Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter

Accepted Manuscript Title: Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter Author: D´ebora Alvares Leite Figueiredo Paola Cristina Branco Douglas Amaral dos Santos Andrews Krupinski Emerenciano Renata Stecca Iunes Jo˜ao Carlos Shimada Borges Jos´e Roberto Machado Cunha da Silva PII: DOI: Reference:

S0166-445X(16)30256-9 http://dx.doi.org/doi:10.1016/j.aquatox.2016.09.010 AQTOX 4482

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

14-6-2016 12-9-2016 13-9-2016

Please cite this article as: Figueiredo, D´ebora Alvares Leite, Branco, Paola Cristina, Santos, Douglas Amaral dos, Emerenciano, Andrews Krupinski, Iunes, Renata Stecca, Borges, Jo˜ao Carlos Shimada, Silva, Jos´e Roberto Machado Cunha da, Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2016.09.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter Débora Alvares Leite Figueiredoa* Paola Cristina Branco a Douglas Amaral dos Santos a Andrews Krupinski Emerenciano a Renata Stecca Iunes a João Carlos Shimada Borges b c José Roberto Machado Cunha da Silva a a – Department of Cell and Developmental Biology, Institute of Biomedical Science, University of São Paulo, Av Prof. Lineu Prestes, 1524, CEP 05509-900, São Paulo, SP, Brazil b – Metropolitan United Faculties, Scholl of Veterinary Medicine, Rua Ministro Nelson Hungria, 541, São Paulo, SP, Brazil. c – Universidade Paulista, Av. Marquês de São Vicente, 3001 - Água Branca - São Paulo Brazil * corresponding author e-mail: [email protected] , phone number 11 55 3091 7223

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Highlights

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Ocean acidification was evaluated in sea urchins’ immunity Two sea urchins’ species were analyzed L. variegatus and E. lucunter Both species were affected and OA altered Phagocytic Capacity and cell spreading area Only E lucunter presented a decreased cell spreading and coelomocytes proportion alteration A recovery test demonstrated that such effects may be reversible after a short exposure.

Abstract The rising concentration of atmospheric CO2 by anthropogenic activities is changing the chemistry of the oceans, resulting in a decreased pH. Several studies have shown that the decrease in pH can affect calcification rates and reproduction of marine invertebrates, but little attention has been drawn to their immune response. Thus this study evaluated in two adult tropical sea urchin species, Lytechinus variegatus and Echinometra lucunter, the effects of ocean acidification over a period of 24h and 5days, on parameters of the immune response, the extracellular acid base balance, and the ability to recover these parameters. For this reason, the phagocytic capacity (PC), the phagocytic index (PI), the capacity of cell adhesion, cell spreading, cell spreading area of phagocytic amebocytes in vitro, and the coelomic fluid pH were analyzed in animals exposed to a pH of 8.0 (control group), 7.6 and 7.3. Experimental pH’s were predicted by IPCC for the future of the two species. Furthermore, a recovery test was conducted to verify whether animals have the ability to restore these physiological parameters after being re-exposed to control conditions. Both species presented a significant decrease in PC, in the pH of coelomic fluid and in the cell spreading area. Besides that, Echinometra lucunter showed a significant decrease in cell spreading and significant differences in coelomocyte proportions. The recovery test showed that the PC of both species increased, also being below the control values. Even so, they were still significantly higher than those exposed to acidified seawater, indicating that with the re-establishment of the pH value the phagocytic capacity of cells tends to restore control conditions. These results demonstrate that the immune system and the coelomic fluid pH of these animals can be affected by ocean acidification. However, the effects of a short-term exposure can be reversible if the natural values are re-established. Thus, the effects of 2

ocean acidification could lead to consequences for pathogen resistance and survival of these sea urchin species. Keywords Ocean acidification, climate change, innate immunity, sea urchin, Lytechinus variegatus, Echinometra lucunter.

1. Introduction Anthropogenic activities are causing profound changes in nature. According to the Intergovernmental Panel on Climate Change (2007) the atmospheric CO2 concentration increased about 100 ppm in the last 250 years. This increase, leads to changes in the oceans’ chemistry since atmospheric CO2 diffuses passively in the ocean surface forming carbonic acid (H2CO3) which dissociates into hydrogen (H+) and bicarbonate (HCO3-) reducing pH; (Miles et al. 2006, Fabry et al. 2008). The pH of the ocean surface decreased by 0.1 units since the pre-industrial era; and is expected to decrease 0.3 to 0.4 units by 2100 and 0.7 units until 2300 (Caldeira & Wickett 2003, IPCC 2007). Changes in the oceans’ chemistry, especially rapid modifications such as ocean acidification, could have biological, physiological and evolutionary consequences for the marine biota and ecosystem processes in varying degrees. (The Royal Society 2005, Bibby et al. 2008). Since 1993 the literature on impacts of climate change has increased, but the most emphasized topics are related to temperature (Harley et al. 2006). The studies related to ocean acidification are focused mainly in how calcifying organisms will be affected. Studies conducted so far show alterations on coral reefs (Kleypas et al. 1999), plankton (Riebesell et al. 2000) mussels (Bibby et al. 2008, Michaelidis et al. 2005), gastropods (Bibby et al. 2007) and fishes (Dixson et al. 2010). Studies conducted with sea urchins are mostly related to embryonic development and processes related to fertilization (Kurihara et al. 3

2004a; O’Donnell et al. 2009, 2010, Byrne et al. 2010, Moulin et al. 2011, Ericson et al. 2012, Kurihara et al. 2013). However little attention has been paid to other biological process (Kurihara et al. 2004b), particularly to CO2 sensitivity in immune defense functions (Bibby et al. 2008), which are extremely important for the survival of organisms. Sea urchins are exclusively marine benthic animals that belong to the echinoid class of echinoderms, they are widely distributed in all oceans and depths and are important for nutrient cycling and are important grazers in the marine environment (Valverde & Meurer et al. 2007). These animals are also used as bioindicators for environmental monitoring (Kobayashi & Okamura 2005). Regarding their immunity, their perivisceral coelom possess cells called coelomocytes. Echinoids have four types of coelomocytes. Phagocytic amoebocytes (PA), red sphere cell (RSC), colorless sphere cells (CSC), and vibratile cells (VC), (Figure -S1) (Mangiaterra & Silva 2001, Borges et al. 2005, Smith et al. 2006). PA’s constitute the majority of coelomocytes. They are involved in different immune functions such as phagocytosis, graft rejection, chemotaxis, encapsulation and agglutination (Branco et al. 2014, Smith et al. 2006, Gross et al. 1999). These functions show that this cell type is the main effector of the echinoderm immune system (Ramirez – Gomez & Garcia-Arrarás 2010). According to Silva (2013) they are used as a tool to evaluate the immune response of sea urchins exposed to biotic and abiotic factors (Borges et al., 2010). In ecotoxicological studies, the topics related to the immune response of invertebrates are increasing (Coteur et al. 2004). These animals have only an innate immune response to defend themselves and any disorder that impairs the recognition and destruction of pathogens by the phagocytic cells can endanger their survival. However, there are very few data available on the impact of ocean acidification on animal immune systems and survival (Dupont & Thorndyke 2012). Another important point is to know how this immune system acts when facing chemical and physical stressors such as pH, which could alter their internal pH and harm several cellular functions. Studies related to hypercapnia (elevated CO2 concentration) conducted with nonaquatic animals has shown alterations in the immune system. In Drosophila hypercapnia leads to an alteration in gene expression: while the expression of metabolic genes is upregulated the reproductive and immune genes (mainly antimicrobial peptides genes) are down-regulated (Helenius et al. 2009). In human and mouse macrophages stimulated with 4

LPS hypercapnia inhibits TNF (tumor necrosis factor) and IL-6 (interleukin-6) expression and also inhibits phagocytosis by these cells, indicating that the immune system cells are critical targets affected by alterations in CO2 concentration (Wang et al. 2010). Thus the aim of this study was to investigate the impact of ocean acidification on parameters of the immune system and in the coelomic fluid pH of two echinoderm species Lytechinus variegatus and Echinometra lucunter at two different pH’s ( 7.6 and 7.3) and at two different time periods (24h and 5 days). These species were chosen because they inhabit different tidal zones on the coast: L. variegatus inhabits zones deeper than 5 meters and sandy soil, and E. lucunter inhabits the rocky shore and is closer to the surface regularly being exposed to tidal variations. 2. Materials and Methods 2.1. Sea urchins collection and maintenance Adult sea urchins Lytechinus variegatus (n= 35) and Echinometra lucunter (n=35) males and females were collected from sites near the laboratories of the Marine Biology Center (CEBIMar-USP) at Barequeçaba beach, São Sebastião – São Paulo- Brazil ( 23º49.530´S; 0.45º26.394´W; 35±1.0%0 at 24 ±1ºC) during the summer of 2012 at depths of 3-10 m. The mean weight of the animals were 232.65 ± 53g for L. variegatus and 104.31 ± 31g for E. Lucunter. Animals were collected under permit from SISBIO (authorization number: 30422), and the research organisms did not require animal ethics approval. The animals were maintained in a glass fiber tank (500 liters) with renewed seawater pumped daily in the vivarium of CEBIMar and were acclimated 5 days before experiments began. After the acclimation period, animals (n=5 per group) were transferred to experimental tanks (100 liters – 20 liters per animal), where they remained during the experimental period. Animals were not fed during the experiments, and they were maintained under natural photoperiod using 12h/12h cycle of light/dark. 2.2 Seawater parameters Water was replaced (70% of the tank) every day after being previously adjusted with the respective experimental pH and temperature (25.2 ± 0,4ºC). Tanks were equipped with a submersed B500 pump (Sarlobetter®), and aerators in order to maintain the levels of

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oxygen constant (11 ppm). Tests of pH, salinity (35,1 ± 0,4 psu), nitrite (ppm) (Nutrafin Test®) and oxygen (LabconTest®) were performed every day in all groups analyzed. 2.3. Acidified seawater experiment 2.3.1 pH variation Animals were divided into five groups (n=5) per species, the variation and maintenance of pH was achieved by bubbling CO2 in water through the automatic feedback system Sera Seramic pH controller® which measured pH to an accuracy of 0.01 pH units. Animals were placed in the tank with the control pH (8.0) and the exposure occurred by lowering the control pH until the expected values (7.6± 0.03 and 7.3 ± 0.03) in a six-hour period avoiding exposure shock. Both species were exposed over a period of 24 hours and for 5 days. These pH’s were chosen because they were predicted by IPCC for the years of 2100 and 2300, respectively, and the time of exposure was defined in order to verify the acute responses occurring at two different periods. 2.3.2. Collection and counting of perivisceral coelomic fluid Coelomic fluid was collected from each test animal via the peristomial membrane with a needle (12.7 x 0.33mm) and a syringe of 1 mL without anticoagulant. The needle was introduced diagonally to reach the coelom while avoiding puncturing gonads and gut. Differential counting of cells was performed in a Neubauer chamber without dilution under an interference phase contrast microscope (JENAMED 2 - Zeiss). Coelomic fluid was drawn two times for each animal. In the first draw, one mL of coelomic fluid was used for the cell viability assay, differential cell counting (PA, SCS, RSC and VC) and for phagocytosis assay. In the second draw, two mL of coelomic fluid were used for measuring coelomic fluid pH and for determining cell adhesion / spreading. The period between the samples was one hour. 2.3.3. Cell viability assay The trypan Blue (0.4%) exclusion technique was used to determine cell viability. This vital dye interaction with the cell is absent unless a membrane lesion is present. All cells present in four quadrants (Neubauer chamber) were counted. Cells which presented exclusion towards the dye were considered viable (Freshney 1987). 6

2.3.4. Fungal viability assay Commercial yeast Saccharomyces cerevisae (Itaiquara®) with a viability greater than 90% was used. The yeast viability was tested at different pH’s (8.0, 7.6 and 7.3). A solution of fluorescein (Sigma®) in acetone (5 mg/mL) and ethidium bromide (Sigma®) in seawater was used to identify live and dead yeast. Live yeast emitted green fluorescence and dead yeast emitted red fluorescence, when observed under fluorescence microscopy (Zeiss standard 25 with fluorescence and U-MWU filter) (Silva, 2000). 2.3.5. Measurement of coelomic fluid pH A sample (3ml) of coelomic fluid was extracted at each sampling time, from each individual sea urchin, and analyzed with a pH meter (pHep+ HI 96108 Hanna Instruments® ). Measurements were made after the phagocytosis assays to avoid animal stress and alterations on immune parameters. 2.3.6. Phagocytosis assay Aliquots of 100µl of coelomic fluid were collected and added to glass slides for 1 hour for cell spreading while in a humid chamber at the same temperature as the water tanks. After this period, a suspension of yeast diluted in seawater was added. Coelomocytes were exposed to yeast for a 1h period. After that, cells were observed by an interference phase contrast microscope (JENAMED 2 - Zeiss) for evaluation of phagocytic indexes. During the entire experiment, the glass slides were maintained in a humid chamber and at the same temperature as the water tanks. After incubation, slides were washed twice in filtered seawater to remove free yeast (Borges et al. 2005). One hundred amoebocytes were counted on each slide, and the phagocytic indexes were calculated according to Silva & Peck (2000) based on the formula:

Phagocytic capacity: PC = no. of phagocytic amoebocyte phagocytosing

x 100

Total number of phagocytic amoebocytes Phagocytic index: PI 7

= total number of phagocytosed yeast no. of phagocytic amoebocyte phagocytosing

To prepare the yeast solution approximately 100µg of yeast were diluted in seawater and counted in a Neubauer chamber in order to obtain a stock solution. The yeast concentration used was calculated based on the amoebocyte number of each animal, so there is no general value. The calculation was done so that the relationship between yeast and amoebocyte was 10: 1 (Borges et al. 2002).

2.3.7. Adhesion cell capacity determination An aliquot of 100µl of coelomic fluid was placed in glass slides for different time periods (5, 10, 15 and 30 min), in a humid chamber at the seawater temperature. After these periods glass slides were washed in filtered seawater, observed and photographed in an interference phase contrast microscope (Axioplan 2 imaging - Zeiss). The number of adhered cells was counted in 10 random fields per slide. (adapted from Borges et al. 2002). 2.3.8 Spreading capacity determination An aliquot of 100µl of coelomic fluid was placed in glass slides for different time periods (15, 20, 30 and 60min) in a humid chamber at the seawater temperature. After these periods glass slides were washed in filtered sea water, observed and photographed in an interference phase contrast microscope (Axioplan 2 imaging - Zeiss). The percentage of cell spreading was determined from counts of 100 cells. Cells were considered spread when they presented lamelipodia (Figure 3), and were in an elongated shape (Branco et al. 2012). 2.3.9. Measure of cell spreading area The pictures used to evaluate the capacity of adhesion and spreading were also used to measure the total area of spreading of PA after 60 minutes of incubation in a humid chamber. The measurements were made using Image J software Inc.

®

and the cell

spreading area was measured with a tool to delimit the cell surface. The results were presented as the mean ±SD of all cell measurements (µm2) found in the five photos 8

2.4. Recovery Test The recovery capacity of some parameters of these animals were evaluated. For this purpose, new animals were collected and divided into two groups (n=5): a control group and a pH 7.3 group. Animals were exposed for five days to acidified seawater (pH 7.3) After exposure, following the same protocol as the previous tests, a differential cell count of coelomocytes (PA, CSC, RS and VC), a phagocytosis assay and the pH of coelomic fluid were evaluated (the coelomic fluid of each animal was collected and the animal was returned to the experimental tank). After the exposure period, the injection of CO2 immediately stopped and the pH was slowly increased (over a six-hour period) through the renewal of the tank water to avoid exposure shock, animals were kept in the tank for five more days (recovery period) in the control condition. After this period the tests mentioned above were evaluated again. 2.5. Statistical analysis The data were registered as mean±SD. Three replicates were performed per experiment in two different biological replicates. The data were analyzed by one-way ANOVA using the assumptions of the Kolmogorove-Smirnov test (normality test) and Bartlett test (homogeneity of variances). To compare the means, Tukey’s post-test (GraphPad Software Inc.) was performed. Significant differences were considered at P < 0.05. 3. Results 3.1. Acidified seawater experiment 3.1.1. Coelomocyte counting The total coelomocyte count of E. lucunter showed no difference, but in L. variegatus the number of coelomocytes increased in the group exposed for 24h at pH of 7.3. (Figure 1). When analyzing the cell type proportion, there were no significant differences for L. variegatus, but in E. lucunter there were significant differences (p<0.05) in two cellular types at the lower pH in the acute period (Table 1). Phagocytic amoebocyte concentration decrease 18% in the 24h pH 7.3 group. In contrast, there was a significant increase (77%) 9

in vibratile cell concentration in the 24h pH 7.3 group with no differences in the other cell types. 3.1.2. Cell and yeast viability assays Cellular viability of coelomocytes remained higher than 98% in all animals with no significant differences between groups. The yeast viability also remained higher than 98%. 3.1.3. Phagocytic Indexes For both species a reduction in phagocytic index was observed in animals exposed to acidic water. Different pH treatments and exposure time affected such indexes. A reduction in PC was observed for pH 7.6 after 5 days of exposure, while animals exposed to pH 7.3 demonstrated a compromised PC in spite of exposure time. In the species E. lucunter the lowest values were observed in the groups with the lowest pH (Figure 2A). For L. variegatus a decrease was observed of 40% and 50% in the groups exposed for five days in a pH of 7.6 and 7.3 respectively (Figure 2B). There were no significant differences in the phagocytic index for either species at any pH and for any test period. 3.1.4. Coelomic fluid pH For both species a significant decrease was observed in the coelomic fluid pH. For L. variegatus all experimental groups showed significant differences compared to the control group and as with the phagocytic capacity of these animals, there were significant differences between experimental groups. The lowest value was found in the group exposed for 24h at a lower pH (Figure 2C). The species E. lucunter showed significant differences in all experimental groups compared to the control (p<0.001) but show no differences between experimental groups. The lowest values were found in the acute period (24h) of both experimental pH’s. After five days of exposure the coelomic fluid pH started to rise, but still remained significantly lower than the control group (Figure 2D). Another important observation was that in the groups exposed for 5 days, the spines started falling and braking after 3 days of exposure, at both experimental pH’s and in both species.

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3.1.5. Cell Adhesion No significant differences were found in cell adhesion in any time analyzed (5, 10,15 and 30 min) for either species and at any pH tested . 3.1.6. Cell Spreading In the species L. variegatus no significant differences were observed for cell spreading at any time analyzed, but for E. lucunter, there was a significant decrease in cell spreading at 60 minutes. The 5d pH 7.6 experimental group showed significant differences when compared to all the experimental groups and the control group (Figure 3). For both species the cells in all experimental groups were slightly spread after 60 minutes compared to the control group, with reduced lamellipodia and filopodia (Figure 4). 3.1.7. Cell spreading area Results showed a significant decrease in PA spreading area of E. lucunter and L. variegatus in all groups compared to the control (p<0.05; Figure 5). 3.2. Recovery test 3.2.1. Coelomocytes counting The proportion of cell types showed no difference within L. variegatus but in E. lucunter the proportion of colorless and red sphere cells significantly increased after the recovery period (Table 1), and these same red sphere cells showed significant differences between the control group and the group exposed to acidified seawater. The proportion of phagocytic amoebocytes was still significantly lower than the control group both in the exposure to acidified seawater period as well as after the recovery period. 3.2.2. Phagocytosis assay The phagocytic capacity showed a significant increase after the recovery period, for both species, compared to the groups exposed to acidified seawater, but it was still significantly lower than the control group (Figure 6); showing a tendency towards a recovery of these parameters in these animals. 3.2.3. Coelomic fluid pH 11

The coelomic fluid pH of E. lucunter showed no difference after the recovery period, but for L. variegatus as observed for the phagocytic capacity, there was a significant increase in the coelomic fluid pH after the recovery period but this increase was still lower than the control group (Figure 6). 4. Discussion After exposure to acidified seawater the proportion and types of coelomocytes in our results agreed with the literature for both species (Mangiaterra & Silva 2001, Faria & Silva 2007, Borges 2005 et al., Smith et al. 2006), that is PA’s being the predominant type in all tests performed. Although the distribution of cell types varies among individuals, alterations in the cell type distribution can bring important information about the physiological state of the animal and can be a tool to evaluate and monitor changes in the environment being used as biomarkers (Branco et al. 2014). The total cell count only increased for L. variegatus in the 24h, pH 7.3 treatment. However, these results showed that pH was not a contributing factor to significant differences in the L. variegatus cell proportion. In E. lucunter the total cell count was unchanged but there was both a significant reduction in the concentration of PA, and a significant increase in the VC proportion within the same experimental group. In the polar sea star, L. Polaris, Dupont & Thorndyke (2012) also described a relative increase in the total coelomocyte count. For the polar sea urchin S. droebachiensis, the authors described an increase in the proportion of PA and a decrease in VC proportion after exposure to elevated pCO2 for 5 days. Althougt it is important to note that these observations were made for two polar species and the differences between them could be a consequence of alterations in metabolic rates for both species (Borges et al. 2012). Contrary data were observed in the sea star Asterias rubens, in which a significant decrease in the total number of coelomocytes was observed after one week and six months of exposure to acidified seawater (Hernroth et al. 2011). The cell type distribution is variable among species and even in individuals of the same species (Smith et al. 2006, Ramirez-Gomez and Garcia-Arrarás 2010). It is possible that the differences in the proportion of PA and VC found here were an acute response to the lowered pH because after 5 days no differences in these cell types were found. In the 12

literature, the function of VC is still controversial. Some authors believe it is related to the movement of the coelomic fluid (Smith et al. 2006), however, since its function is still unknown it is not possible to relate the increase in concentration of this cell type with a specific stress response. As described for Heliocidaris erythrogramma (Brothers et al. 2016), no difference was found here in the proportion of red sphere cells and colorless sphere cells in the first experiment, however the red sphere cells have increased in different stress situations such as in increased temperatures (Branco et al. 2012) and anthropogenic pollution (Matranga et al. 2000); (Borges et al. 2010). As cited above, neither Hernroth (2011), nor Dupont & Thorndyke (2012) found differences in these cell types during the exposure period. Apparently these cells are not involved in the stress response caused by decreased pH. In the recovery test, however, RSC and CSC presented significant increases after the exposure period and mainly after the recovery period, exclusively in E. lucunter. Just as with the VC, the CSC function remains unknown. In fact, some authors believe that both sphere cells are actually different stages of maturation of the same cell type (Branco et al. 2013, Matranga et al. 2000), this would explain the increase observed. The phagocytic capacity refers to the ability of cells to engulf foreign material (Figure 7), an essential mechanism in immune responses. In both species tested, this parameter was significantly altered when compared to the control and also between the experimental groups. Similar results were observed in the sea star A. rubens, in which the phagocytic capacity decreased by 30% following short and long term exposure to ocean acidification (Hernroth et al. 2011) and in the sea urchin Heliocidaris erythrogramma in which the phagocytic capacity was depressed at all time periods tested (Brothers et al 2016). In the mussel, M. edulis, a decline in phagocytic capacity in decreased pH seawater was also observed (Bibby et al. 2008). In vertebrate macrophages, the literature presents controversial data. While Riemann et al. (2016) and Kong et al. (2013) described that the extracellular acidosis increased the phagocytic activity in mice and human macrophages. Wang et al. (2010) showed that the elevated CO2 inhibited phagocytosis by macrophages. In these cases, we must take into account that they are different model systems and different organisms. Although echinoderms and vertebrates share many common features (e.g. both are deuterostomes), it is not surprising that there are significant differences in their cell physiology and responses facing the same stressor. 13

Dupont and Thorndyke (2012) estimated the cellular immune response through the total and differential coelomocyte count in two polar echinoderm species, and suggested a positive correlation between pH and cellular immune response in sea urchins. In our experiment, E. lucunter showed a significantly decreased in PA at the lowest pH tested. However, we cannot associate this decrease in cell proportion with a decrease in cellular immune response efficiency since the phagocytic capacity was also significantly reduced in L. variegatus and the proportion of PA was not altered in this species. There are probably altered mechanisms involved in these cells that are impairing their phagocytic function such as cytoskeleton rearrangement or cell migration. After the recovery period, the phagocytic capacity increased significantly, in both species in very similar way. It is important to note that the values found after the recovery period were still lower than the control group, but this test showed that with the reestablishment of the pH value the phagocytic capacity of cells tended to increase. Perhaps with a longer recovery period these cells could reach the control values. The phagocytes are cells related to the recognition and elimination of invading organisms and committed tissues, and are considered important defense mechanisms (Secombes & Fletcher 1992). As describe by Bertheussen (1981) and Tagima et al. (2007), our results showed that the only cell type capable for phagocytosis was PA, and ocean acidification can disrupt its function. For the phagocytic index, no differences were found neither after the period of exposure, nor after the recovery period. Borges et al. (2002) observed an increase in the phagocytic index as well as an increase in the phagocytic capacity in the polar sea urchin, Sterechinus neumayeri, at polar temperatures. In the same species, Branco et al. (2012) found, no significant differences in this parameter when exposing tropical species to increased temperatures. The phagocytic index and the phagocytic capacity bring different information about phagocytosis. While the first represents the amount of yeast phagocytosed per amoebocyte, the second refers to the ability of the cells to perform phagocytosis: the essential mechanism of defensive reactions in the body. The number of yeast phagocytosed was not altered, but the capacity to phagocytose was. This is the first time that this parameter was analyzed concerning ocean acidification and the results suggest that it is not affected by the reduction in pH contrary to what occurs with the phagocytic capacity. 14

Phagocytosis involves several complex steps such as chemotaxis, adhesion, opsonization, and ingestion of the invasive particle (Matranga 2005). Thus, in the search for mechanisms that may be involved in the decreased phagocytic capacity, we assessed the ability of adhesion and spreading of phagocytic amoebocytes. This spreading could reflect a cytoskeletal disorder, which would justify our previous results (decreased phagocytosis). Cell adhesion presented no significant differences in the treatments for either species, and as for cell spreading, just E. lucunter presented significant differences after 60 minutes. However, it was observed, for both species, after the exposition period, that cells were attached and started the process of spreading but failed to spread like the control group cells did. Besides that, the cells presented with reduced lamellipodia and filopodia, therefore we analyzed the cell spreading area. The results obtained showed that this reduction was significant in the two analyzed species. The reduction in the number of lamellipodia and filopodia could be due to alterations in the components of the cytoskeleton, since they are structures composed by actin filaments which are the cellular engineers that drives cell motility (Lambrechts et al. 2004).Thus alterations in actin or other cytoskeletal regulating proteins could be affected. Cofilin, a regulator of actin filament dynamics at the leading edge of migrating cells (Yamaguchi & Condeelis 2007) seems to act as a pH sensor involved in mediating the pH-dependent actin filament dynamic (Frantz et al. 2008). Indeed, after exposure to acidified seawater cytoskeletal actin I, along with other cytoskeletal proteins, was found downregulated in Acropora millepora (Kaniewska et al. 2012). Cell motility is crucial to phagocytize and for organism defense in pathological situation once immune cells can invade infected tissue to eliminate pathogens. For migration, cells undergo several steps such as (1) cell polarization, (2) protrusion of lamellipodia, (3) formation of attachment sites and (4) retraction of cell’s rear (Lambrechts et al. 2004). As the cellular viability presented here shows values above 98%, it is probable that problems in the migrating mechanisms of the cells are to blame for their inability to migrate and consequently to phagocytize yeasts. This could explain the significant reduction in the phagocytic capacity. The pH of the coelomic fluid was significantly affected by pH, in both time of exposure and pH values tested in the two species, our data is supported by previous studies (Miles et al. 2007, Hernroth et al. 2011, Stumpp et al. 2012 and Kurihara et al.

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2013). In L. variegatus, the most significant decrease was in the acute period in the lowest pH whereas E. lucunter presented the lowest values in the acute period in both pH’s. Similar to results evidenced by Spicer et al. (2011) after 5 days of exposure E. lucunter presented an increase in coelomic fluid pH when compared to groups exposed for 24h in both pH’s, but values were still significantly lower than the control. In L. variegatus this phenomenon occurs only in the lowest pH. A total compensation of the coelomic fluid pH was found in S. droebachiensis after five days of exposure, (Dupont & Torndyke 2012) but in the same species another study described a complete compensation of the coelomic fluid pH only after 10 days of exposure (Stumpp et al. 2012). This may be explained by the differences found between species along with prior intra-specific differences (Dupont 2010, 2012). Maybe with a longer exposure period we could observe the same phenomenon. The compensation of the coelomic fluid pH could happen due to a partial compensatory mechanism with bicarbonate observed in the sea urchin P. miliaris and in S. droebachiensis. In these species an accumulation in the concentration of bicarbonate in the coelomic fluid was observed after exposure to acidified seawater (Miles et al. 2006, Stumpp et al. 2012). This mechanism, however, has a price: some authors point out that this increase in bicarbonate ions may be due to shell dissolution of the animals (Miles et al. 2006, Spicer et al. 2007,2011, Stump et al. 2012 and Dupont &Thorndyke 2012). Another study suggests that bicarbonate can be obtained by intestinal reabsorption (Holtmann et al. 2013). The effect of excess of bicarbonate on innate immune parameters have not yet been evaluated, but it certainly deserves attention in future studies. In larval sea urchins, after exposure to under high CO2 conditions, O’Donnell et al. (2010), described changes in the expression of a variety of genes including acid-baseregulation, mineralization, skeletogenesis and genes related to metabolism. A bicarbonate transporter (Slc4a3), for example, was induced . An increase in the expression of genes related to acid-base regulation may indicate an attempt to normalize coelomic fluid pH. Also, a decrease in the expression of genes related to energy metabolism (especially in mitochondrial function) could be associated with decreased cell spreading since energy is needed to carry out this process. Future studies are needed in this area to better understand the relation between gene expression patterns and the alterations found here.

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The recovery test demonstrated that the pH of coelomic fluid increased and presented a tendency to reach values obtained for the control group. Long-term studies are now necessary to establish how many days are necessary for recovery. For both species, after 3 days of exposure, spine falling was registered, with this phenomenon becoming more pronounced at 5 days of exposure. Spine fragility was also observed after exposure to high CO2 water in sea urchins S. droebachiensis. This fragility can occur due to contact with acidic water which can cause small fractures in the epithelium covering of the spines, leaving them in direct contact with acidic water and, leading to their dissolution (Holtmann et al. 2013). It is not possible to state if this spine falling is due to carapace dissolution or by a decrease in gene expression as reported in S. purpuratus in response to elevated CO2 (Todgham & Hofmann 2009). These authors describe that genes involved in biomineralization, cellular stress response and energy metabolism accounted for 50% of the overall reduction in gene expression. Similar results were found by Liu et al. (2012), who demonstrated a down regulation of calcification related genes due to declined pH in the Oyster Pinctada fucata. Beyond their role in locomotion, the spines are also defensive structures for sea urchins, and their fragility can be detrimental to the survival of these animals Most of the studies related to the impacts of ocean acidification are focused in singlespecies experiments, and probably the response between species could differ due to differences in the environmental conditions (Dupont et al. 2010). As a consequence, the aim of this study was to evaluate the immune parameters of two urchin species that inhabit two tidal zones. E lucunter lives in the intertidal zone regularly covered and uncovered by the movement of the tides, and since the organisms who live in this zone are subject to a cycle of immersion and emersion, they are often also exposed to different abiotic factors such as temperature and salinity (Hofmann 1999). L. variegatus is not exposed to tidal variation nor to strong changes in these abiotic factors. Therefore we suspected that E. lucunter would present a better response to ocean acidification compared to L. variegatus. However, both species presented similar responses to ocean acidification; phagocytic capacity and pH of the coelomic fluid were similarly affected. In addition, E. lucunter presented differences in the proportions of PA, VC and cell spreading while L. variegatus presented differences in the total cell count.

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It is not possible to affirm if these differences imply that one species is more resistant or less affected than the other, since little is known about the effects of ocean acidification in these organisms. However, the differences found here could represent an attempt to adapt to the acidified environment, or it could indicate that E. lucunter may be able to tolerate variations in temperature but not in pH. 5. Conclusion This work studied the immune, cellular and physiological responses, such as cell spreading and coelomic fluid pH to induced seawater acidification, in two tropical sea urchin species that live in different environmental conditions (L. variegatus and E. lucunter. Acidification affected the coelomic fluid pH as well as the amoebocyte phagocytic capacity in both species analyzed. These changes may alter different metabolic processes occurring in their coelomic fluid as well as their ability to destroy invading pathogens, thus affecting their survival in a future acidified ocean, which could lead to serious consequences for the entire marine ecosystem. Acknowledgments The authors would like to thank to CEBIMar - USP (Marine Biology center of the University of São Paulo) for their technical and laboratorial support in all experiments and to CNPQ (158622/2011-1) and FAPESP (2010/04527-5) for the financial support. References Bertheussen, K., 1981. Endocytosis By Echinoid Phagocytes in vitro. I. Recognition of foreign matter. Dev. Comp. Immunol. 5, 241-250. Bibby, R., Cleall-Harding, P., Rundle, S., Widdicombe, S., Spicer, J., 2007. Ocean acidification disrupts induced defences in the intertidal gastropod Littorina littorea. Biol. Lett .3, 699-701 Bibby, R., Widdicombe, S., Parry, H., Spicer, J., Pipe, R., 2008. Effects of ocean acidification on the immune response of the blue mussel Mytilus edulis. Aquat. Biol. 2, 6774

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Figure Captions Figure 1: Total coelomocyte count of (A) L. variegatus and (B) E. lucunter after the exposure period of 24h and 5d in different pH treatments. * Indicates difference to control group (p<0.05) ♦ indicates differences among experimental groups (p<0.05)

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Figure 2: Effect of acidified seawater in the phagocytic capacity of (A) L. variegatus and (B) E. lucunter, and coelomic fluid pH of (C) L.variegatus and (D) E. lucunter after 24h and 5 days of exposure. * indicates differences from control. ♦ indicate differences with the group 24h pH 7.6 (p<0.05). a indicates differences between the experimental groups marked.

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Figure 3: E. lucunter cellular spreading on glass slides after 60 minutes in the experimental group 5d pH 7.6. *** refer to statistical differences (P<0.001) from the control groups and the other experimental groups. Values expressed in percentage.

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Figure 4: Coelomocytes of L variegatus (A, B and C) and E. lucunter (D, E and F) after 24h and 5d of exposure to pH 7.3, photographed after the spreading period of 60 minutes. The arrows indicate phagocytic amoebocytes. It is possible to note that cells are less spread and the number of filopodia and lamelipodia is reduced in these cells after their exposure to acidified seawater. A, D – control groups, B, E – 24h exposure, C, F 5d exposure

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Figure 5: Cellular spreading area of (A) L. variegatus and (B) E. lucunter amoebocytes after 24 hours and 5 days exposure to acidified seawater. * indicates differences from control group: *p<0.05 **p<0.01 and *** p<0.001

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Figure 6: The phagocytic capacity of (A) L. variegatus and (B) E. lucunter and the coelomic fluid pH of (C) L. variegatus and (D) E. lucunter in the recovery test. * indicates differences from the control. ♦ indicate differences with the 5d pH 7.3 group.

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Figure 7: Yeast phagocytosis by phagocytic amoebocytes in the sea urchin Lytechinus variegatus after the incubation period of 1h. The arrows indicate the yeast inside the phagocytic amoebocyte. Scale bar 10 µm.

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Table 1: Coelomocyte percentages of L. variegatus and E. lucunter exposed to acidified seawater in different pH’s and time periods. Values quoted are mean ± SD PA phagocytic amoebocyte, SCS colorless sphere cell, RSC red sphere cell, VC vibratile cell. Period of exposure 24h (acute test) and 5 days. * a

refer to statistical difference to control group (P<0.05). refer to statistical difference between experimental groups

Specie Treaatments (n)

Control 5

Animals exposed to acidified seawater Lytechinus variegatus 24h pH 7.6 5d pH 7.6 24h pH 7.3 5d pH 7.3 Control 5 5 5 5 5

% of PA

75.3 ± 10.17

69.83 ± 5.18

76.80 ± 9.12

73.56 ± 6,7

73.79 ± 6.15

75.31 ± 7.27a

68.95 ± 6.29

69.63 ±10.33

61.16 ± 3.07a

63.38 ± 6.09

% of CSC % of RSC

6.5 ± 4.92 4.3 ± 2.18

10.36 ± 2.73 5.34 ± 1.28

8.82 ± 2.42 3.56 ± 1.18

7.25 ± 2.18 6.38 ± 3.7

9.78 ± 6.29 3.80 ± 2.32

3.31 ± 2.40 3.40 ± 3.41

3.21 ± 0.5 3.40 ± 1.88

3.68 ± 2.78 2.88 ± 2.22

3.37 ± 1.07 3.59 ± 1.49

5.0 ± 3.73 6.09 ± 3.23

% of VC

14.0 ± 4.27

14.48 ± 7.46

10.82 ± 7.03

12.81 ± 4.0

12.63 ± 4.96

17.97 ± 6.72a

24.43 ± 4.6

23.81 ± 8.62

31.88 ± 3.89a

25.53 ± 4.59

24h pH 7.6 5

Echinometra lucunter 5d pH 7.6 24h pH 7.3 5 5

5d pH 7.3 5

Recovery test Specie Treaatments (n) % of PA % of CSC % of RSC

Control 5

Lytechinus variegatus 5d pH 7.3 5

Recover 5

69.0 ± 5.04

69.52 ± 6.35

76.03 ± 7.67

14.2 ± 4.58 7.3 ± 1.73

13.21 ± 2.95 8.12 ± 2.89

9.69 ± 4.31 5.0 ± 2.54

Control 5

Echinometra lucunter 5d pH 7.3 5

Recover 5

75.4 ± 7.61

64.8 ± 3.27*

63.76 ± 6.65*

a

8.36 ± 2.48*a 10.75 ± 3.11*

2.6 ± 1.08 2.6 ± 1.91

3.89 ± 1.49 8.63 ±4.44*

1

% of VC

9.4 ±1.97

9.15 ± 2.07

9.27 ± 3.13

19.4 ± 6.67

22.62 ± 6.25

17.13 4.48

2