Effects of ocean acidification and warming on the covering behavior of sea urchins Glyptocidaris crenularis and Strongylocentrotus intermedius 14 ISJ 15: 14-18, 2018 ISSN 1824-307X SHORT COMMUNICATION Effects of water temperature and pH value on covering behavior of the sea urchin Glyptocidaris crenularis MF Yang#, SB Luo#, JN Sun, DT Shi, JY Ding, YQ Chang, C Zhao # These authors contributed equally to this work Key Laboratory of North Mariculture and Stock Enhancement, Ministry of Agriculture, Dalian Ocean University, Dalian, China, 116023 Accepted November 28, 2017 Abstract As marine calcifying organisms, sea urchins are sensitive in the changing ocean. More information is needed about the effects of pH value and water temperature on behaviors of sea urchins. Here, we reported that pH value and water temperature significantly affected the covering behavior of sea urchins Glyptocidaris crenularis. Lower pH value (pH = 7.4) significantly reduced the time to first covering (p = 0.026), while significantly decreased number of covered sea urchins (p = 0.029) and number of shells used for covering (p = 0.007) in G. crenularis. Water temperature did not significantly affect the time to first covering (p = 0.180) or number of covered sea urchins (p = 0.157), though significantly affected number of shells used for covering (p = 0.042). The present study provides preliminary information on behavioral ecology of sea urchins. Notably, the effects of CO2 induced acidification should be further investigated in future. Key Words: sea urchin; covering behavior; pH value; water temperature Introduction Over the past 250 years, Atmospheric carbon dioxide (CO2) levels increased by approximately 40 % (Solomon et al., 2007), one third of which has been taken up by the oceans (Sabine et al., 2004). Due to absorbing increasing amounts of CO2, ocean warming and acidification have been identified as the greatest anthropogenic threat to marine ecosystems (Halpern et al., 2008; de Madron et al., 2011). This highlights the risks to marine calcifying organisms in future (Orr et al., 2005). As marine calcifying organisms, sea urchins are sensitive in the changing ocean (Dupont et al., 2013). Ocean warming and acidification have showed greatly impacts on reproduction (Reuter et al., 2011), gamete health (Morgan and Galione, 2007), larval development (Brennand et al., 2010; Stumpp et al., 2011a, b) calcification (Byrne et al., 2011) and gene expression (O'Donnell et al., 2010) of sea urchins. Effects of pH value and water temperature on ___________________________________________________________________________ Corresponding author: Chong Zhao Key Laboratory of North Mariculture and Stock Enhancement Ministry of Agriculture Dalian Ocean University Email: chongzhao@dlou.edu.cn behaviors of sea urchins remain largely unknown, although the fragility has showed in various of behaviors of marine fish (Munday et al., 2009; Cripps et al., 2011; Simpson et al., 2011) and bivalves (Chan et al., 2011). Covering behavior refers to echinoids using their tube feet and spines to manipulate environmental objects, such as shells, stones and algae fragments, to put onto their dorsal surface (Verling et al., 2002). Covering behavior has been well proposed to have multiple functions of protection from lights (Verling et al., 2002), predation (Agatsuma, 2001), floating sand (Richner and Milinski, 2000) and wave surge (Dumont et al., 2007). Recently, effects of elevated temperature and acidification have been investigated separately (Challener and McClintock, 2013; Zhao et al., 2014; Brothers and McClintock, 2015; Zhang et al., 2017). The effects of water temperature and pH value on covering behavior, however, were not simultaneously investigated. Acidification can be experimentally imitated by CO2 (for example, Dupont et al., 2013) or HCl (for example, Yamada and Ikeda, 1999) in laboratory. A comparative study indicates that HCl-induced acidification showed lower acute toxicity to aquatic animals than that induced by CO2 (Zhang et al., 2011). mailto:chongzhao@dlou.edu.cn 15 Table 1 Characteristics of sea urchins and covering materials Water temperature 15°C 25°C pH value 7.4 8.2 7.4 8.2 Glyptocidaris crenularis Test diameter (mm) 16.55 ± 1.98 16.44 ± 1.76 16.58 ± 2.04 16.50 ± 1.89 Test height (mm) 7.87 ± 1.14 8.20 ± 0.98 8.09 ± 0.98 8.16 ± 1.23 Body weight (g) 2.04 ± 0.73 2.10 ± 0.71 2.10 ± 0.74 2.06 ± 0.74 Mytilus galloprovincialis Shell length (mm) 11.82 ± 0.81 11.91 ± 0.85 11.74 ± 0.84 11.59 ± 0.76 Shell height (mm) 19.84 ± 2.61 20.31 ± 1.45 19.96 ± 1.39 19.45 ± 2.02 Shell weight (g) 0.12 ± 0.02 0.13 ± 0.02 0.12 ± 0.02 0.12 ± 0.02 (Mean ± SD, N = 36 for Glyptocidaris crenularis, N = 40 for Mytilus galloprovincialis) Thus, HCl-induced acidification can be used to provide preliminary information on behavioral response of sea urchins to ocean acidification. The aim of the present study is to investigate the effects of water temperature and pH value on the covering behavior of the sea urchin Glyptocidaris crenularis. Materials and Methods Sea urchins Glyptocidaris crenularis were originally reared at Dalian Haibao Seafood Company in Lvshun of Dalian, China. Individuals from one cohort were transported to Key Laboratory of North Mariculture and Stock Enhancement, Dalian Ocean University, China. All sea urchins were acclimated for 7 days at 19 - 21 °C of water temperature, 30 - 31 ‰ of salinity and 8.2 of pH before the behavioral experiment. There was no significant difference of test diameter, test height and body weight among experimental groups in G. crenularis (p > 0.05, Table 1). Experimental design Geochemical models indicates that ocean acidification will be over 1.4 pH units in the next 300 years (Caldeira and Wickett, 2003). Consequently, two pH values (7.4 and 8.2) were comparatively studied in the coral Oculina patagonica (Fine and Tchernov, 2007). We accordingly set pH = 7.4 and 8.2 in the present study. The low pH value (pH = 7.4) was simply produced by adding hydrochloric acid to the natural seawater (pH = 8.2). To maintain water temperatures, the behavioral experiments were carried out in buckets (diameter of the bucket bottom = 22 cm) bathed in temperature-controlled tanks, where two water temperatures (15 °C and 25 °C) were set. Forty shells of Mytilus galloprovincialis were randomly selected and put into each bucket according to our previous study (Zhao et al., 2014). There were no significant differences in shell length, shell width and body weight of M. galloprovincialis among experimental groups (Table 1, p > 0.05). We then placed 144 sea urchins into the 12 buckets (12 individuals per bucket, 4 treatments, 3 replicates). Because the experimental duration was relatively short, pH values were well maintained. Time to first covering refers to the time of covering by the first sea urchin that covered, indicating the behavioral reaction. The number of covered sea urchins and number of shells used for covering were measured every 10 min during 90 min, indicating the behavioral capability. Fig. 1 Time to first covering of Glyptocidaris crenularis at different water temperatures and pH values (N = 3, mean ± SD). 16 Fig. 2 Number of covered Glyptocidaris crenularis at different water temperatures and pH values (N = 3, mean ± SD). Statistical analysis All original data were tested for normal distribution and homogeneity of variance. Repeated measured ANOVA was used to detect the differences of number of covered sea urchins and number of shells used for covering among experimental groups. Time to first covering was analyzed using Mann-Whiney U test because the data showed heterogeneity of variance. All analysis was carried out using SPSS 13.0. A probability level of p < 0.05 was considered statistically significant. Results Time to first covering Time to first covering was significantly lower in sea urchins exposed to lower pH (p = 0.026, Fig. 1). However, water temperature did not significantly affect time to first covering, although G. crenularis showed obviously quicker reaction at 25 °C than those at 15 °C (p = 0.180, Fig. 1). Number of covered sea urchins and number of shells used for covering Lower pH significantly decreased number of covered sea urchins (p = 0.029, Fig. 2) and number of shells used for covering (p = 0.007, Fig. 3). Water temperature did not significantly affect number of covered sea urchins (p = 0.157, Fig. 2), though significantly affected number of shells used for covering (p = 0.042, Fig. 3). Interaction between water temperature and pH value showed no significant effect on number of covered sea urchins (p = 0.752) or number of shells used for covering (p = 0.989). Observational duration significantly affected both number of covered sea urchins (p < 0.001) and number of shells used for covering (p = 0.01). Both number of covered sea urchins and number of shells used for covering increased in the first 20 or 30 min. and then obviously decreased in lower pH groups (Figs 2, 3). Discussion Water temperature and pH value significantly affected calcification (Byrne et al., 2011), growth (Albright et al., 2012) and behavior (Cripps et al., 2011) of marine organisms, ultimately impacting the entire ecosystem (Strandberg et al., 2012). Considering their roles in structuring marine benthic communities, it is critical to understand ecological response of sea urchins to water temperature and pH value. The present study investigated the behavioral response of sea urchins to water temperatures and pH values. It provides preliminary information on behavioral ecology of sea urchins. In the present study, we found that lower pH value (pH = 7.4) significantly affected covering behavior of G. crenularis. This result is in agreement with previous reports on the behavioral response of marine organisms to pH values. In reef fish Pseudochromis fuscus, for example, ocean acidification significantly disturbed its prey detection (Cripps et al., 2011). Lower pH value significantly decreased the number of covered sea urchins and number of shells used for covering. This result is not in agreement with the finding of Challener and McClintock (2013) that ocean acidification did not significantly impact covering behavior of juvenile sea 17 Fig. 3 Number of shells used for covering of Glyptocidaris crenularis at different water temperatures and pH values (N = 3, mean ± SD). urchins Lytechinus variegatus. The disagreement is probably due to the difference of behavioral response to ocean acidification between the two species. Interestingly, we found that lower pH value significantly increased the speed of covering reaction. A reasonable explanation is that the acute stress increased the covering reaction. Consistently, Brothers and McClintock (2015) found that acute exposure to elevated temperature significantly increased covering behavior of L. variegatus, compared to chronical exposure. Water temperature did not significantly affect the time to first covering and number of covered sea urchins. Crook (2003) found that Paracentrotus lividus covered more quickly during summer time and obviously less in winter time. These results confirm the difference of the covering behavior among different species (Verling et al., 2004). Interestingly, we found number of shells used for covering was significantly different between sea urchins at 15 °C and 25 °C, although the p value was very close to 0.05 (p = 0.042). This result is not consistent with our previous study, in which we found no significant difference of number of shells used for covering between G. crenularis in 15 °C and 25 °C (Zhao et al., 2014). This disagreement might be due to limited sample size in both studies and strongly highlights the individual variation of covering behavior of sea urchins. Considering the multiple functions of covering behavior, our finding indicates that lower pH value could probably trigger a series of indirect ecological impacts on sea urchins (at least on G. crenularis). Notably, the present study used HCl rather than CO2 to set the pH value. The effects of CO2 induced acidification should be further investigated. Acknowledgements This work was supported by the National Natural Science Foundation of China (41506177) and the Chinese National 863 Project (2012AA10A412). We thank H Zhou, X Tian and W Feng for their assistance. All authors have no conflict of interest. References Agatsuma Y. Effect of the covering behavior of the juvenile sea urchin Strongylocentrotus intermedius on predation by the spider crab Pugettia quadridens. Fish. Sci. 67: 1181-1183, 2001. Albright R, Bland C, Gillette P, Serafy JE, Langdon C, Capo TR. Juvenile growth of the tropical sea urchin Lytechinus variegatus exposed to near-future ocean acidification scenarios. J. Exp. Mar. Biol. Ecol. 426: 12-17, 2012. Brennand HS, Soars N, Dworjanyn SA, Davis AR, Byrne M. Impact of ocean warming and ocean acidification on larval development and calcification in the sea urchin Tripneustes gratilla. PLOS ONE 5(6): e11372, 2010. Brothers CJ, McClintock JB. The effects of climate-induced elevated seawater temperature on the covering behavior, righting response, and Aristotle's lantern reflex of the sea urchin Lytechinus variegatus. J. Exp. Mar. Biol. Ecol., http://apps.webofknowledge.com/OneClickSearch.do?product=WOS&search_mode=OneClickSearch&colName=WOS&SID=3FgAk9B6H7k4bjahI3b&field=AU&value=Soars,%20N http://apps.webofknowledge.com/DaisyOneClickSearch.do?product=WOS&search_mode=DaisyOneClickSearch&colName=WOS&SID=3FgAk9B6H7k4bjahI3b&author_name=Byrne,%20M&dais_id=1963402 18 467: 33-38, 2015. Byrne M, Ho M, Wong E, Soars NA, Selvakumaraswamy P, Shepard-Brennand H, et al. Unshelled abalone and corrupted urchins: development of marine calcifiers in a changing ocean. Proc. R. Soc. Lond. B Biol. Sci. 278: 2376-2383, 2011. Caldeira K, Wickett ME. Anthropogenic carbon and ocean pH. Nature 425: 365, 2003. Chan KYK, Grunbaum D, O'Donnell MJ. Effects of ocean acidification on swimming performance in larval sand dollars and oysters. Integr. Comp. Biol. 51: E22, 2011. Challener RC, McClintock JB. Exposure to extreme hypercapnia under laboratory conditions does not impact righting and covering behavior of juveniles of the common sea urchin Lytechinus variegatus. Mar. Freshw. Behav. Physiol. 46: 191-199, 2013. Cripps IL, Munday PL, McCormick MI. Ocean acidification affects prey detection by a predatory reef fish. PLOS ONE 6(7): e22736 (1-7), 2011. Crook AC. Individual variation in the covering behaviour of the shallow water sea urchin Paracentrotus lividus. Mar. Ecol. 24: 275-287, 2003. De Madron X, Durrieu, Guieu C, Sempere R, et al. Marine ecosystems' responses to climatic and anthropogenic forcings in the Mediterranean. Prog. Oceanogr. 91: 97-166, 2011. Dumont CP, Drolet D, Deschênes I, Himmelman JH. Multiple factors explain the covering behaviour in the green sea urchin, Strongylocentrotus droebachiensis. Anim. Behav. 73: 979-986, 2007. Dupont S, Dorey N, Stumpp M, Melzner F, Thorndyke M. Long-term and trans-life-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis. Mar. Biol. 160: 1835-1843, 2013. Fine M, Tchernov D. Scleractinian coral species survive and recover from decalcification. Science 315: 1811, 2007. Halpern BS, Walbridge S, Selkoe KA, Kappel CV, Micheli F, D’Agrosa C, et al. A global map of human impact on marine ecosystems. Science 319: 948-952, 2008. Morgan AJ, Galione A. Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca2+ stores in sea urchin eggs. J. Bio. Chem. 282: 37730-37737, 2007. Munday PL, Dixson DL, Donelson JM, Jones GP, Pratchett MS, Devitsina GV, et al. Ocean acidification impairs olfactory discrimination and homing ability of a marine fish. Proc. Natl. Acad. Sci. USA 106: 1848-1852, 2009. O'Donnell MJ, Todgham AE, Sewell MA, Hammond LM, Ruggiero K, Fangue NA, et al. Ocean acidification alters skeletogenesis and gene expression in larval sea urchins. Mar. Ecol-Prog. Ser. 398: 157-171, 2010. Orr JC, Fabry VJ, Aumont O, Bopp L, Doney SC, Feely RA, et al. Anthropogenic ocean acidification over the twenty-first century and its impact on organisms. Nature 437: 681-686, 2005. Reuter KE, Lotterhos KE, Crim RN, Thompson CA, Harley CDG. Elevated pCO2 increases sperm limitation and risk of polyspermy in the red sea urchin Strongylocentrotus franciscanus. Global Change Biol. 17: 163-171, 2011. Richner H, Milinski M. On the functional significance of masking behaviour in sea urchins-an experiment with Paracentrotus lividus. Mar. Ecol. Prog. Ser. 205: 307-308, 2000. Sabine CL, Feely RA, Gruber N, Key RM, Lee K, Bullister JL, et al. The oceanic sink for anthropogenic CO2. Science 305: 367-371, 2004. Simpson SD, Munday PL, Wittenrich ML, Manassa R, Dixson DL, Gagliano M, et al. Ocean acidification erodes crucial auditory behaviour in a marine fish. Biol. Lett-UK 7: 917-920, 2011. Solomon S, Qin D, Manning M, Chen Z, Marquis M. et al. Climate Change 2007: The physical science basis: Contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change. New York: Cambridge Univ. Press, 2007. Strandberg M, Damgaard C, Degn HJ, Bak J, Nielsen KE. Evidence for acidification-driven ecosystem collapse of danish erica tetralix wet heathland. Ambio. 41: 393-401, 2012. Stumpp M, Dupont S, Thorndyke MC, Melzner F. CO2 induced seawater acidification impacts sea urchin larval development II: Gene expression patterns in pluteus larvae. Comp. Biochem. Physiol. 160A: 320-330, 2011a. Stumpp M, Wren J, Melzner F Thorndyke MC, Dupont ST. CO2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay. Comp. Biochem. Physiol. 160A: 331-340, 2011b. Verling E, Crook A, Barnes D. Covering behaviour in Paracentrotus lividus: is light important? Mar. Biol. 140: 391-396, 2002. Yamada Y, Ikeda T. Acute toxicity of lowered pH to some oceanic zooplankto n. Plankton Biol. Ecol. 46: 62-67, 1999. Zhang DJ, Li SJ, Wang GZ, Guo DH. Comparative study on the acute toxicity of ocean acidification driven by CO2 and HCl on several marine copepods. J. Xiamen Univ. 50: 631-636, 2011. Zhang L, Zhang L, Shi D, Wei J, Chang Y, Zhao C. Effects of long-term elevated temperature on covering, sheltering and righting behaviors of the sea urchin Strongylocentrotus intermedius. Peer J. 5:e3122, 2017. Zhao C, Luo S, Zhou H, Tian X, Chang Y. Effect of temperature on covering behavior of the sea urchins Glyptocidaris crenulars and Strongylocentrotus intermedius. 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