Effects of eutrophic seawater and temperature on the physiology and ...

Ecotoxicology (2015) 24:1040–1052 DOI 10.1007/s10646-015-1444-6

Effects of eutrophic seawater and temperature on the physiology and morphology of Hypnea musciformis J. V. Lamouroux (Gigartinales, Rhodophyta) Caroline de Faveri1 • E´der C. Schmidt1,2 • Carmem Simioni1 • Cintia D. L. Martins3 Jose´ Bonomi-Barufi3 • Paulo A. Horta3 • Zenilda L. Bouzon1

•

Accepted: 2 March 2015 / Published online: 8 March 2015 Ó Springer Science+Business Media New York 2015

Abstract As both food and source of a kappa-carrageenan, Hypnea musciformis represents a species of great economic interest. It also synthesizes substances with antiviral, anti-helminthic and anti-inflammatory potential and shows promise for use as a bioindicator of cadmium. In this study, we investigated the combined effects of seawater from three urbanized areas (area 1: natural runoff, NRA; area 2: urbanized runoff and sewage with treatment, RTA; area 3: urbanized runoff and untreated sewage, RUS) and three different temperatures (15, 25 and 30 °C) on the growth rate, photosynthetic efficiency, photosynthetic pigments and cell morphology of H. musciformis. After 4 days (96 h) of culture, the biomass of H. musciformis showed differences that fluctuated among the areas and temperature treatments. Specifically, the specimens cultivated in 35 °C had low values of ETRmax, aETR, bETR, and Fv/Fm photosynthetic parameters, as well as changes in cell morphology, with reduction in photosynthetic pigments and drastic reduction in growth rates. When combined with the extreme temperatures, high concentrations of ammonium ion in seawater effluent caused an inhibition of photosynthetic activity, as well as significant variation in & E´der C. Schmidt [email protected]; [email protected] 1

Plant Cell Biology Laboratory, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, CP 476, Floriano´polis, SC 88049-900, Brazil

2

Postdoctoral Research of Postgraduate Program in Cell Biology and Development, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, CP 476, Florianopolis, SC 88049-900, Brazil

3

Phycology Laboratory, Department of Botany, Federal University of Santa Catarina, Floriano´polis, SC 88010-970, Brazil

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chlorophyll a and carotenoid contents. As observed by light microscopy, the synergism between different temperatures and pollutants found in eutrophic waters caused changes in cellular morphology with increased cell wall thickening and decreased floridean starch grains. H. musciformis also showed important changes in physiological response to each factor independently, as well as changes resulting from the synergistic interaction of these factors combined. Therefore, we can conclude that extreme temperature combined with the effect of eutrophic waters, especially RUS, caused distinct morphological and physiological changes in the red alga H. musciformis. Keywords Eutrophic seawater Temperature Hypnea musciformis Photosynthetic efficiency Photosynthetic pigments Morphology

Introduction Previous decades have been characterized by the urbanization of coastal areas around the planet, leaving a very distinctive ecological footprint (Vitousek et al. 1997; Lotze et al. 2006). In developing countries, this process has been followed by the discharge of extraordinary volumes of sewage into coastal waters. These pollutants, mainly inorganic nutrients and suspended solids, have resulted in changes in water quality. Moreover, urbanization has changed entire habitats, in particular because pollution is followed by the input of fresh water related to the sealing of soils by artificial surfaces (Scherner et al. 2012a). In southern Brazil, we identified a model environment characterized by urbanization with concomitant pollution of coastal waters. In particular, the population of Santa Catarina Island Bay has increased, producing a conurbation

Effects of eutrophic seawater and temperature

of nine municipalities. Nowadays, around 1,000,000 people live in the Bay area, and this number usually increases threefold with the arrival of tourists during the summer months. A large majority of this population lives in the central area of the bay, where high nutrient concentrations, caused by urbanization, have been reported (Pagliosa et al. 2005, 2006; Pagliosa and Barbosa 2006). On a daily basis, this population produces around 8 billion liters of sewage drained into the adjacent marine environments, with only 20 % of this volume being treated (Tucci et al. 2001; Moraes et al. 2009). All untreated waste and nutrient remnants in the treated sewage are discharged directly into the environment, acting, in turn, as potential stressors. These stressors, singly or combined, produce important shifts in the intertidal community structure, reducing richness and diversity of species in tropical and subtropical marine ecosystems (Mangialajo et al. 2008; Martins et al. 2012; Scherner et al. 2013). These changes, as observed through descriptive ecology, are related to the ecophysiological differential ability of dominant groups to respond to, or survive in, urbanized environments, as observed by Scherner et al. (2012a), b. While opportunistic species, such as Ulva spp. Linnaeus (Chlorophyta) benefit from polluted conditions, canopyforming groups, such as those represented by Sargassum C. Agardh or Cystoseira amentacea (C. Agardh) Bory de Saint-Vincent (Phaeophyceae), are negatively impacted (Mangialajo et al. 2008; Scherner et al. 2012a). These anthropogenic scenarios (Steffen et al. 2011) are particularly worrisome if we consider their potential interactions with environmental factors related to climate change (Horta et al. 2012). Since 1961, the average temperature of global oceans has increased, which has produced oceanographic and biological changes observed in the extension of some species’ distribution range (De Faveri et al. 2010) or even in the health or physiological behavior of other marine organisms (Turra et al. 2013). During the last year, marine heat waves (HW) have produced dramatic changes in the physiognomy of phytobenthic communities, causing the local extinction of canopyforming species, as observed in Western Australia (Smale and Wernberg 2013). Besides the obvious negative ecological impact, these changes can have economic implications if a given species with potential for cultivation can be extirpated from these areas. Furthermore, these increasingly frequent extreme conditions require technological and management adjustments in algae cultivation systems. Among the 50 species described in the genus Rhodophyta along tropical and subtropical coasts (Masuda et al. 1997), Hypnea musciformis can be found in mesolittoral and subtidal regions, on rocky shores, or as an epiphyte on Sargassum spp. as the main substrate

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(Schenkman 1989; Reis et al. 2006). This species can also take advantage of eutrophic environments, sharing space with other opportunistic species (Orfanidis et al. 1998). Hypnea is a source of kappa carrageenan and phycocolloids throughout the world, presenting significant economic importance (Reis et al., 2008). In Brazil, H. musciformis is the only source of kappa-carrageenan from native algae and has been exploited for decades in the northeastern states (Oliveira 1998; Reis et al. 2008). In addition, H. musciformis synthesizes substances with antiviral (Santos et al. 1999), anti-helminthic and anti-inflammatory potential (Schenkman 1989) and shows promise for use as a cadmium bioindicator (Rathinam et al. 2010). Knowledge about the ecophysiology of this species will help to explain its broad distribution where it shows tolerance to wide ranges of temperature, salinity and light intensity (Yokoya and Oliveira 1992a, 1992b). In addition, different Hypnea species have shown great efficiency in accumulating nitrogen sources, thus enabling increased growth rates under high ammonium availability. Overall, therefore, the bioremediation potential of H. musciformis reinforces the traditional application (Reis et al. 2006). In this study, we investigated the effect of different temperatures and seawater with different sources, representing areas that receive sewage with or without conventional treatment, in H. musciformis ecophysiology.

Materials and methods Site characterization Seawater used in the experiments was collected from three sites on Santa Catarina Island, each with different physical and chemical characteristics. Three sample replicates of water were manually collected in each area for the evaluation of ammonia, nitrate, nitrite and phosphate concentrations, which were measured by colorimetric methods (Grasshoff et al. 1983). Seawater temperature and pH were measured in the field using a portable pH meter (206Lutron, Taiwan). Turbidity and salinity from each area were measured in the laboratory through turbidimeter (Alfakit, Brazil) and refractometer (Alfakit, Brazil), after standard calibration procedures. Ponta das Canas (area 1 = natural runoff, NRA) (27°230 3400 S; 48°260 1000 W) was used as the experimental control condition site, as it represents the most preserved, i.e., least polluted and most pristine area among the selected sites. The nutrient concentrations of seawater samples utilized in the experiments were 1.22 lM (±0.10 standard deviation, SD) of ammonia, 0.19 lM (±0.04 SD) of nitrate, 0.12 lM (±0.04 SD) of nitrite and 0.33 lM (±0.06 SD) of phosphate. The sea surface temperature

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(SST) in the field was 25 °C during the survey, reaching 30 °C on the surface during the summer and 15 °C during the winter. The turbidity, pH and salinity were 3.4 ntu, 7.9 and 36 psu, respectively. Hercilio Luz Bridge (area 2 = urbanized runoff and sewage with treatment, RTA) (27°350 3800 S; 48°330 4600 W) was chosen as the site of intermediate pollution by the presence of the disposal of partially treated sewage effluent, as described by Scherner et al. (2012b). The nutrient concentrations of seawater samples utilized in the experiments were 3.74 lM (± 0.39 SD) of ammonia, 0.25 lM (±0.18 SD) of nitrate, 0.14 lM (±0.01 SD) of nitrite and 0.41 lM (±0.21 SD) of phosphate. The water temperature in the field was 27.2 °C during the survey, reaching 32 °C on the surface during the last summer and 16.4 °C during the winter. The turbidity, pH and salinity were respectively 8.07 ntu, 5.87 and 31 psu. Saco dos Limo˜es (area 3 = urbanized runoff and untreated sewage, RUS) (27°360 5300 S; 48°320 4400 W) is an area adjacent to the most urbanized region on Santa Catarina Island, receiving anthropogenic untreated sewage inputs, as described by Bouzon et al. (2006). The nutrient concentrations of seawater samples utilized in the experiments were 39.9 lM (±7.25 SD) of ammonia, 5.82 lM (±0.83 SD) of nitrate, 1.18 lM (±0.20 SD) of nitrite and 4.21 lM (±0.71 SD) of phosphate. The water temperature in the field was 27.2 °C during the survey, reaching 35 °C on the surface during the last summer and 16.4 °C during the winter. The marine HW observed during last January of the experimental period supported this SST for 1 week. The turbidity, pH and salinity were respectively 8.2 ntu, 8.2 and 29 psu. Algal material Hypnea musciformis specimens used in the experiments were only collected from intertidal zone of Ponta das Canas beach, during low tide in the morning in March, April and May, 2011. Samples were transported in dark containers to the Plant Cell Biology Laboratory (Federal University of Santa Catarina, Floriano´polis, Santa Catarina, Brazil) and were meticulously cleaned with filtered seawater to remove sediment and epibionts. The apical portions were immersed and maintained in seawater enriched with von Stosch 50 % (Edwards 1970), salinity 34 psu and 24 °C (±2 °C) with a 12 h photoperiod and photosynthetically active radiation (PAR) of 80 lmol photons m-2 s-1 (Li-cor light meter 250,USA) for an acclimation period of 14 days before their utilization in experiments. Experimental Conditions Apical thalli portions of H. musciformis (1.0 ± 0.05 g) were cultivated for 96 h (4 days) in Erlenmeyer flasks with

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250 ml of seawater from Area 1 (NRA), 2 (RTA) or 3 (RUS) at three different chambers, adjusted to 3 temperatures (15, 25 and 35 °C). These temperatures were chosen to represent the temperatures of upwelling and winter surface waters, the most frequent values in the region and the highest summer values observed during the low tides of HW periods, respectively. The Erlenmeyers were shaken and had their positions randomized every 3 h during the light phase of the photoperiod. The experiments were carried out in growth chambers (347 CDG; Fanem, Brazil) with PAR of 80 lmol photons m-2 s-1 provided by fluorescent lights (Philips C-5 Super 84 16 W/840, Brazil) and 12 h of photocycle (starting at 8 h), with continuous aeration. Three replicates were made for each experimental group, and the experiments were all repeated three times. Growth rate (GR) Biomass was calculated at the beginning and end of experiments, and the growth rate (RGR) was calculated using the following equation: GR [% day-1] = [(Wt/Wi) - 1] * 100/t, where Wi = initial wet mass, Wt = wet mass after 96 h, and t = internal time in days (Penniman et al. 1986). Photosynthetic performance Measurements of physiological parameters (chlorophyll a fluorescence) were performed at 24 and 96 h after incubation. The chlorophyll a (Chl a) fluorescence parameters were measured with a pulse amplitude-modulated fluorometer (Diving-PAM underwater fluorometer; Walz, Effeltrich, Germany). Measurements were taken after algae were kept in the dark for 10 min. It allowed calculation of maximum quantum yield, Fv/Fm = (Fm-Fo)/Fm, where Fm is the maximum fluorescence emitted after the first saturation pulse and Fo is basal amount of fluorescence under measuring light, previously to saturating pulse application. Photosynthesis irradiance curves were generated based on relative ETR (rETR), and its parameters were calculated according to Platt et al. (1980): photosynthetic efficiency (aETR), maximum photosynthesis rate (ETRmax) and photoinhibition (bETR). ETR can be estimated from the effective quantum yield; therefore, we followed the formula rETR = DF/Fm’ 9 irradiance (PAR) 9 0.15 9 0.84, where DF/ Fm’ = (Fm0 - Ft)/Fm0 , Fm0 is the maximum fluorescence of an illuminated sample, and Ft is the transient fluorescence (Schreiber et al. 1994). The following assumptions were made: (1) 0.15, as the factor of quanta of light trapped by chlorophyll a, for photosystem II in Rhodophyta (Figueroa et al. 1997); (2) ETR-0.84, as a factor based on the average light which is absorbed by plants and other seaweeds with similar thallus construction (Figueroa et al. 2009), value


utilized in others works with the same taxon (Fernandes et al. 2012); and (3) PAR, as the PAR (Diving-PAM Underwater Fluorometer Handbook of Operation, Heins Walz GmbH 1998). Photosynthetic pigments At the end of the experiments, samples were frozen by immersion in liquid nitrogen and kept at -40 °C until ready for use. All pigments were extracted in quadruplicate (Schmidt et al. 2010a) for chlorophyll a, total carotenoid and phycobiliprotein determination. Chlorophyll a (Chl a) and total carotenoids Samples were extracted from approximately 1 g of tissue with 3 ml of dimethylsulfoxide (DMSO, Merck, Darmstadt, FRG) at 40 °C for 30 min, using a glass tissue homogenizer (Hiscox and Israelstam 1979; Schmidt et al. 2010b). Pigments were quantified in a spectrometer (Hitachi, Model 100-20; Hitachi Co., Japan) at 630, 647 and 664 nm. The quantification of chlorophyll concentration was performed according to Jeffrey and Humphrey (1975). Carotenoids were read in the spectrophotometer at 480 nm, following Wellburn (1994). Phycobiliproteins About 1 g of algal material was ground to a powder with liquid nitrogen and extracted at 4 °C in darkness in 0.05 M phosphate buffer, pH 6.4. The homogenates were centrifuged at 20009g for 20 min. Levels of allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE) were determined by spectrophotometry (Hitachi, Model 100-20; Hitachi Co., Japan) at 498, 615 and 651 nm, and the concentration was calculated using the equations of Kursar et al. (1983). Light microscopy (LM) and cytochemistry Control samples (initial stage, collected in NRA) and treated plants of H. musciformis were fixed in 2.5 % paraformaldehyde in 0.1 M (pH 7.2) phosphate buffer overnight. Subsequently, samples were dehydrated in increasing series of ethanol aqueous solutions (Schmidt et al. 2009). After dehydration, samples were infiltrated with historesin (Leica Historesin, Heidelberg, Germany). Sections 5 lm in length were stained with different cytochemical techniques and investigated with an epifluorescent (Olympus BX 41) microscope equipped with Image Q Capture Pro 5.1 software (Qimaging Corporation, Austin, TX, USA). LM sections were stained as follows: Periodic Acid-Schiff (PAS) used to identify neutral polysaccharides

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(Schmidt et al. 2009) and Toluidine Blue (TB-O) 0.5 %, pH 3.0 (Merck Darmstadt, Germany) used for acid polysaccharides through a metachromatic reaction (Schmidt et al. 2010a). Controls consisted of applying solutions to sections without the staining component (e.g., omission of periodic acid application in the PAS reaction). Data analysis The eventual differences among the experiments were evaluated with unifactorial ANOVA (considering repetitions as factor) for all descriptors. There are not initial analyses for physiological variables considering that the H. musciformis was collected same locality (control area). Data from different quantitative descriptors were analyzed after homogeneity of variance was tested with the Levene’s test and factorial Analysis of Variance (ANOVA), followed by Tukey post hoc analysis. Statistical analyses were performed using the Statistica software package (Release 10.0).

Results Growth rates (GRs) After 4 days (96 h) of cultivation H. musciformis, as analyzed by two-way ANOVA (Table 1), showed statistical differences between GRs of algae cultivated with seawater from different areas (Ponta das Cana Beach, Hercı´lio Luz Bridge and Saco dos Limo˜es Beach) and exposed to three temperatures (15, 25 and 35 °C). The algae exposed to 35 °C showed some bleaching and necrosis of the apical segments, as well as weight loss (Fig. 1). Photosynthetic performance Hypnea musciformis showed high values of such photosynthetic parameters as maximum ETR (ETRmax, Fig. 2a), photoinhibition values (bETR, Fig. 2b) photosynthetic

Table 1 RGR of H. musciformis (n = 9, ±SD) during 4 days of cultivation with seawater from three different areas at three different temperatures (15, 25 and 35 °C) Treatments 15 °C

25 °C

35 °C

NRA

1.6 ± 0.74a

1.3 ± 0.61a

-1.0 ± 1.32c

RTA

0.4 ± 0.77b

1.2 ± 0.26a

-5.5 ± 0.85c

RUS

1.6 ± 0.87a

1.4 ± 0.49a

-4.8 ± 1.1b

Letters indicate significant differences according to Tukey’s test (p \ 0.05)

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Fig. 1 Morphological structure of H. musciformis during 4 days of cultivation with seawater from three different areas at three temperatures (15, 25 and 35 °C). The arrows show thallus bleaching

efficiency (aETR, Fig. 2c), and potential quantum yield of photosystem II (Fv/Fm, Fig. 2d) in plants cultivated at 25 °C with seawater from all areas. On the other hand, plants cultivated at 35 °C showed low values of these same photosynthetic parameters (Fig. 2a–d), irrespective of water quality. Fv/Fm also showed significant differences among sites, with additional significant interaction among sites and temperature values (Table 2). Fv/Fm was higher at 25 °C in all treatments, but significantly lower values occurred at higher temperatures in RUS just in the first 24 h. Photosynthetic pigments The interaction between temperatures and seawater pollution produced significant variation in chlorophyll a (Fig. 3a; Table 3), reducing its concentration when higher temperatures were combined with enriched waters, especially RUS. Carotenoid concentration also presented significant variation as a result of interaction among the experimental conditions, e.g., higher values were observed in RUS when exposed to intermediate temperatures (Fig. 3b; Table 3). On the other hand, phycobiliprotein contents (APC, PC and PE) showed significant differences when compared according to temperature and urban effluents (Fig. 3c–e; Table 3), with higher values observed in RTA treatments.

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Observations under light microscopy and cytochemistry The cortical region of H. musciformis is formed by two or three small cell layers, which are surrounded by a thick cell wall (Fig. 4a). In the outer layer, the cortical cells are elongated, while the cells of the second and third layers are more spherical. All cortical cells are surrounded by thick mucilage. Subcortical cells are more vacuolated when compared to cortical cells (Fig. 4a). TB-O staining in control cells showed a metachromatic reaction in the cell wall, indicating the presence of acidic polysaccharides, such as carrageenan (Fig. 4a). This reaction was more intense in the mucilage that coats the cortical cells. The cell wall of subcortical and medullary cells showed lenticular thickness. In the treated plants, we observed changes in cell morphology and reduction in volume (Fig. 4b–j). In particular, an increase in cell wall thickness of cortical and subcortical cells was observed. Treated plants showed the presence of endophytic cells with a metachromatic reaction (Fig. 4b–j). Control samples of H. musciformis stained with PAS exhibited a strong reaction in the cell wall and mucilage layer, indicating the presence of cellulosic compounds and neutral polysaccharides (Fig. 5a). In the cytoplasm, a positive reaction for neutral polysaccharides was detected,


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Fig. 2 Photosynthetic parameters of H. musciformis (n = 9, ±SD) after 4 days of cultivation with seawater of three different areas at three temperatures (15, 25 and 35 °C). Lowercase and uppercase letters indicate significant differences according to Tukey’s test

Table 2 Bifactorial ANOVA of photosynthetic parameters of H. musciformis (n = 9) after 4 days of cultivation with seawater from three different areas (NRA, RTA and RUS) at three different temperatures (15, 25 and 35 °C)

(p B 0.05) to temperature and sites, respectively. Parameter settings: ETRmax = maximum electron transport rates, Fv/Fm = maximum quantum yield; photoinhibition = bETR and aETR = photosynthetic efficiency

bETR

ETRmax

aETR

Fv/Fm

F

p

F

p

F

p

F

P

84.832

0.000*

37.892

0.000*

63.680

0.000*

117.52

0.000*

Site (2)

0.676

0.510

1.2179

0.2988

1.628

0.199

3.303

0.040*

Time (3)

2.102

2.102

2.6980

0.1026

0.432

0.511

2.091

0.150

192

2.200

0.0719

1.8390

0.1245

1.247

0.293

2.599

0.039*

193 293

1.780 0.014

0.172 0.985

0.7399 0.1237

0.4789 0.8837

2.096 1.293

0.126 0.277

0.171 1.341

0.842 0.264

19293

3.891

0.005*

2.0388

0.0920

1.690

0.155

0.661

0.619

Temp. (1)

ETRmax maximum photosynthesis rate, bETR photoinhibition, aETR photosynthetic efficiency, Fv/Fm quantum yield potential * significant differences

especially with the presence of many floridean starch grains, the main reserve substance of red algae (Fig. 5a). When the treated plants were stained with PAS, they showed a decrease in the density of starch grains in the

cortical and subcortical cells (Fig. 5b–j) and an increase in cell wall thickness (Fig. 5b–j). Endophytic cells also showed a positive reaction with PAS, indicating the presence of cellulosic compounds and starch grains (Fig. 5b–j, arrows).

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Fig. 3 Changes of photosynthetic pigments (microgram per gram (FW)) of H. musciformis (n = 9, ±SD) after 4 days of cultivation with seawater of three different areas and different temperatures (15,

25 and 35 °C). Lowercase and uppercase letters indicate significant differences according to Tukey’s test (p B 0.05) to temperature and sites, respectively

Discussion

showed important changes in physiological response by the effect of each individual factor, but also by shifts promoted by the synergistic interactions among all factors. Extreme temperature stimulated by HW was the main stressor causing damage to photosystems of H. musciformis. The specimens cultivated in 35 °C chambers had low values of ETRmax, aETR, bETR, and Fv/Fm photosynthetic parameters,

Based on the results of the present study, the red alga H. musciformis cultivated with different sources of urbanization effluent (Ponta das Cana Beach, Hercı´lio Luz Bridge and Saco dos Limo˜es Beach, Santa Catarina Island, Brazil) and exposed to three temperatures (15, 25 and 35 °C)

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Table 3 Bifactorial ANOVA of photosynthetic pigments and carotenoids of H. musciformis (n = 9) after 4 days of cultivation with seawater from three different areas (NRA, RTA and RUS) at three different temperatures (15, 25 and 35 °C) Chl a F Temp. (1)

Car p

F

APC p

F

PC p

F

PE p

F

p

12.49

0.000*

16.462

0.000*

25.981

0.000*

28.085

0.000*

29.940

0.000*

Site (2)

222.68

0.000*

0.754

0.4799

2.024

0.1516

1.472

0.247

2.514

0.099

192

211.27

0.000*

15.929

0.000*

6.576

0.000*

5.987

0.001*

6.018

0.001*

Chl a chlorophyll a, Car carotenoids, APC allophycocyanin, PC phycocyanin, PE: phycoerythrin * significant differences

Fig. 4 Light microscopy of thallus transversal sections of H. musciformis stained with TB-O control (a) and during 4 days of cultivation with seawater from three different areas at three temperatures 15 (b–d), 25 (e–g) and 35 °C (h–j). Note the

metachromatic reaction of cell wall (CW) of cortical cell (CC) and subcortical cell (SC). The arrows indicate the presence of endophytic cells

changes in cell morphology, as well as reduction in photosynthetic pigments and a drastic reduction in growth rates, when compared with samples of H. musciformis cultivated at 15 and 25 °C. The physical and chemical analysis of the seawater the three collection sites showed important differences. Saco

dos Limo˜es Beach showed high concentrations of ammonia, nitrate, nitrite and phosphate, indicating that is the most polluted than Ponta das Cana Beach and Hercı´lio Luz Bridge. Bouzon et al. (2006) describe this area as one of the most polluted sites of Florianopolis Bay. Eutrophication is a common phenomenon in coastal marine areas and can be

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Fig. 5 Light microscopy of thallus transversal sections of H. musciformis stained with PAS control (a) and during 4 days of cultivation with seawater from three different areas at three temperatures 15 (b–d), 25 (e–g) and 35 °C (h–j). Positive PAS

reaction evidencing floridean starch grains (S) in the cortical (CC) and subcortical (SC) cells and positive reaction with the cell wall (CW). Arrows indicate the presence of endophytic cells

characterized by high concentrations of dissolved inorganic nutrients. Because substances such as phosphorus, nitrate and ammoniacal nitrogen are nutrients for biological processes, excess in the sewage and industrial effluents lead to the eutrophication of natural waters (de Jonge et al. 2002). A recent study performed by Martins et al. (2012) in southern Brazil showed that anthropogenically altered environments have high ammoniacal nitrogen values when compared to preserved environments, leading to the increase of opportunistic algae. For growth rate of H. musciformis cultivated during 4 days (96 h), an increase was seen in the units cultivated at 15 °C and 25 °C, obtaining maximum values (1.6 % per day) with NRA and RUS treatments at the lowest temperature. Schaffelke and Klumpp (1998) observed positive responses to the addition of nutrients for short periods in the growth of Sargassum baccularia (Mertens) C. Agardh. The largest increases in growth rate were observed in a concentration range of 8/1–20/2 mM ammonium/phosphate, i.e., extreme nutrient concentrations led to lower

growth rates, indicating optimal amounts of nutrients for the growth of macroalgae. However, for NRA, RTA and RUS seawater treatments at 35 °C, the specimens showed a negative growth rate and a process of bleaching and necrosis in the apical segments. According to Yokoya et al. (2007), the brown strain of H. musciformis (same lineage as that used in the present study) is more sensitive to high temperatures (above 30 °C), showing a significant decrease in biomass. The authors of that study found that the highest rates of growth occurred at temperatures of 20 and 25 °C, confirming the findings observed in this study and, hence, supporting the idea that environmental factors, such as changes in water temperature, are the main contributors to the increase or decrease in biomass of this species (Reis and Yoneshigue-Valentin 1998). Kim et al. (2007) evaluated the effect of temperature and nutrients in several species of Porphyra spp. and observed that temperature significantly affected the growth rates of all specimens. The growth rate of Porphyra leucosticta was higher at 10 °C and 15 °C compared to 20 °C (p = 0.01).

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Porphyra linearis and P. umbilicalis had higher growth rates at 10 °C. The low availability of nutrients also significantly influenced the growth rate of specimens. The growth rates of P. linearis and P. umbilicalis were significantly higher in media with 250 lM L-1 of ammonia, relative to the medium with 25 lM L-1 of nutrient (p = 0.022). However, Martins and Yokoya (2010) showed that the long-term effect of ammonia may become toxic, as based on the correlation between increased nutrient and growth rates in two brown strains of H. musciformis. The extreme temperature of 35 °C with high concentrations of ammonium ion, as seen in the present study, may have caused an inhibition of photosynthetic activity with concomitant reduction in biomass because treatment with RTA seawater at 35 °C decreased from 0.99 % at the end of the experiment. Within this context, it is possible to infer that the increased sensitivity of marine macroalgae may be related to modifications in the environment resulting from eutrophication processes (Ralph 1999). The ETRmax, bETR, aETR, and Fv/Fm photosynthetic parameters of H. musciformis showed significant changes relative to temperature. According to Bautista and Necchi (2007), several algae have photosynthetic sensitivity to extreme temperatures that later act as a stressful and limiting growth factor for these organisms. At all temperatures tested (15, 25 and 35 °C), photoinhibition was observed during cornering light; however, for the higher temperature, photoinhibition was more pronounced when compared to the temperature of 25 °C. Environmental stresses can affect the efficiency of PSII, causing the characteristic decrease in the rate of electron transport. This drop in values of Fv/Fm, according to Maxwell and Johnson (2000), can be considered as diagnostic photoinhibition which induces an imbalance between energy intake and utilization. In general, the increase in temperature and the excess of organic matter promote the accelerated depletion of dissolved oxygen, which can cause mass mortality of the biota (Boynton et al. 1982; Abreu et al. 1995). In this sense, the photosynthetic pigments that allowed the capture of light at photosystem II are also related to photosynthetic efficiency. Algae have developed different mechanisms for capturing sunlight and can regulate their pigment content in response to quantity and quality of light, in addition to nutrient supply and temperature variation, such that normal function of these algal physiological mechanisms ensures survival of the species (Lo´pez-Figueroa and Niell 1990). Chlorophyll a levels decreased drastically in H. musciformis after exposure to a temperature of 35 °C in RTA and RUS seawaters. To explain, treatments with a temperature of 35 °C most likely promoted a reduction in enzymatic activity (Stobart et al. 1985; Xia et al. 2004) or promoted the

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deficiency of Mg and Fe in the biosynthesis of chlorophyll a (Greger and Ogren 1991; Xia et al. 2004). Besides chlorophyll, carotenoids also play an important role in maintaining photosynthetic efficiency, growth and acclimation to various environmental conditions, as well as protecting the photosynthetic apparatus in macroalgae (Go´mez-Pinchetti et al. 1992). The exposure of H. musciformis increase in the synthesis of carotenoids in most treatments, which may have resulted from the stress suffered in treatments by the combined effect of pollution and temperature. The stimulation of carotenoid synthesis in most treatments of H. musciformis may have been a consequence of the stress caused by the synergistic action of factors acting on the photoprotective apparatus of chloroplasts and causing both the extinction of reactive oxygen species and the absorption and dispersion of excess energy (Torres et al. 2008; Martins et al. 2011). Apart from chlorophyll and carotenoids, red algae have accessory pigments known as phycobiliproteins which are efficient in transferring energy to the reaction center of photosystem II. These pigments are related to protein synthesis. On the other hand, a decrease in pigment content may occur by the availability of nutrients, such as nitrogen and phosphate. In the present study, incubation experiments on H. musciformis were conducted in natural water with high nutrient concentrations. Therefore, the possible influence of these high concentrations on the synthesis of phycobiliproteins was found in comparison to the control area subjected to temperatures of 15 and 25 °C. These temperatures may have influenced pigment synthesis by acting as a potentiator for the incorporation of nutrients. Another factor that can be attributed to pigment induction in H. musciformis is related to higher turbidity values recorded for the RTA and RUS areas during the study period, which may have contributed to the reduction of brightness in the middle, leading to algae pigments synthesizing more specifically for low incidence of light, such as phycobiliproteins, instead of chlorophyll a (Lo´pez-Figueroa and Niell 1990). Indeed, the synergistic interaction between high degrees of urbanization (high nutrient concentration) and extreme temperatures promoted changes in pigment concentration of H. musciformis. During the analysis H. musciformis LM images, the increase in cell wall thickness was evident, as verified by cytochemical methods for acidic and neutral polysaccharides. It is likely that the stress presented in H. musciformis as a result of varying temperatures combined with differing qualities of seawater caused changes in the cellular metabolism of algae. The increase of cell wall thickness in treated plants of H. musciformis can be interpreted as a physical defense mechanism against the concentration of nutrients in the medium. However, this mechanism of cell wall thickness did not prevent the growth and development of endophytic

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cells in the cell wall. The presence of endophytic algae has also been reported in H. musciformis when treated with different cadmium concentrations (Bouzon et al. 2012). Associations between algae are common in nature. In this context, numerous species of filamentous algae have been reported among the living host cell walls. Experimental studies have established that the number of endophytic algae can induce tumor lesions or degradation in host Rhodophyta (Correa 1994). By changing the mucilage layer lining the stalk of the treated plants, the number and depth of endophytes were observed. Thus, we suggest that this region has an important role in protecting the mucilage; in addition, this region is easily changed in adverse environmental conditions. The cellular metabolism of treated plants was modified with a decrease in floridean starch grains observed through PAS staining. This alteration may be related to a change in the route of biosynthesis of starch enzymes in the Calvin cycle, possibly by activating the degradation pathway. Degradation pathways may be utilized to activate the biosynthesis of defense compounds. Inhibition of the synthesis of floridean starch grains, by means of UDP-glucose, diverts synthesis of the production of cell wall components, such as carrageenan, that also use UDP-glucose as a precursor for biosynthesis of this polysaccharide. This phenomenon, as observed with LM, also resulted in the increase of the cell wall in H. musciformis. Similar results were observed in H. musciformis treated with cadmium (Bouzon et al. 2012) and with ultraviolet radiation-B (Schmidt et al. 2012).

Conclusion In summary, the present study demonstrates that H. musciformis ecophysiology was strongly impacted by the combination of polluted seawater and extreme temperatures (15 and 35 °C). Commitments of physiology observed with environmental health degradation reinforce the threats faced by key primary producers as Hypnea and marine ecosystems in scenarios of climate changes. When combined with the extreme temperatures, high concentrations of ammonium ion in polluted seawater caused an inhibition of photosynthetic activity, as well as significant variation in chlorophyll a and carotenoid contents. As observed by light microscopy, the synergism between different temperatures and pollutants found in eutrophic waters caused changes in cellular morphology with increased cell wall thickening and decreased floridean starch grains. Therefore, the documented impacts observed in Hypnea, reinforce demands of sanitation coastal regions enabling eventual sustainable coastal management and conservation of different environmental goods and services.

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C. Faveri et al. Conflict of interest of interest

The authors declare that they have no conflict

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