FANG Jinghui, ZHANG Jihong, JIANG Zengjie, ZHAO Xuewei, JIANG Xu,DU Meirong, GAO Yaping, and FANG Jianguang, *

?

Tolerance, Oxygen Consumption and Ammonia Excretion ofin Different Temperatures and Salinities

FANG Jinghui1), ZHANG Jihong1), JIANG Zengjie1), ZHAO Xuewei2), JIANG Xu1),DU Meirong1), GAO Yaping1), and FANG Jianguang1), *

1),,,266071,2)..,116503,

There are more than 2000 species of brittle stars in the world. For most of them, many scientific questions including basic characteristics of eco-physiology are still unknown. In the present study,acclimated at 15℃, salinity 31, were assessed for temperature and salinity tolerance. Its oxygen consumption and ammonia excretion were studied at different temperatures (5, 10, 15, 20, 25℃) and salinities (25, 30, 35).could tolerate 0–24℃ and no brittle star was dead in the salinity range of 19–48 in the experimental situation. Two-way ANOVA showed that the oxygen consumption and ammonia excretion normalized with both dry mass and wet mass,10,which is used to describe the temperature sensitivity of respiration, and moisture content were significantly affected by temperature and salinity, and the combined effects of the two factors were significant. Stepwise multiple regression analysis revealed that logarithmic oxygen consumption and ammonia excretion showed a significant positive relationship with logarithmic temperature and salinity. The logarithmic moisture content of the brittle stars showed an inverse relationship with logarithmic salinity, but a positive relationship with logarithmic temperature. This suggests that the tolerance of temperature and salinity of brittle stars is closely related to their living environment, and that the effects of temperature on oxygen consumption are more significant at higher salinity, and that the ammonia excretion is less affected by salinity at lower temperatures.

;tolerance; oxygen consumption; ammonia excretion; temperature; salinity

1 Introduction

There are more than 2000 species of brittle stars in the world and all of them are marine ones. They are important to the ocean ecosystem because of their wide distribution and their occurrence in numerous individuals (Stancyk, 1974; Pawson, 2007; Liao and Xiao, 2011; St?hr and O’Hara, 2011). Some of them feed on sediment, thereby promoting the breakdown of organic wastes and improving the oxygen content of the bottom environment (Liao, 2004). Brittle stars are even an important diet of several carnivorous fishes (Xue, 2010a, 2010b). Some species, such asand, contain trace elements, eicosapentaenoic acid (EPA, 20: 5n?3) and alpha-linolenic acid (18: 3n?3), and are considered as a functional food for improvement of human health (Wang, 2010; Zhang, 2011). However, brittle stars have not been well studied and only a little has been known about them. Most of the reports about brittle stars are focused on taxonomy and many species were only reported when they were found (Chang, 1948; Clark, 1917; Clark, 1955). In the past 50 years, brittle stars received comparatively little attention (St?hr and O’Hara, 2011). Many scientific questions including basic characteristics of eco-physiology in brittle stars still need to be clarified.

Although brittle stars are marine animals, their tolerances of salinity are different and some of them can endure lower salinities (Thomas, 1961; Liao, 2004).can live at salinity 7.7 (Thomas, 1961), which is the lowest recorded salinity within the geographic range of any echinoderms (Binyon, 1966). Evenlives in estuaries with lower salinity (Liao, 2004). Moreover, some brittle stars are sensitive to the fluctuation of temperature.can not endure the slow changes (more than 6 days) of temperature from 0℃ to 2℃ or 3℃ (Peck, 2009). However, for most brittle stars, the tolerances of salinity and temperature are still unknown, and their metabolism at different temperatures and salinities has seldom been explored. It is not known if there is any relationship between the tolerances and metabolism.

Djakonov, 1954 is abundant in the Yellow Sea area of China, the northern East China Sea, Sea of Japan and Sea of Korea.lives in water about 40–60m deep (Liao, 2004). They can live on several kinds of sediment with higher densities. Currently, there are a few reports about this species. Their numerous populations show that they are of some importance in marine ecosystem. Sometimes,appears also in bottom mariculture areas. Their numerous individuals may consume a lot of oxygen and excrete much ammonia, which may affect water quality near the bottom. However, their effects on bottom mariculture and their ecological functions are still largely unknown. The records of salinity and temperature at the place where the experimental animals were collected had the values of 31–32 and ?1–22℃ during the year, respectively.

In the present study, the tolerances ofto temperature and salinity were determined in a preliminary experiment. The effects of temperature and salinity on the metabolism ofwere also studiedThe results would be important to explain the distribution, abundance, and ecological role of this species.

2 Materials and Methods

2.1 Source of Animals and Acclimation

individuals were collected in the north of the Yellow Sea (39?02.514′N, 122?44.089′E). The active and integral animals were taken to the laboratory and cultured in two glass tanks (50cm×50cm×40cm) with a water recirculation system. The water temperature was maintained at 15℃ and controlled by a chiller (HXL HLS1000, Dalian Huixin Titanium Equipment Development Co., Ltd, China). The salinity was 31, pH was 8.0–8.1. The brittle stars were fed once a day at 8:00 with the feed for Japanese flounderThe acclimation period lasted for 7 days.

2.2 Experimental Design and Management

2.2.1 Preliminary experiment

There were five treatments: high temperature (HT), low temperature (LT), high salinity (HS), low salinity (LS) and the control (C). The changes of temperature and salinity were made according to the water quality conditions during acclimation (, 15℃;, 31). Temperature and salinity were decreased or increased 1 unit per day. The salinity was kept stable at 31 in temperature treatments, and the temperature was kept stable at 15℃ in salinity groups. The high temperature was controlled by separate electric heaters (MX300 IC, WEIPRO Aquarium Equipment Co. Ltd, China). Low temperature was controlled by a chiller able to cool the water down to 2℃. Ice made from the same seawater in the corresponding aquarium was added to the water to achieve 0℃ and 1℃. Sea salt (Qingdao Goe Haida Sea Salt Co., Ltd., China) was added into the seawater to achieve higher salinities. Seawater was mixed with distilled water to obtain lower salinities. The control treatment was kept at 15℃ and salinity 31. There were three replications of each treatment. Twenty brittle stars starved for 24h were selected for each replication. So there were 300 brittle stars (body mass (1.00±0.16)g, disk diameter (13.48±0.66)mm) used in the tolerance experiment.

The aquarium size for each group was 20cm×30cm×20cm. During the experiment, the water was changed entirely at 9:00 every day. Light aeration was provided continuously. A simulated natural photoperiod (14h light: 10h dark) was used during the experiment. The temperature was measured using a thermometer (0.1℃, WLB, http:// puteyibiao.nhw100.com/, China). Salinity was checked with a hand refractometer (LH-T100, Lohang Biological Co. Ltd, China). The activity and the survival of brittle stars were recorded at 9:00 every day. An animal was considered as dead when it did not move after being touched with a glass stick or if the arms were totally inflexible. The test variables, temperature and salinity, were changed every day until all animals had died. The results of the preliminary experiment were used in designing the temperature and salinity gradient in metabolism experiment.

2.2.2 Respiration and excretion experiment

According to the results of the tolerance experiment, five temperatures (5, 10, 15, 20, 25℃) and three salinities (25, 30, 35) were chosen to be tested in the metabolism experiment. Brittle stars with similar size (body mass (0.98±0.11)g, disk diameter (13.01±0.25)mm) were divided into three groups with different salinities after acclimation. The salinity was changed 1–2 units per day to the target salinity values. There were 48 brittle stars in each salinity group, 16 for 25℃, and 8 for each of the other four temperature treatments. The water temperature was decreased or increased by 1–2℃ per day. The temperature and salinity managements were the same as in the preliminary experiment. All animals were acclimated for five days to the corresponding states.

To determine the metabolism, oxygen consumption and ammonia excretion were quantified using a closed respirometer. Four individuals ofwere placed separately in four 250mL respirometers (Yang, 2006), and the exact volume of each respirometer was measured and marked on its outer covering. The optimal size of the respirometer was determined by a pre-expe- riment to be 250mL. The time of metabolic rate measurement was pre-calculated to ensure the dissolved oxygen in each respiration chamber remaining above 5.0mgL?1. The animals were maintained in the respirometers with light aeration for 3h for acclimation prior to measurements. After acclimation, the water in the chambers was exchanged with the same water of the corresponding treatment and the respirometers were sealed under water to avoid remaining air bubbles. Three chambers withoutwere used as controls for each treatment. The chambers were immersed into the corresponding treatment water for about 3h. The final level of the dissolved oxygen for each chamber was measured with a DO meter (YSI-5000, USA), and measurements of ammonia excretion were taken simultaneously, which were determined by the oxidizing reaction of sodium hypobromite (GB/T 12763.4-2007). After the measurements, brittle stars were removed from the water and blotted with sterile gauze to remove external water (Battaglene,1999), and then weighed. Finally they were dried separately in an oven at 70℃ for 48h to determine their dry weights.

Oxygen consumption rate (O2) (unit: μmolO2h?1g?1) was calculated using the following equations:

,

wherecandeare the DO levels (mgL?1) of control and experimental chambers, respectively,is the volume (L) of chamber,is the experimental time (h), 32 is the molecular weight of O2, andDandWare the dry and wet body masses (g) of the measured, respectively.

Ammonia excretion rate () (unit: μmolNh?1g?1) was calculated using the following equations:

,

wherecandeare the ammonia N levels (mgL?1) of control and experimental chambers, respectively,is the volume (L) of chamber,is the experimental time (h), 14 is the atomic mass of N, andDandware the dry and wet body masses (g) of the measured, respectively.

Given the widespread usage of10in modeling and to facilitate comparisons across published studies,10values were used to describe the temperature sensitivity of respiration. They were calculated separately for each of the four measured temperature intervals (5–10℃, 10–5℃, 15–20℃, 20–25℃) based on the equation from Tjoelker(2001):

where(T)is oxygen consumption rate at temperature,0, the oxygen consumption rate at a reference temperature0(5, 10, 15 or 20℃), and10, the ratio of rates given a 10℃ change in temperature.

The experiments were conducted from November 15, 2012 to December 28, 2012.

2.3 Data Analysis

Statistical analysis of the data was performed with a statistical package (SPSS 11.0 for Windows, SPSS Inc., Richmond, CA, USA). The assumption of homogeneity of variances was tested for all data, which were log- transformed if necessary. The influence of and interaction between temperature and salinity on oxygen consumption, ammonia excretion, moisture content and10were tested using two-way analysis of variance (ANOVA). Statistically significant influences proved by ANOVA were followed by a Duncan’s multiple comparison to determine the within treatment effects. The effects of temperature and salinity on oxygen consumption, ammonia excretion and moisture content were tested using stepwise multiple regression analysis. The differences were considered statistically significant at<0.05.

3 Results

There were no deadin the control during the period of the experiment. In the respiration and excretion experiments, there remained 4, 10 and 8 brittle stars alive at salinities 25, 30 and 35, respectively, in the 25℃ treatments after acclimation.

3.1 Temperature Tolerance at Normal Salinity

Under the conditions of the experiment,could tolerate a temperature range of 0–24℃ at the salinity of 31 (Fig.1). Dead animals appeared at 25℃ and all individuals died at 27℃.

Fig.1 Mortality of Ophiopholis sarsii vadicola at different temperatures (T).

3.2 Salinity Tolerance at the Temperature of Acclimation

Under the conditions of the experiment,could tolerate a salinity range of 19–48 (Fig.2). At the salinities of 20 or 46, the brittle stars began to lose their arms. Dead animals appeared at salinities 18 and 49, respectively. All brittle stars died at salinities 16 and 52 at the LS and HS treatments, respectively.

Fig.2 Mortality of Ophiopholis sarsii vadicola at different salinities (S).

3.3 Oxygen Consumption

The two-way ANOVA analysis showed that both temperature and salinity significantly affected the oxygen consumption related to dry body mass (O2-D,:=676.202,<0.001;:=22.110,<0.001) and wet body mass (O2-W,:=774.642,<0.001;:=51.074,<0.001). The interaction between the two factors was significant (Table 1,O2-D:=11.735,<0.001;O2-W:=10.581,<0.001). Temperature affected oxygen consumption more than salinity itself or the combination of the two factors did. The oxygen consumption ofincreased significantly when the temperature and salinity increased respectively (<0.05, Fig.3).

Table 1 Two-way ANOVA table of VO2-MD, VO2-Mw, TAN-MD, TAN-Mw and moisture for Ophiopholis sarsii vadicola at different temperatures (T) and salinities (S)

Notes:?O2-DandO2-Ware the oxygen consumption ofnormalized with dry mass and wet mass, respectively.??-Dand-Ware the ammonia excretion ofnormalized with dry mass and wet mass, respectively.

Fig.3 Oxygen consumption of Ophiopholis sarsii vadicola normalized with dry mass (VO2-MD) and wet mass (VO2-MW) at different temperatures (T) and salinities (S). Different letters denote significant differences among treatments at the same salinity (P<0.05).

Stepwise multiple regression analysis revealed a significantly positive relationship between logarithmic oxygen consumption (O2-DandO2-W) and logarithmic temperature () plus logarithmic salinity () as shown by the regression equations:

(=180.905,<0.001,2=0.864,=60),

(=159.203,<0.001,2=0.848,=60).

3.410Coefficients

There was a significant increase in10in the tem- perature range of 20–25℃ at salinity 25. The values of10increased with the increasing temperature at salinity 30. There was no obvious relationship between10and temperature at salinity 35, and the values were higher when the temperature was below 15℃ (<0.05). Two- way ANOVA analysis showed that both temperature and salinity affected10significantly (<0.05). The interaction was significant between the two factors (<0.05, Table 2).

Table 2 Q10 of Ophiopholis sarsii vadicola for temperature (T,℃) intervals of 5℃ at different salinities (S) (mean±S.E.)

(to be continued)

Notes:?O2-DandO2-Wwere the oxygen consumption ofnormalized with dry mass and wet mass, respectively.??andwere from the results of Two-way ANOVA for10. Data with different letters at the same salinity were significantly different (<0.05).

3.5 Ammonia Excretion

As to the oxygen consumption, two-way ANOVA analysis showed that both temperature and salinity significantly affected the ammonia excretion normalized with dry mass (-D,:=241.227,<0.001;:=13.446,<0.001) and wet mass (-W,:=245.116,<0.001;:=22.914,<0.001). The interaction was significant between the two factors (-D:=52.850,<0.001;-W:=56.470,<0.001). Temperature affected the ammonia excretion more significantly than did salinity and the interaction (Table 1). Similar to the oxygen consumption theofincreased significantly with increasing temperatures within 5–20℃ (<0.05), but it decreased at 25℃. Moreover, there was a trend that it increased with increasing salinity in the interval 5–20℃, and the most significant effect was observed at 20℃. However, ammonia excretion decreased with the increasing salinity at 25℃. The highestwas observed at 20℃, salinity 35 (Fig.4).

Fig.4 Ammonia excretion of Ophiopholis sarsii vadicola normalized with dry mass (VO2-MD) and wet mass (VO2-MW) at different temperatures and salinities (S). Different letters denote significant differences among treatments at the same salinity (P<0.05). S25, S30 and S35 are salinities 25, 30 and 35, respectively. Error bars represent 1 S.E.

Stepwise multiple regression analysis illustrated a significantly positive relationship among logarithmic ammonia excretion (-Dand-w), logarithmic temperature () and logarithmic salinity (). The liner relationship is shown by the regression equations:

(=57.374,<0.001,2=0.668,=60),

(=61.156,<0.001,2=0.682,=60).

3.6 Moisture Content

Two-way ANOVA analysis showed that the moisture content ofwas significantly affected by temperature and salinity (:=16.896,<0.001;:=64.308,<0.001), and the interaction was significant between the two factors (=4.088,<0.01). Salinity affected moisture content ofmore significantly than did temperature and the interaction (Table 1). The moisture content was highest at salinity 25 (<0.05, Fig.5).

Fig.5 Moisture content of Ophiopholis sarsii vadicola at different temperatures (T) and salinities (S). Different letters denote significant differences among treatments at the same salinity (P<0.05). S25, S30 and S35 are salinity of 25, 30 and 35, respectively. Error bars represent 1 S.E.

Stepwise multiple regression analysis revealed a significantly positive relationship between logarithmic mois- ture content () and logarithmic temperature (), and inverse relationship with logarithmic salinity (). The linear relationship is shown by the regression equation:

(=25.479,<0.001,2=0.472,=60).

4 Discussion

In previous studies, the temperature and salinity tolerances of brittle stars were only based on the conditions of their distribution (Fell, 1961; Liao, 2004). Few reports were focused on studying their tolerances of temperature and salinity experimentally (Peck, 2009). However, from studying the tolerances of some other echinoderms it was found that they could not regulate the osmotic concentration of coelomic fluid (Binyon, 1972a, b). The osmotic conditions in echinoderms could change within one day following salinity fluctuation (Stickle and Denoux, 1976). Dong(2008) reported that the osmotic pressure ofchanged gradually at salinities such as 20, 25, 30, and 40, and stabilized by 6 h. In the present study, the acclimation time lasted for five days. The brittle stars had time to adjust their osmotic pressure.Nevertheless, they could not recover their moisture content at salinity 25. It means thatcannot regulate the osmotic pressure of the coelomic fluid, which may be the main cause of their low salinity tolerance in the experiments. Low salinity is a stressor in juvenile(Dong, 2008; Wang, 2013), which is similar to the result in the present study. Furthermore,can live at a low salinity of 10. However, it can not endure fresh water. Because they live at estuary where the salinity is above 10 (Liao, 2004), the tolerance of temperature and salinity of echinoderms may correlate to their living environment. The collectedin the present study lives at salinities 31?32. It can not acclimatize itself to a wide salinity range. Peck(2009) reported that thewhich lived blew 0℃ could not tolerate a slow temperature increase to 2 or 3℃. In the waters where thewas collected, the temperature ranges ?1–22℃ in the whole year, which is close to the tolerance temperature offound in present experimentsSo the salinity and temperature of the living environment affect the tolerance a lot in. However, whether this phenomenon is common to most brittle stars is unknown. More experiments with different species and environmental conditions will be conducted to reveal the possible differences and underlying mechanisms.

It was demonstrated that both temperature and salinity affected the metabolism of aquatic animals significantly (Claireaux and Lagardère, 1999; Resgalla, 2007; Yu, 2012). Nevertheless, these parameters have seldom been involved in studying the eco-physiology of brittle stars. One previous study found that there is a positive relationship between the oxygen consumption ofand temperature(Christensen, 2011). Sometimes the oxygen consumption is depressed with animals under suitable temperature (Hokanson, 1977; Jobling, 1994; Bao, 2010). In the present study, the oxygen consumption ofincreased with increasing temperature even up to 25℃. There was no depression in oxygen consumption in this temperature range although first mortalities occurred at 25℃ in the tolerance test. A similar result has been found in seacucumberswith their respiration increased up to 25 and 27℃ which is no longer suitable for them (Dong, 2005; Bao, 2008). However, the ammonia excretion of sea cucumber also increases with increasing temperature (Bao, 2008), which is not consistent with the present study. The ammonia excretion ofwas already depressed at 25℃. It is an interesting phenomenon inwith higher oxygen consumption and lower ammonia excretion at the same temperature, which has seldom been reported in other echinoderms. The physiological system ofobviously began to change at 25℃. Maybe it decreased the metabolic turnover to avoid exhaustion or exponential increase of oxygen consumption which could not be covered by respiration.

The value of10is a parameter to describe the sensitivity of organisms to temperature increase. Higher values of10at a certain temperature range mean that the respiration of the organism reacts sensitively at these temperatures by increasing rapidly (Rao and Bullock, 1954). Former studies reported that the value of10is not only affected by temperature but also correlates with the size, season, nutrition and habitat acclimation temperature of poikilotherms (Rao and Bullock, 1954; McPherson, 1968). In this study, the value of10was significantly affected by salinity. The changes of10from 5℃ to 25℃ at the three salinities were different, which was only detected by invoking both temperature and salinity simultaneously.

Salinity is more important to echinoderms because they have noorgan to regulate the osmotic pressure of coelomic fluid (Binyon, 1972a). The change of osmotic pressure is probably one main factor for metabolism fluctuations of aquatic organisms caused by different salinities. When water is isotonic to the normal coelomic fluid, aquatic organisms spend less energy to regulate osmotic pressure, which results in a lower metabolism level. Several reports support this theory (Talbot and Lawrence, 2002; Yuan, 2006). Furthermore, Diehl (1986) reported that the osmotic pressure of echinoderms would be equilibrated with the surrounding water within 8–24h. In the present study, the animals had enough time (the acclimation time >24h) to regulate the osmotic pressure. However, the oxygen consumption and ammonia excretion were lower at salinity 25, which was not the optimal salinity, except for the ammonia excretion at 25℃. It seems inconsistent with the former theory. However, there are some similar reports about four echinoderms which show lower metabolism at lower salinity (Giese, 1966; Sabourin and Stickle, 1981; Shirley and Stickle, 1982). Maybe different echinoderms species is the main cause.

The effects of temperature and salinity on the metabolism of echinoderms and interactions between them haven’t been well studied. In the present experiments, the effects of temperature and salinity and their combined effect on the metabolism were proved to be significant. The effects of temperature on oxygen consumption were stronger at higher salinities. The ammonia excretion was less affected by salinity at lower temperatures. The investigation reveals that temperature and salinity are both important to the metabolism of

Acknowledgements

This study was supported by the National Basic Research Program of China (Grant No. 2011CB409805); the National Science & Technology Pillar Program (Grant No. 2011BAD13B06); Special Scientific Research Funds for Central Non-profit Institutes, Yellow Sea Fisheries Research Institute (20603022013042); the National Science and Technology Planning Project of China (Grant No. 2011BAD13B05).

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(Edited by Qiu Yantao)

10.1007/s11802-015-2513-4

(October 11, 2013; revised December 10, 2013; accepted September 25, 2014)

. Tel: 0086-532-85822957 E-mail: fangjg@ysfri.ac.cn

ISSN 1672-5182, 2015 14 (3): 549-556

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015