2004b). In addition, several

studies have shown a decreased macroalgae growth

rate (Wood, 1987) and reduced primary productivity

(Worrest, 1983). The photosynthetic process is also

potentially affected by inhibiting the activity of the

1,5 di-phosphate carboxylase/oxygenase (Rubisco)

D1 protein of the photosystem II reaction center

(Lesser & Shick, 1994) and by altering the thylakoid

membrane composition of chloroplasts (Grossman

et al., 1993).

One of the strategies used by macroalgae to

survive exposure to high levels of UVR is the

synthesis and accumulation of photoprotective com-

pounds, such as mycosporine-like amino acids

(MAAs) and carotenoids, which directly or indirectly

absorb UVR energy (Karsten & Wiencke, 1999;

Karsten et al., 1999, 2000; Sommaruga, 2001;

Sonntag et al., 2007). Nonetheless, photosynthetic

pigmentation is a main target of ultraviolet-B radi-

ation. Several studies suggest that changes have

occurred in the concentrations of chlorophyll a in

such red macroalgae species as Leptosomia simplex

L. (Dohler, 1998), Mastocarpus stellatus and Chon-

drus crispus (Roleda et al., 2004b), Palmaria pal-

mata, and Phycodrys rubens (Bischof et al., 2000).

Phycobiliprotein content has also been altered, as

demonstrated in studies by Eswaran et al. (2001)

reporting on K. alvarezii cultivated in long line and

Ahnfeltiopsis concinna (J. Agardh) PC Silva & De

Cew (Beach et al., 2000). Finally, some papers have

reported changes in the ultrastructure of macroalgae

exposed to UVBR (Poppe et al., 2002, 2003; Garbary

et al., 2004; Holzinger et al., 2004, 2006; Holzinger

& Lutz, 2006; Schmidt et al., 2009). These changes

mainly occur in the chloroplasts, modifying the

quantity, size, organization, as well as the number

of thylakoids (Talarico & Maranzana, 2000). At the

same time, however, other studies have not shown

ultrastructural damage in green algae, such as Zyg-

nema C. Agardh exposed to PAR ? UV-A ? UV-B

during 24 h (Holzinger et al., 2009) or Urospora

penicilliformis (Roth) J.E. Areschoug (Roleda et al.,

2009).

It is true that the species Kappaphycus alvarezii

(Doty) Doty ex P. Silva has been reported in several

studies in Brazil. These studies, particularly those

representing the States of Sao Paulo, Rio de Janeiro,

and Santa Catarina (Florianopolis), have reported on

growth rates, both within the confines of aquaculture

and in vitro, carrageenan analyses, and strain selec-

tion (Paula et al., 1999; Hayashi et al., 2007a, b). To

date, however, no study has focused on the effects of

UVBR on K. alvarezii, which is a red macroalgae that

presents several colors strains, including red, brown,

yellowish, and different gradations of green (Areces,

1995). It exists in reef environments of the Indo-

Pacific, China, Japan, the islands of Southeast Asia,

and the East Africa region to Guam (Doty et al.,

1987; Paula & Pereira, 1998). According to Hayashi

et al. (2007a), K. alvarezii is an important source of

kappa carrageenan, a hydrocolloid that has been

widely used in industry as a gelling and thickening

agent. Because of the significant economic impact of

this understudied macroalgae, we undertook this

172 Hydrobiologia (2010) 649:171–182

123

investigation to study in vitro the potentially damag-

ing effects of UVBR on the ultrastructure, growth

rates, and photosynthetic pigments of two different

strains (green and red) of K. alvarezii.

Materials and methods

The two strains (green and red) of K. alvarezii were

taken from a culture collection at LAMAR-UFSC

(Macroalgae Laboratory, Federal University of Santa

Catarina, Florianopolis, SC, Brazil).

Culture conditions

The apical thalli portions were selected (±50 mg)

from each of the two strains and cultivated in the

culture room of LAMAR for 28 days in 250-ml

beakers with 200-ml natural sterilized seawater, ±34

practical salinity units (p.s.u.), and salinity enriched

with 0.8-ml von Stosch medium (Edwards, 1970).

Culture room conditions were 24�C temperature,

continuous aeration, illumination from above with

fluorescent lights (Philips C-5 Super 84 16 W/840,

Brazil), photosynthetically active radiation (PAR) at

80 lmol photons m-2 s-1 (Li-cor light meter 250,

USA), and 12 h photocycle (starting at 8 o’clock).

The UVBR was provided through a Vilber Lourmat

lamp (VL-6LM, Marne La Vallee, France) with peak

output at 312 nm. The intensity of UVBR was

1.6 W m-2 (Radiometer-model IL 1400A; Interna-

tional Light, Newburyport, MA, USA). In order to

avoid exposure of UV-C radiation, a cellulose

diacetate foil with thickness of 0.075 was utilized.

The apices were subjected to 3 h of UVBR exposure

per day in a culture room, from 12 to 15 h. During

these 3 h, the air flow was increased into uplift the

thallus close to the air–water interphase effectively

exposing the apices to the UVBR.

A random rotation of the beaker positions was

carried out so that all apices would receive the same

irradiation during the experiment. Apical thalli con-

trols were evaluated using PAR alone, while exposed

apical thalli were cultivated under PAR ? UVBR.

Samples for transmission electron microscope (TEM)

were fixed directly on day 28, the last day of

experimentation, and samples of photosynthetic pig-

ments were frozen by immersion in liquid nitrogen on

day 28, after the final exposure to UV-B at 15:00 h.

Medium was replaced weekly. Four replicates

were made for each experimental group.

Transmission electron microscope

For observation under the transmission electron

microscope, samples *5 mm in length were fixed

with 2.5% glutaraldehyde in 0.1 M sodium cacodyl-

ate buffer (pH 7.2) plus 0.2 M sucrose overnight. The

material was post-fixed with 1% osmium tetroxide for

4 h, dehydrated in a graded acetone series, and

embedded in Spurr’s resin. Thin sections were

stained with aqueous uranyl acetate followed by lead

citrate, according to Reynolds (1963). Four replicates

were made for each experimental group; two samples

per replication were then examined under TEM (Jeol

JEM1011 at 80 kV). Similarities based on the

comparison of individual treatments with replicates

suggested that the ultrastructural analyses were

reliable.

Growth rates (GRs)

Growth rates for treatment groups and control were

calculated using the following equation: GR

(% day-1) = [(Wt/Wi)1/t - 1]*100, where Wi is the

initial wet weight, Wt the wet weight after 28 days,

and t internal time in days (Penniman et al., 1986).

Pigments analysis

The content of photosynthetic pigments (chlorophyll

a and phycobiliproteins) of green and red strains of

K. alvarezii was analyzed between treatment group

and control. Samples (fresh weight) were frozen by

immersion in liquid nitrogen and kept at -40�C until

the analyses. The photosynthetic pigments were

extracted according to Aguirre-von-Wobeser et al.

(2001), using 0.100 g of each sample. All pigments

were extracted in quadruplicate samples.

Chlorophyll a (Chl a)

Chlorophyll a was extracted from *0.100 g of tissue

in 1 ml of dimethylsulfoxide (DMSO, Merck, Darms-

tadt, FRG) at 40�C, during 30 min, using a glass

tissue homogenizer (Hiscox & Israelstam, 1979).

Pigments were quantified spectrophotometrically

according to Wellburn (1994).

Hydrobiologia (2010) 649:171–182 173

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Phycobiliproteins

About 0.100 g algae material was ground to a powder

with liquid nitrogen and extracted at 4�C in darkness

in 0.1 M phosphate buffer, pH 6.4. The homogenates

were centrifuged at 2,000g for 20 min. Phycobilipro-

tein levels [allophycocyanin (APC), phycocyanin

(PC), and phycoerythrin (PE)] were determined by

UV–Vis spectrophotometry, and the equations of

Kursar et al. (1983) were used for calculations.

Data analysis

Data were analyzed by unifactorial Analysis of

Variance (ANOVA) and the Tukey a posteriori test.

All statistical analyses were performed using the

Statistica software package (Release 6.0), considering

P B 0.05. Analyses were performed to evaluate the

effect on growth rates and concentration of photosyn-

thetic pigments between treatment group and control.

Results

Observations under TEM

When observed by transmission electron microscopy

(TEM), the cells of the cortical region of the two

strains were surrounded by a thick cell wall of 3.0–

4.5 lm. This wall was formed by concentric micro-

fibrils embedded in an amorphous matrix which

consisted of sulfated polysaccharides such as carrag-

eenans (Fig. 1a, b). In both strains of K. alvarezii, the

chloroplasts were large and elongated. The chloro-

plasts assumed the typical internal organization of the

red algae with unstacked, evenly spaced thylakoids.

The chloroplasts apparently consisted of an individ-

ual and flat thylakoid surrounded by a single periph-

eral thylakoid (Fig. 1c, d). Plastoglobuli were

observed between the thylakoids (Fig. 1d).

After exposure to UVBR for 3 h per day during a

28-day period, the two strains of K. alvarezii were

Fig. 1 Transmission

electron microscopy (TEM)

micrographic images of

PAR-only K. alvarezii.a, c Green strain; b, d red

strain. a Detail of cortical

cells showing the cup-shape

(heads). b Magnification of

cortical cells showing the

cup-shape. Observe the

starch grains (c) and detail

of the chloroplast of a

cortical cell (d). Observe

the evident thylakoid

(arrows) and plastoglobuli.

C chloroplast, CC cortical

cell, CW cell wall,

P plastoglobulus, S starch

grain

174 Hydrobiologia (2010) 649:171–182

123

observed to undergo some ultrastructural changes,

including an increase in the thickness of the cell wall

of the first cortical cells, with a corresponding

increase in the number of concentric microfibrils

(Fig. 2a, b). The first cortical cells lost their cup-

shape and showed an irregular outline (Fig. 2a). Cells

showed a reduction in the volume of vacuoles, but

organelles grew in number, filling the cytoplasm

(Fig. 2b). Chloroplasts of the cortical and subcortical

cells also showed visible changes in ultrastructural

organization with irregular morphology (Fig. 2c, d).

The number of plastoglobuli increased in the chlo-

roplasts (Fig. 2c–e). The two strains of K. alvarezii

exposed to UVBR showed the same changes, i.e.,

increase in the thickness of the cell wall, contour

alteration, and organization of chloroplasts.

Growth rates

After 28 days in culture, the green and red strains of

K. alvarezii showed statistical differences (P B 0. 05)

in growth rates (GRs) between thalli cultured under

PAR (control condition) and thalli grown under a com-

bination of PAR ? UV-B (Fig. 3a, b). UV exposure

caused a significant reduction in growth rates for both

strains. A significant decrease in GRs was, however,

detected in both control strains during days 21–28. In

addition, from day 21 onward, the exposed red strain

showed a depigmentation and a partial necrosis of the

apical segments (Fig. 4). This process ultimately led to

weight loss.

Pigments

Changes in the levels of photosynthetic pigments in

green and red strains of K. alvarezii algae exposed to

UVBR are shown in Fig. 5a–d. The UV-B-exposed

algae of both strains of K. alvarezii showed a mean

increase of chlorophyll a level compared with control

algae. While the values of Chl a concentration were

not significantly different (P = 0.82) in the exposed

green strain, the red strain did show significant

difference in the concentration of Chl a.

After UVBR exposure, phycobiliprotein levels

(APC, PC, and PE) were reduced in both strains.

Phycobiliprotein levels were statistically different

between PAR-exposed and PAR ? UV-B-exposed

algae in both strains. Phycobiliprotein levels in the

Fig. 2 TEM micrographic images of PAR ? UV-B of

K. alvarezii. a, c Green strain; b, d, e red strain. a Cortical

cell with an irregular outline (arrows). Observe detail of cortical

cell showing the thickening of cell wall (arrowheads). b Detail

of reduction in the volume of vacuoles. c Detail of disrupted

chloroplast with some intact thylakoids (arrow). Mitochondria

close to the chloroplast. d, e Detail of chloroplasts of the

cortical cell with some preserved thylakoids (arrows) and large

plastoglobuli. C chloroplast, CC cortical cell, CW cell wall, Mmitochondria, P plastoglobulus, S starch grain

Hydrobiologia (2010) 649:171–182 175

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red strain were higher than those found in the green

strain. However, the green strain showed higher

levels of Chl a compared to the red strain.

The red strain presented higher concentrations of

red pigments compared to the green strain. PE

content was found in highest concentrations in both

strains of K. alvarezii, irrespective of exposure.

Generally, however, the ratio between the photosyn-

thetic pigments of PAR-exposed and PAR ? UV-B-

exposed algae of K. alvarezii decreased after expo-

sure to UVBR (PE/Chl a, APC/Chl a, PC/Chl a, and

PC/APC) (Table 1). The ratio of PE/APC showed a

subtle increase in the exposed green strain, but a

decrease in the PAR ? UV-B-exposed red strain,

while the ratio of PE/PC significantly increased in

both PAR ? UV-B-exposed strains.

Discussion

When analyzed under TEM, the two control strains of

K. alvarezii showed microfibrils structured in con-

centric layers with different degrees of compression.

The increase in the thickness of the cell wall of the

two PAR ? UV-B-exposed strains of K. alvarezii

could be interpreted as a defense mechanism against

Fig. 3 Growth rates (GRs) of K. alvarezii under different

light treatments. a Green strain; b red strain. Vertical bars

represent ±SD for means (n = 4). Letters indicate significant

differences according to Tukey test (P \ 0.05). Filled squarePAR exposed, open square PAR ? UV-B exposed

Fig. 4 Morphological

response of K. alvareziiafter 28 days of exposure to

different light treatments.

a, c Green strain; b, d, e red

strain. a, b Detail of

dichotomy in apical

segments of PAR-only

(arrows). c, d Observe

reduction of dichotomy in

apical segments of

PAR ? UV-B (arrows).

e Detail of bleaching that

occurs in apical segments

(arrow)

176 Hydrobiologia (2010) 649:171–182

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exposure to ultraviolet radiation. According to Hol-

losy (2002), an increase in leaf thickness has been

interpreted as a protective mechanism against dam-

age caused by UV radiation. In Audouinella saviana

(F.S. Collins) Woelkerling, cultured under different

light regimes, changes in morphology were observed

mainly in the branching of the thalli, thickness of the

cell wall, the number of grooves in the cell surface,

and the formation of reproductive structures. There

were adaptations apparent at the cell walls as the light

intensity was increased and vice versa. These changes

in thickness and grooving may be interpreted as a

mechanism by which the alga increases the surface

area of the cell wall which it presents to the incident

light, as well as a defense mechanism against excess

light (Talarico, 1996).

In red algae, the thylakoids not associated to one

another are free in chloroplasts. The chloroplasts of

both PAR-exposed (control) strains of K. alvarezii

show a typical structure of red algae with one

peripheral thylakoid surrounded by parallel thylak-

oids. The number of parallel thylakoids is variable,

and this number mainly depends on the spatial

location of the cell in the algae. When compared to

controls, the chloroplasts of both strains exposed to

PAR ? UV-B show significant structural changes,

including modification in the quantity, size, and

organization of thylakoids when exposed for 3 h per

day during a 28-day period. However, studies on red

macroalgae exposed to PAR ? UV-A ? UV-B radi-

ation also showed ultrastructural changes in chloro-

plasts manifested as dilation and disorganization of

Fig. 5 Content of

photosynthetic pigments

(lg/g) of the green and red

strains of K. alvarezii under

different light treatments.

a Chlorophyll a,

b allophycocyanin,

c phycocyanin,

d phycoerythrin. Vertical

bars represent ±SD for

means (n = 4). Lettersindicate significant

differences according to

Tukey test (P \ 0.05).

Filled square PAR exposed;

open square PAR ? UV-B

exposed

Table 1 Photosynthetic pigments ratio of K. alvarezii under UVBR

Treatment PE/Chl a APC/Chl a PC/Chl a PE/APC PE/PC PC/APC

Control algaea 1.0 0.88 0.48 1.13 2.1 0.54

Exposed algae 0.36 0.26 0.04 1.38 7.8 0.18

Control algaeb 2.67 2.30 0.91 1.15 2.91 0.40

Exposed algae 1.68 1.57 0.35 1.06 4.8 0.22

a Green strainb Red strain

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thylakoids and the formation of translucent vesicles

between thylakoids. These species included Palmaria

palmata (Linnaeus) Kuntze exposed during 48 h with

intensity of 0.85 W m-2, P. decipiens (Reinsch)

Ricker exposed during 23 h with intensity of

0.85 W m-2, Phycodrys austrogeorgica Skottsberg

exposed during 12 h with intensity of 0.85 W m-2,

and Bangia atropurpurea (Roth) C. Agardh exposed

during 96 h with intensity of 0.85 W m-2 (Poppe

et al., 2003). In Ph. austrogeorgica, after 12 h of

exposure to PAR ? UV-A ? UV-B radiation, phy-

cobilisomes became detached from the thylakoid

membranes (Poppe et al., 2003). However, despite

differences in UVBR exposure time of the various

species mentioned above, we see that all species

presented ultrastructural changes similar to those

observed in both strains of K. alvarezii.

Meanwhile, in green algae, such as Prasiola crispa

(Lightfoot) Kutzing exposed to 24 h PAR ? UV-

A ? UV-B radiation, the chloroplasts showed only

mild changes in thylakoids, with a decrease in

plastoglobuli and the appearance of numerous elec-

tron-dense cells (Holzinger et al., 2006). The elec-

tron-dense lipid droplets described in the chloroplasts

of K. alvarezii are plastoglobuli and are interpreted as

lipid material with a reserve role. The number of

plastoglobuli increased when exposed to UVBR in

these two strains of K. alvarezii. Similar results

were reported with the formation of plastoglobuli in

P. palmata and Odonthalia dentata (Linnaeus)

Lyngbye (Holzinger et al., 2004) and with zoopores

of the brown macroalgae Laminaria hyperborea

(Gunnerus) Foslie (Steinhoff et al., 2008) after

exposure to UV radiation. This increase in the number

of lipids can be considered as a change in metabolism,

which resulted in reduction of cell proliferation and

decrease in growth rates. Other studies, however, did

not show ultrastructural damage in green algae,

including Zygnema exposed to PAR ? UV-A ?

UV-B during 24 h (Holzinger et al., 2009) and

Urospora penicilliformis (Roleda et al., 2009).

The first days of K. alvarezii cultivation provided a

period of acclimation for the algae exposed to UVBR.

During that time, they showed lower GRs when

compared to control algae. Altamirano et al. (2000)

also reported that Ulva rigida C. Agardh showed a

decrease in GRs during the first days of exposure to

UVBR, but with no significant differences after

20 days. In our study, the control algae also showed

a decrease in GRs at the end of the experiment.

However, this reduction may have been the result of

space and nutrient limitations in the cultures.

According to Altamirano et al. (2000), intertidal

macroalgae are the most likely to suffer from the

effects of enhanced UVBR. Hanelt & Roleda (2009)

describe how photosynthetic activity of marine

macrophytes, which grow in the intertidal and upper

sublittoral zone, is often depressed on sunny days in a

typical diurnal pattern when more energy is absorbed

than required by its metabolism. The reduction in the

GRs of the two strains of K. alvarezii that we

observed in the first days of UVBR exposure may

therefore indicate that UVR is the key factor limiting

growth. According to van de Poll et al. (2001),

growth reduction results from the combined effects of

damage to several cellular components, such as

proteins from PSII reaction centers and DNA. These

processes are directly affected by UVR, and the

ability to repair or prevent damage eventually deter-

mines the UV tolerance of species. Thus, to assess

their responses to these new light conditions, it is

necessary to understand the ability of macroalgae to

acclimate to UVR stress.

The DNA molecules absorb 50% of the incident

UVBR and are the primary targets of photodamage.

The reduction in growth observed in the green and

red strains studied in this report may also be related to

the delay in the process of cell division, resulting

from the formation of pyrimidine dimers (Buma

et al., 1995; van de Poll et al., 2001). However, DNA

damage was examined in two carrageenophytes,

Mastocarpus stellatus and Chondrus crispus, exposed

to natural solar radiation was not detected (Roleda

et al., 2004a, b).

The decrease in GRs observed in the two strains of

K. alvarezii studied may therefore be related to the

use of energy for activation of mechanisms of

adaptation and repair of damage induced by UVBR.

For example, chlorophyll and other pigments in algae

and photosynthetic organisms may significantly con-

tribute to shielding of the DNA from ultraviolet

radiation (Lao & Glaser, 1996).

Specifically, with increasing UVBR, as previously

described, the failure of protective mechanisms can

cause changes and cellular imbalances (Bowler et al.,

1992). These imbalances lead to conformational

changes in DNA molecules and a resultant break-

down in replication, transcription, and translation

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(Lao & Glaser, 1996; Buma et al., 2000). Such

interference with these processes ultimately leads to

decreased macroalgae growth rates (Wood, 1987)

and, finally, increased mortality (Franklin & Forster,

1997). UV-mediated growth inhibition and the

simultaneous occurrence of DNA damage indicate

that growth is halted at the higher doses because

DNA damage is not effectively repaired (Buma et al.,

2000). Moreover, the depigmentation and the partial

necrosis of the apical segments of the red strain of

K. alvarezii during the first 21 days of the present

study could have been the result of changes in

metabolism and DNA damage caused by the forma-

tion of pyrimidine dimers. Under similar conditions,

tissue deformation and necrosis were observed in

Laminaria ochroleuca Bachelot de la Pylaie (Roleda

et al., 2004a). Finally, many reports demonstrated

that UVBR affects growth rates as observed in Alaria

esculenta (Linnaeus) Greville, Saccorhiza dermato-

dea (Bachelot de la Pylaie) J.E. Areschoug (Roleda

et al., 2005), as well as Laminaria digitata,

L. saccharina, L. solidungula, and L. hyperborea

(Roleda et al., 2006a, b). Above we cited Hollosy

(2002) with respect to leaf thickness as a protective

mechanism, and we also noted that the increase in the

thickness of the cell wall of the two PAR ? UV-B-

exposed strains of K. alvarezii could be interpreted as

a defense mechanism against exposure to ultraviolet

radiation. In fact, the green strain of K. alvarezii did

have the highest GRs for both PAR-exposed and

PAR ? UV-B-exposed cultures, thus showing better

adaptation to radiation stress as compared to the red

strain of K. alvarezii.

Our results are corroborated by findings based on

studies carried out on other species of algae, which

had been exposed to UVBR for 3 h daily. These

include Delesseria sanguinea (Hudson) J.V. Lamou-

roux (Pang et al., 2001), Phyllophora pseudocerano-

ides (S.G. Gmelin) Newroth & A.R.A, Rhodymenia

pseudopalmata (J.V. Lamouroux) P.C. Silva, Phy-

codrys rubens (Linnaeus) Batters, and Polyneura

hilliae (Greville) Kylin (van de Poll et al., 2001).

In the present study, UVBR stimulated the

synthesis of chlorophyll a in both strains of

K. alvarezii, compared to control algae. After 8 h of

exposure under UVBR, similar studies with the red

macroalgae Leptosomia simplex L. also detected an

increase in Chl a level (Dohler, 1998), and the

increased Chl a level under UVBR was also observed

in other carrageenophytes, specifically, Mastocarpus

stellatus and Chondrus crispus (Roleda et al., 2004b).

Many other investigations of red macroalgae have,

however, shown a decrease in Chl a concentration

after UVBR exposure, such as Eucheuma strictum F.

Schmitz cultivated in vitro during 16 days of expo-

sure (Wood, 1989); K. alvarezii cultured in long line

and incubated with UVBR during 30, 60, 90, 120,

150, and 180 min (Eswaran et al., 2001); C. crispus

cultivated in vitro and subjected to 20 h of UVBR

exposure (Yakovleva & Titlyanov, 2001), Palmaria

palmata, and Phycodrys rubens (Bischof et al., 2000).

The levels of phycobiliproteins decreased in both

strains of K. alvarezii exposed to UVBR. The

phycobiliproteins are located in the phycobilisomes

outside of the thylakoid of chloroplasts. In red algae,

PE is used during the acclimation process; therefore,

it is located more externally in the phycobilisomes

(Talarico, 1996). Our results demonstrated that

phycobiliprotein levels, including APC, PC, and PE,

in strains of K. alvarezii decreased after exposure to

UVBR. These molecules absorb solar energy, trans-

ferring it to the reaction center of photosystem II,

where Chl a is excited by the flow of electrons (Gantt,

1981). We found a decrease in the phycobiliprotein

levels similar to the findings of Eswaran et al. (2001)

with K. alvarezii cultivated in long line and incubated

with UVBR during 30, 60, 90, 120, 150, and

180 min. According to the same authors, PE, which

is responsible for the major light harvesting function

([90%), was seriously affected by UVBR. This

indicates that UVBR strongly inhibited the accumu-

lation of phycobiliproteins. Eswaran et al. (2002)

reported a drastic decline in the absorbance of

phycobilisomes with increasing UVBR exposure

time. Similarly, studies with the cyanobacterium

Anabaena Bory de Saint-Vincent revealed a decrease

of phycobilisomes with increasing UVBR (Sinha

et al., 1995).

Phycoerythrin is the first pigment affected by UV

radiation, followed by PC, APC, the carotenoids, and,

finally, Chl a, which is the most resistant (Gerber &

Hader, 1993, Sinha et al., 1995). This pattern of

destruction of pigments in chloroplasts was observed

in the red macroalgae Ahnfeltiopsis concinna

(J. Agardh) PC Silva & De Cew (Beach et al.,

2000). Similar results were observed with the cyano-

bacterium Anabaena and Nostoc Vaucher ex Bornete

& Flahault, where the concentration of PE decreased

Hydrobiologia (2010) 649:171–182 179

123

upon UVR exposure (Sinha et al., 1995). In the

present study, both strains of K. alvarezii gave

evidence that PE was the phycobiliprotein which

suffered the largest reduction in concentration, i.e.,

by 27% in the red strain and by 68% in the green

strain. The green strain of K. alvarezii showed even

lower levels of PE when compared with the red

strain.

In summary, the present study demonstrates that

high UVBR negatively affects the intertidal macro-

algae. This became obvious after only 3 h of daily

exposure to UVBR over a 28-day experimental

period and through the ultrastructural damage

changes observed primarily on the internal organiza-

tion of chloroplasts. Moreover, this exposure can

have caused photodamage and photoinhibition of

photosynthetic pigments, leading to a decrease in

photosynthetic efficiency and a corresponding

decrease in growth rates. However, the stress suffered

by algae exposed to UVBR did not lead to degrada-

tion of Chl a, and one can plausibly argue that the

increase of this pigment is therefore a form of

adaptation to radiation.

Acknowledgments The authors would like to acknowledge

the Central Laboratory of Electron Microscopy (LCME),

Federal University of Santa Catarina, Florianopolis, SC,

Brazil, for the use of their transmission electron microscope.

We also wish to thank Prof. Pio Colepicolo Neto, University of

Sao Paulo, for supplying the UV lamp. This study was part of

the Master’s thesis of the first author.

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