Physiological energetics and gross biochemical composition of the ascidian Styela clava cultured in suspension in a temperate bay of Korea

Aquaculture 319 (2011) 168–177

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Physiological energetics and gross biochemical composition of the ascidian Styela clava cultured in suspension in a temperate bay of Korea Chang-Keun Kang a,⁎, Eun Jung Choy b, Won Chan Lee c, Nam Jung Kim a, Hyun-Je Park a, Kwang-Sik Choi d a

POSTECH Ocean Science & Technology Institute, Pohang University of Sciences and Technology, Gyeongbuk 790-784, Republic of Korea Korea Polar Research Institute, Korea Ocean Research and Development Institute (KORDI), Incheon 406-840, Republic of Korea Marine Environment Research Division, National Fisheries Research and Development Institute (NFRDI), Busan 619-705, Republic of Korea d Faculty of Marine Biomedical Science, Jeju National University, 66 Jejudaehakno, Jeju 690-756, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 10 January 2011 Received in revised form 9 April 2011 Accepted 11 June 2011 Available online 2 July 2011 Keywords: Styela clava Suspended culture Physiological energetics Biochemical composition

a b s t r a c t The ascidian Styela clava has recently become a common species for suspended aquaculture in Korea. Because of the ecological and commercial importance of this species, it is important to understand seasonal variations in its physiological energetics and gross biochemical composition. The purpose of this study was to determine fundamental biological traits for the cultivation of S. clava. Physiological processes (food consumption, feces production, ammonia excretion and respiration), and gross biochemical composition (protein, lipids, and carbohydrates) of the ascidian were measured monthly from April 2008 to April 2009 under in situ environmental conditions in a mariculture region on the temperate coast of Jindong Bay, Korea. Changes in the physiology and somatic growth of S. clava were largely influenced by seasonal variation in water temperatures. The reduced importance of food availability in explaining their physiological adjustments seems to be due to low seston concentrations in water column of the bay. Seasonal variations in the ingestion and respiration rates of the ascidians were mismatched, resulting in an energy imbalance (i.e. an increased metabolic energy cost and lowered ingestion rate; and vice versa). This mismatched activity resulted in negative scope for growth (SFG) values during spring–summer, followed by rapid exhaustion of energy reserves and flesh weight loss. Weight loss during this period was also related to spring spawning. During autumn–winter, the ascidians had a positive SFG as a result of decreased oxygen consumption and elevated ingestion rates, showing accumulation of nutrient reserves and weight gain. The ascidians showed positive SFG, even in cold conditions below 12 °C in the present study. In this respect, an autumn–winter culturing period is recommended to maximize ascidian production in long-line suspended culture under the natural environmental conditions occurring in the study area. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The sessile suspension-feeding ascidian Styela clava is ubiquitous in coastal rocky habitats worldwide. This ascidian is an efficient filterfeeder that is capable of depleting concentrations of suspended particles in the water column. Therefore, its role as a troublesome competitor for settlement space and food with other suspension feeders, such as mussels and oysters, is well known (Thompson and MacNair, 2004). Large populations of ascidians can also have a negative effect on coastal aquaculture systems through phytoplankton consumption, nutrient cycling and biodeposition (Jiang et al., 2008c). In contrast, S. clava has long been one of the favorite types of Korean seafood, and thus is a common aquaculture species in Korea. Suspended culture of the ascidian was initiated in 2001 and the annual production reached about 15,000 t in 2001, but declined to

⁎ Corresponding author. Tel.: + 82 54 279 9503; fax: + 82 54 279 9519. E-mail address: [email protected] (C.-K. Kang). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.06.016

3500 t in 2009 because of mass mortality of mature ascidians and a local shortage of healthy larvae (unpubl. data, Southeast Sea Fisheries Research Institute, National Fisheries Research and Development Institute of Korea). Seasonal cycle for the suspended culture of the ascidian is presently unclear compared to the bivalve cultures since the farming cycle has been determined by the fishermen themselves. Traditionally, the ascidian seeds for culture have been naturally collected using a nylon net collector of 50 mm mesh in shallow subtidal from May to September. The attached spats to the collector are utilized as seeds for the commercial culture. Then the net collectors are transplanted to the subtidal farming ground and hung with long lines in water column. The ascidians are cultured in suspension until next April–May when the marketable-size ascidians are harvested. Scientific information on biological cycle may be needed for more effective culturing activity. Because of its ecological and commercial importance, interest in the biological traits of the ascidian has increased (Bourque et al., 2007; Jiang et al., 2008b). Temperate coastal areas such as the Korean peninsula where S. clava occurs are characterized by great seasonal fluctuations in

C.-K. Kang et al. / Aquaculture 319 (2011) 168–177

environmental variables such as temperature and nutritional condition (Lee et al., 2001). Accordingly, ascidians can acclimatize to a wide range of environmental conditions and survive in particular environmental conditions by physiological adjustments for maintenance, growth, and reproduction. In general, seasonal measurement of physiological processes, such as food consumption, feces production, respiration, and excretion has been known to provide insight into seasonal adaptive strategies in the individual physiological components of marine mollusks (Bayne et al., 1985; Bayne and Newell, 1983). In addition, the integration of these processes by means of physiological energetics can provide further insight into the growth phase of an organism, giving information on acquisition and expenditure of energy and the efficiencies of energy transformation from the standpoint of the individual organism (Bayne and Newell, 1983). Little information is available about seasonal variation in physiological energetics and biochemical composition of the ascidian. Indeed, while most of the physiological studies of filter-feeding animals have been done on bivalves, few studies have focused on the reproductive biology (Bourque et al., 2007), food selectivity (Jiang et al., 2008c), oxygen consumption (Jiang et al., 2008a; Zhang and Fang, 2000), ammonia excretion (Zhang et al., 2000) and physiological energetics (Jiang et al., 2008b) of the ascidian S. clava. Most of these measurements have been made under controlled laboratory conditions, demonstrating that each physiological activity of S. clava is closely related to exogenous as well as endogenous factors. In general, the physiological measurements are represented by an allometric relationship between the rates of physiological processes and body size. Using an allometric equation, seasonal physiological responses of a single standard-sized group can be estimated and compared for a given combination of natural environmental conditions in the field (Bayne et al., 1985; Farías et al., 2003; Navarro and Thompson, 1996). Therefore, in relation to seasonal changes in energy balance caused by variations in field-ambient temperatures and nutrient concentrations, monthly physiological measurements are expected to describe the seasonal variation in bioenergetics of S. clava in more detail, and allow us to further understand its adaptive responses to optimize energy balance. In this paper, we detail the seasonal variation in physiological energetics and gross biochemical composition of S. clava cultured in suspension in Jindong Bay, Korea. Rates of consumption (i.e. ingestion) of food and feces production, ammonia excretion and oxygen consumption were measured monthly during an annual cycle and related to the individual body mass to analyze allometric relationships. Scope for growth (SFG) was then determined to evaluate the integrated

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physiological responses of S. clava to seasonal changes in environmental conditions. Seasonal patterns of biochemical components of S. clava were also simultaneously measured. The aims of the present study were to highlight physiological strategies to optimize energy gains for growth of S. clava, and to better understand seasonal cycles of storage and utilization of energy reserves to gain basic information about optimum culture conditions in natural temperate environments. 2. Materials and methods 2.1. Study site Jindong Bay is a small shallow bay located on the southern coast of Korea at about 35° 8′N and 128° 3′E with a mean water depth of 8.83 m, and is well known as an area for culturing suspension-feeding bivalves and tunicates (Fig. 1). The tide is semidiurnal with a maximum tidal range of 2.0 m on spring tide. The water column is well mixed because of its shallow depth. Of the total bay area of 2160 ha, 333.5 ha has been exploited for the cultivation of oysters, mussels, ark shells and ascidians. Annual production of cultured species in the bay has dropped dramatically because of the occurrence every summer of red tide and hypoxia events (Kim et al., 2001; Kim and Kim, 2003). For this reason, cultivation of bivalves has been recently reduced. Styela clava has previously been considered a fouling organism in culture systems, but is now one of the most dominant cultivated species in the bay. The ascidians are cultured in suspension using the nylon net in water column of the bay. One of the ascidian-culturing beds was selected to collect specimens and measure physiological processes for in situ conditions at the central part of the bay. 2.2. Field experimental conditions and biometric measurement of animals The experiments were performed monthly on board of a barge from April 2008 to April 2009. For the in situ experiments, a 5 × 10 m barge was deployed at the collection site of the animals. For each experiment, 30 ascidians were randomly collected from a depth of 1 to 5 m below the water surface. For each specimen, a section of rope 50 mm long and 3 mm thick was cut to avoid physiological disturbance caused by collection, and individual ascidians attached on the rope were used for the experiments. After collection, specimens were immediately cleaned of any epibionts in a container filled with in situ seawater, and individual animals were then held in

44o

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Masan

42 o

o

35 20'

Jinhae

40 o

KOREA y

38o

ae

35o 00'

nh

124o 126o 128o 130o 132o E

Jindong Bay

Ji

34o

Ba

36o

128o 50' Fig. 1. Map showing the sampling and experimental site (■) in Jindong Bay, Korea.

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500 ml grazing chambers and flow-through closed chambers, respectively. Unfiltered seawater was pumped directly from a depth of about 3 m under the barge into a mixing chamber equipped with a magnetic stirrer, and then flowed into the experimental chambers. Ten chambers were used for grazing and respiration experiments, respectively, of which nine were filled with one individual and one was a control without an ascidian. Grazing and respiration chambers were half or absolutely immersed, respectively, in water baths into which seawater was pumped in situ and continuously supplied in order to maintain field ambient temperature. Flow rates were controlled between 30 and 50 ml min − 1, allowing the reduction of seston concentrations between the inflow and outflow to be maintained at 20–40% (Navarro and Thompson, 1996), and oxygen concentrations in the respiration chamber were kept at over 80% of the saturated concentration (Bougrier et al., 1995). All physiological measurements were done for 24 h after an acclimation period of 12 h in the experimental chambers, through which in situ seawater flowed from the mixing chamber. The ascidian specimens were transported to the laboratory immediately after physiological measurements. They were placed overnight in filtered seawater to evacuate their gut contents and then carefully dissected. The ascidian tunic was dried at 60 °C for approximately 72 h and the tunic dry weight was recorded. Tissues dissected for biochemical analysis were freeze-dried for 72 h and the tissue dry weight (DW) recorded. The dried tissues were ground to a powder with a mortar and pestle, and an aliquot was heated at 450 °C for 24 h to determine the ash weight. The remaining dry tissue was stored at − 20 °C in a refrigerator for later biochemical analysis. Powdered tissue samples (5–10 mg) were utilized to analyze biochemical composition. Protein content was determined using the method of Lowry et al. (1951) after alkaline hydrolysis with 0.5 N NaOH at 30 °C for 24 h. Carbohydrate and glycogen were extracted in 15% trichloroacetic acid and determined following the phenol– sulfuric acid method of Dubois et al. (1956). Glycogen was quantified after precipitation with 100% ethanol. Extraction of total lipid was performed in a mixture of chloroform and methanol (Bligh and Dyer, 1959), and the lipid content determined following the method of Marsh and Weinstein (1966). 2.3. Environmental conditions and seston analyses For each experiment, water temperature was measured using a CTD meter (Sea-Bird Electronics Inc., Bellevue, WA). Six water samples (every 4 h over a 24-h period) were taken during each experimental period. Water samples for suspended particulate matter were screened through a 180 μm Nitex mesh to eliminate zooplankton and large particles, collected in acid-washed plastic bottles, and then kept on ice in the dark. The water was filtered onto precombusted Whatman GF/F glass fiber filters (47 mm, 0.7 μm pore size), and the filters were dried at 60 °C for 48 h and placed into a desiccator. Total suspended particulate matter (SPM) was determined by weighing the filter before and after filtration of a known volume of water. The water was also filtered in the same manner for the analyses of biochemical composition (proteins, lipids, and carbohydrate) and chlorophyll a of particulate matter, and the filters were stored at −20 °C in a refrigerator until later analysis. Biochemical composition of the particulate matter was analyzed by the same procedure as described for animal tissue analyses (see Section 2.2). Chlorophyll a concentration was determined from acetone extracts (for 24 h in the dark at −20 °C) using a fluorometer (Turner Designs, Sunnyvale, CA) according to the method of Holm-Hassen et al. (1965). Additionally, the water was filtered onto 25 mm precombusted Whatman GF/F filters to analyze particulate organic carbon and nitrogen (POC and PN). The filters were dried at 60 °C for 48 h and wrapped in a tin disk. POC and PN were then determined after combustion at high temperature (1010 °C) using a CHN elemental analyzer (EuroVector,

Milan, Italy). The instrument was calibrated with an acetanilide standard (C, H, N = 71.09, 6.71, 10.36%). 2.4. Measurement of physiological rates and scope for growth Consumption (filtration) rate (C) was estimated by measuring the removal of SPM by individual ascidians, calculated from the reduction between the outflow from the control chamber and the outflow from each experimental chamber containing individual specimens. SPM during each experiment was collected several times (three to five) from the outflows of all the chambers using the same method as described for seawater samples (see Section 2.3). Protein, carbohydrate and lipid of the filtered SPM were analyzed and the rate was then converted into energy equivalents (J d − 1) using the conversion factors: 1 mg protein, carbohydrate and lipid = 24.0, 17.5 and 39.5 J, respectively (Gnaiger, 1983). During all experiments, little rejection of pseudofeces was found. Accordingly, ingestion rate (I) was estimated as C = I in this study. Feces (F) produced by individual ascidians were collected with a Pasteur pipette, transferred into a 5 ml precombusted and preweighed glass tube, rinsed with distilled water, freeze-dried, and then weighed. Protein, carbohydrate and lipid of the fecal materials were also analyzed using the same methods as described for animal tissue analyses (see Section 2.2). The rate of feces production was then converted into an energy equivalent (J d − 1) using the conversion factors as described above. The rate of ammonia excretion (U) was calculated from the increase in ammonia concentrations between the outflow from the control chamber and the outflow from each experimental chamber containing individual specimens. Water samples for ammonia analysis during each experiment were collected several times, simultaneously with SPM collection. The ammonia concentration was analyzed by the phenol–hypochlorite method (Widdows, 1978) and the result was transformed to an energy equivalent (J d − 1) using the conversion factor of 1 mg NH4–N = 24.83 J (Elliot and Davison, 1975). Oxygen consumption (R) was determined as the rate of decrease of oxygen concentration inside the measurement chamber, as recorded by oxymetric probes fitted with a stirring rod (Orbisphere Laboratories, Maurepas, France). Detailed methodology of the continuous monitoring system for oxygen consumption is described elsewhere (Bougrier et al., 1998). The rate of oxygen consumption was also converted into an energy equivalent (J d − 1) using the conversion factor of 1 mg O2 = 14.0 J (Gnaiger, 1983). DW of each ascidian was measured after each physiological measurement. The average DW of all specimens was considered to be the weight of a standard animal. SFG, which is a physiological index of energy balance to estimate production of an animal, was calculated for a standard animal by the standard energy budget equations: I = P + U + F + R; P = I – (U + F + R) = A – R, where I = consumption (i.e. ingestion); P = SFG; U = ammonia excretion; F = feces production; A = assimilation; and R = oxygen consumption. 2.5. Weight standardization of rates and statistical analyses To evaluate the physiological state of S. clava independent of growth, absolute values of DW and biochemical constituents were standardized to an equivalent of 930 mg total dry weight (tan average of all specimens analyzed) and compared for each experiment. Leastsquares regression analyses following logarithmic transformation (base 10) of DW and total (tunic + tissue) dry weight were done for each experiment according to the allometric equation: Y = aW b, where Y = DW, W = total dry weight, a and b = fitted constants representing the intercepts and slopes, respectively, of the regression equations. The same analysis was used to relate gross weights of the biochemical constituents to DW. All regressions were statistically

C.-K. Kang et al. / Aquaculture 319 (2011) 168–177

significant (P b 0.01). Gross biochemical composition was then computed for a given total dry weight by substituting the appropriate values of DW in the regression equations. The results of the biochemical analysis were then expressed as mg per standard animal (see details in Navarro et al., 1989). Physiological rates were also standardized to an equivalent of 165 mg DW, to avoid variation in the rates because of monthly weight changes (Bayne and Newell, 1983; Rueda and Smaal, 2004). The allometric equations between each physiological rate and DW were established using the same method as described above for biochemical composition. The relationships between the physiological measures and DW were then analyzed by linear regression following logarithmic transformation (base 10). Finally, the weightstandardized rate was calculated by substituting the standard weight of the animal in the regression equations obtained for each experimental period. The various sets of regression equations were analyzed by analysis of covariance (ANCOVA) to test the significance of differences in slopes (Sokal and Rholf, 1995), and significant differences among estimates of slopes were tested at a probability of P b 0.05. Pearson product-moment correlation and multiple stepwise regression analyses were carried out to determine the effect of external and internal parameters on the monthly variations of gross biochemical composition and physiological rates of S. clava. A commercially available software package was used to analyze the experimental results (SPSS package, Chicago, IL). 3. Results 3.1. Environmental variables Mean values of environmental variables at the in situ experimental site in Jindong Bay are presented in Table 1. Seawater temperatures varied between 5.0 °C (January 2009) and 26.7 °C (August 2008). Mean chlorophyll a concentration peaked in June (2.71 μg l − 1) and September 2008 (1.51 μg l − 1), with values lower than 1 μg l − 1 in the other months. Mean SPM fluctuated from 2.4 (July 2008) to 9.5 mg l − 1 (May 2008), and mean POM from 0.7 (December 2008) to 3.7 mg l − 1 (November 2008). While no clear seasonal trends were found in both SPM and POM values, energy equivalents of biochemical components of SPM were relatively higher in spring–summer compared to autumn–winter. Mean concentrations of particulates varied from 19.8 (March 2009) to 422.0 J l − 1 (June 2008) for proteins, from 21.3 (March 2009) to 314.1 J l − 1 (August 2008) for carbohydrates, and from 20.9 (September 2008) to 285.7 J l − 1 (June 2008) for lipids. Mean concentrations of POC and PN showed irregular seasonal

171

variations with 81–733 μg l − 1 and 11–92 μg l − 1, respectively. On the other hand, the C:N (by atoms) ratio fell within a small range between 7.0 and 9.3. In contrast, the C:Chl a (by weights) ratio varied greatly from 149 to 1703, but showed no clear seasonal trend.

3.2. Seasonal biometric allometry and gross biochemical composition of flesh Monthly regression analysis of DW against total dry weight (flesh + tunic) in the ascidian is summarized in Table 2. ANCOVA test revealed significant differences between estimates of the slope (b). The b values were higher during the weight loss period (0.993– 1.231, April to September) than during the weight gain period (0.842–0.980, October to March). Further, since the ANCOVA test revealed no significant difference between the b values within the two (weight loss and gain) periods, two common slopes were used to calculate seasonal changes in DW of a standard animal (930 mg total dry weight). The results displayed a clear seasonal trend, with maximum DW in April of both 2008 and 2009 and a minimum in September 2008 (Fig. 2). Ash-free dry weight (AFDW) of a standard animal was also calculated using the same procedure with the flesh dry weight, the seasonal pattern being very similar to that of the flesh weight (detailed procedure not shown). All regressions of biochemical components against DW in S. clava were significant (Table 3), and ANCOVA test revealed significant differences between estimates of the slope for each component, without any seasonal trend. Gross contents (mg) of each biochemical component of a standard animal were calculated using monthly regression equations. Seasonal patterns in the absolute content of each biochemical component paralleled those of DW and AFDW (Fig. 2), with a maximum in April of both years and minimum levels in summer (and also in autumn for carbohydrates and lipids). While flesh tissue growth of the ascidian was accompanied by increased storage of energy reserves during autumn–winter, weight loss in flesh tissue occurred concurrently with the decrease of energy and storage substances during spring–summer. Proteins were calculated to account for about 60% of the spring–summer decrease, and the subsequent autumn–winter increase in DW. On the other hand, carbohydrates and lipids contributed less than 10% to the flesh weight loss and gain. Both substances showed a fast rate of utilization in late spring (April–June) and a slow rate of accumulation in winter (January–April), without any clear mobilization during the summer–autumn period. In addition, little accumulation and exhaustion of glycogen happened over a one-year period.

Table 1 Water temperature (T), chlorophyll a (Chl a), total suspended particulate matter (SPM), suspended particulate organic matter (POM), and particulate protein (PPr), carbohydrate (PCHO), lipid (PLip), particulate organic carbon and nitrogen (POC and PON), C:N and C:Chl a at the in situ experimental site. Month

April 2008 May June July August September October November December January 2009 February March April

T

Chl a

SPM

POM

PPr

PCHO

PLip

POC

PON

C:N

C:Chl a

(°C)

(μg l− 1)

(mg l− 1)

(mg l− 1)

(J l− 1)

(J l− 1)

(J l− 1)

(μg l− 1)

(μg l− 1)

by atoms

by weights

13.3 20.7 22.2 26.4 26.7 25.8 19.9 13.0 10.4 5.0 6.6 10.7 14.8

− 0.23 ± 0.17 2.71 ± 0.52 0.86 ± 0.52 0.43 ± 0.06 1.51 ± 0.28 0.55 ± 0.18 0.97 ± 0.12 0.67 ± 0.30 0.87 ± 0.19 − 0.31 ± 0.11 −

2.55 ± 0.05 9.50 ± 0.14 5.05 ± 1.91 2.40 ± 0.71 3.95 ± 0.64 5.30 ± 1.84 5.35 ± 0.07 7.80 ± 0.14 2.85 ± 1.34 6.50 ± 1.41 − 5.55 ± 1.34 5.95 ± 1.48

0.90 ± 0.14 0.85 ± 0.07 2.30 ± 1.13 0.55 ± 0.50 0.90 ± 0.14 1.35 ± 0.50 2.25 ± 1.48 3.68 ± 0.46 0.65 ± 0.07 1.33 ± 0.53 − 0.83 ± 0.18 1.68 ± 0.32

243.2 ± 47.9 214.0 ± 28.4 442.0 ± 55.8 110.2 ± 5.5 234.9 ± 0.0 46.4 ± 19.8 220.6 ± 47.4 76.7 ± 13.8 26.7 ± 0.8 51.1 ± 18.8 − 19.8 ± 3.0 31.4 ± 0.0

78.9 ± 14.0 103.8 ± 0.1 285.7 ± 125.2 149.3 ± 8.5 138.9 ± 28.2 20.9 ± 12.0 113.8 ± 37.9 30.6 ± 9.2 52.3 ± 12.3 120.7 ± 1.1 − 48.7 ± 14.1 251 ± 45.5

88.2 ± 10.2 81.0 ± 15.4 207.7 ± 48.2 97.5 ± 40.0 314.1 ± 56.8 85.5 ± 8.2 47.5 ± 36.5 44.1 ± 26.6 36.8 ± 10.3 147.5 ± 35.0 − 21.3 ± 7.4 55.5 ± 0.0

81 ± 23 181 ± 64 402 ± 72 224 ± 67 733 ± 205 386 ± 114 262 ± 57 204 ± 165 244 ± 42 153 ± 3 − 215 ± 83 115 ± 13

11 ± 3 25 ± 5 55 ± 12 33 ± 12 92 ± 25 52 ± 19 38 ± 16 30 ± 17 34 ± 3 21 ± 2 − 36 ± 4 13 ± 4

8.81 8.55 8.59 8.00 9.26 8.64 8.10 8.00 8.35 8.34 − 6.95 8.57

− 787 148 260 1705 256 476 210 364 176 − 694 −

All data are mean ± 1 SD values of each experimental period; −, not determined.

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Table 2 Regression coefficients of tissue dry weight (Y, mg) and total (tissue + tunic) dry weight (X, mg) for S. clava following allometric equation Y = aXb. ā, recalculated using common slopes (b) obtained from analysis of covariance (ANCOVA). CI, confidence interval. Results of ANCOVA to test significance of differences in slope are summarized at bottom of table.

a

b

r2

April 2008

30

0.456

1.004

0.896

May

26

0.079

1.231

0.894

June

29

0.154

1.076

0.918

Month

n

b ± 95% CI

a 0.316 0.258

1.059 ± 0.057

0.174

July

15

0.230

0.993

0.975

August

19

0.059

1.175

0.840

0.147 0.137

September

20

0.086

1.097

0.840

0.113

October

18

0.601

0.842

0.978

0.536

November

20

0.700

0.825

0.957

December January 2009

18 20

0.456 0.319

0.896 0.980

0.971 0.960

March

13

0.501

0.948

0.917

April

20

0.296

1.064

0.891

0.557 0.863 ± 0.037

0.553 0.659 0.863

1.059 ± 0.057

0.306

b

Period

Fs

df

Significance

All

4.449

11.224

P < 0.001

April−September

1.391

6.145

NS (P > 0.222)

1.059

October−March

1.905

4.79

NS (P > 0.118)

0.863

3.3. Physiological rates Highly significant regressions were found between physiological rates and DW of ascidians from Jindong Bay at each experiment (P b 0.05, Table 4). However, few physiological data were obtained in May–June 2008 because of the high mortality of experimental animals during the in situ experiments. ANOVA test revealed that the

500

DW AFDW

DW (mg)

400

(a)

3.4. Scope for growth 300

SFG was expressed as the difference between the assimilated ration and metabolic loss (R) of components of the energy budget of ascidians from Jindong Bay (Fig. 3d). SFG of a standard animal of 165 mg DW showed a clear seasonal trend. While negative SFG occurred during the weight loss period of spring–summer, positive SFG occurred during the growth phase of autumn–winter, reflecting seasonal diminishment and accumulation of reserve materials in flesh tissues.

200 100

Protein (mg)

200

(b)

150 100

3.5. Association between different parameters

Lipid (mg)

50

CHO (mg)

coefficients (slope b) from regressions of I, F, N and R against DW of ascidians differed significantly between months, displaying no apparent seasonal trends. Original regression coefficients and intercepts (a values) obtained from experiments under in situ environmental conditions were thus used to calculate physiological rates of a standard animal of 165 mg DW. All physiological rates were then converted into energy values for comparison in this study. Values of ingestion energy of a standard animal fluctuated between 62.1 and 183.5 J d − 1, displaying a clear seasonal pattern with relatively higher levels in autumn–winter compared with lower levels in spring–summer (Fig. 3a). In contrast, no great seasonal fluctuation was found in energy loss values by feces production, which varied between 10.0 and 29.9 J d − 1 (Fig. 3b). Energy loss by ammonia excretion of a standard animal was recorded as being at the lowest level in spring–summer, when the ingestion rate was low and the availability of carbohydrates and lipids as reserve materials in flesh was relatively large (Fig. 3c). An abrupt increase in ammonia excretion occurred in August and peaked in September–October (around 10 J d − 1), followed by a rapid decline from November and then remained low until next spring. Therefore, assimilation energy [A = I − (F + U)] of a standard animal presented a similar seasonal pattern to ingestion energy, with consistently higher values (121.2– 163.0 J d − 1) in the autumn–winter during the period of elevated rates of ingestion compared to those (47.9–86.3 J d − 1) in spring–summer (Fig. 3d). Metabolic energy loss by oxygen consumption of a standard animal also showed a clear seasonal pattern characterized by a remarkable peak (243.7–333.7 J d − 1) in summer when the water temperature was high (Fig. 3d). During the rest of the year, oxygen consumption maintained a steady level with the values lower than the rapidly decreased value (about 100 J d − 1) in October. Consequently, the ratio of oxygen consumption to ammonia excretion (O:N ratio, by atomic equivalents) fluctuated greatly according to the season (Fig. 4). The O:N ratio was considerably higher (N90) from April to June and then declined in August (about 12). The ratio remained at lower levels (b16) from late summer through autumn to winter, when levels of carbohydrate and lipid reserves in flesh were low, and increased after March and peaked again in April.

80 60 40 20 0 40 30 20 10 0

(c)

CHO Glycogen

A M 2008

J

J

(d) A

S

O

N

D

J F 2009

M

A

Month Fig. 2. Seasonal variations in tissue dry weight (DW) and gross weights of biochemical components in a standard individual of 930 mg total dry weight. (a) dry tissue weight (DW) and ash-free tissue dry weight (AFDW); (b) protein; (c) lipid; (d) carbohydrate (CHO) and glycogen. Vertical bars represent 95% confidence interval.

No significant relationships were found between the ingestion rate of a standard animal and the environmental variables (Table 5). Although multiple regression analysis showed a significant negative relationship of ingestion rate with particulate protein concentrations, only 35% of the variation in the ingestion rate was explained by this variable (Table 6). In contrast, the ingestion rate of a standard animal was highly correlated to internal variables of the ascidians, being positive for feces production and ammonia excretion, and negative for dry tissue weight and biochemical components of the flesh. Similar results were obtained for feces production. Multiple regression showed that there was no significant relationship with environmental variables. Oxygen consumption of a standard animal was positively correlated to temperature for external variables and negatively correlated to SFG for internal variables. In a multiple regression analysis, temperature was the only environmental variable that

C.-K. Kang et al. / Aquaculture 319 (2011) 168–177

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Table 3 Regression coefficients of biochemical components (Y, mg) and tissue dry weight (X, mg) for S. clava following allometric equation Y = aXb. Y = mg protein, lipid, carbohydrate, and glycogen. The number (n) of samples was the same as in Table 2. ⁎⁎ significant P = 0.01, and all other regressions P = 0.001. Month

Protein

April 2008 May June July August September October November December January 2009 March April

Lipid

Carbohydrate

Glycogen

a

b

r

a

b

r

a (× 102)

b

r

a (× 103)

b

r

1.114 0.403 0.449 0.507 0.668 0.467 1.652 1.222 1.247 1.528 0.753 0.443

0.877 1.020 1.011 0.974 0.925 0.981 0.791 0.829 0.881 0.798 0.969 0.965

0.982 0.934 0.946 0.990 0.975 0.981 0.791 0.829 0.881 0.798 0.969 0.966

0.139 0.002 0.025 0.106 0.024 0.046 0.159 0.358 0.142 0.136 0.083 0.140

0.946 1.584 1.232 0.922 1.227 1.131 0.862 0.679 0.902 0.957 1.077 0.986

0.950 0.931 0.877 0.920 0.914 0.974 0.942 0.866 0.984 0.978 0.982 0.917

0.138 0.010 0.013 0.033 0.005 0.007 2.495 0.838 0.931 1.291 3.981 1.340

1.537 1.945 1.847 1.829 2.187 2.152 0.732 1.070 1.029 1.131 1.104 1.213

0.905 0.876 0.852 0.915 0.911 0.931 0.791 0.875 0.853 0.906 0.833 0.896

0.468 0.002 0.001 9.661 0.006 0.536 2.512 0.632 0.102 0.191 3.184 5.521

1.572 2.449 2.479 1.116 2.455 1.681 1.070 1.199 1.514 1.510 1.122 1.156

0.765 0.754 0.651⁎⁎ 0.831 0.850 0.767 0.804⁎⁎ 0.688 0.838 0.793 0.834 0.806

Table 4 Regression coefficients of physiological rate (Y, mg) and tissue dry weight (X, mg) for S. clava following allometric equation Y = aXb. Y = J d− 1 ingestion, feces production, ammonia excretion, oxygen consumption. n = 10 for each experiment, ⁎⁎ significant P = 0.01, and all other regressions P = 0.001. Month

Ingestion

April 2008 July August September October November December January 2009 March April

250

Ammonia excretion

b

r

a

b

r

a

b

r

a

b

r

0.142 0.124 0.083 0.561 87.902 46.774 62.230 0.760 0.016 0.220

1.172 1.245 1.365 1.114 0.134 0.230 0.205 1.064 0.161 1.068

0.793⁎⁎ 0.748⁎ 0.860⁎⁎ 0.872⁎⁎ 0.773⁎⁎ 0.773⁎⁎ 0.688⁎⁎ 0.665⁎⁎ 0.833⁎⁎ 0.739⁎

0.001 0.001 0.055 0.018 2.698 1.205 3.041 2.168 0.780 0.001

1.862 2.543 1.287 1.396 0.442 0.607 0.428 0.477 0.635 1.811

0.948⁎⁎ 0.906⁎⁎ 0.651⁎ 0.719⁎ 0.762⁎⁎ 0.664⁎ 0.682⁎ 0.758⁎⁎ 0.693⁎ 0.845⁎⁎

5.03E−08 4.46E-07 0.001 0.087 0.234 0.060 0.182 0.057 0.155 0.042

2.890 2.798 1.924 0.889 0.720 0.841 0.508 0.607 0.595 1.152

0.872⁎⁎ 0.813⁎⁎ 0.786⁎⁎ 0.815⁎⁎ 0.765⁎⁎ 0.771⁎⁎ 0.676⁎ 0.957⁎⁎⁎ 0.791⁎⁎ 0.789⁎⁎

1.452 0.170 0.002 0.175 1.062 1.698 5.047 0.205 4.093 0.107

0.727 1.383 2.257 0.770 0.888 0.678 0.475 1.092 0.539 1.135

0.948⁎⁎ 0.896⁎⁎ 0.877⁎⁎ 0.838⁎⁎ 0.842⁎⁎ 0.824⁎⁎ 0.819⁎⁎ 0.859⁎⁎ 0.853⁎⁎ 0.928⁎⁎

accounted for 86% of the variation in oxygen consumption. Ammonia excretion of a standard animal was highly correlated to internal variables of the ascidians, being positive for ingestion rate and feces production, and negative for dry tissue weight and biochemical components of the flesh. Multiple regressions showed that no environmental variables encountered significance of P ≤ 0.05. SFG was negatively correlated to temperature, POC and PN of the environmental variables, and oxygen consumption of the physiological variables. Multiple regression analysis between the SFG and environmental variables showed that POC and temperature explained 84% of the total variance in SFG.

(a)

150 100 50

F (J d-1) U (J d-1)

0 50 40 30 20 10 0 20 15 10 5 0

(b)

4. Discussion

(c)

A & R (J d-1)

400 300

(d)

As demonstrated previously by manipulative laboratory experiments for the ascidian S. clava and other marine invertebrates (Bayne and Newell, 1983; Hawkins et al., 1985; Jiang et al., 2008b; references therein), in situ physiological experiments under varying field conditions in this study also showed that physiological rates of S. clava were

Oxygen consumption

Assimilation

150 120 90

200 (-) SFG (+) SFG

100 0

Oxygen consumption

a

A M 2008

J

J

A

S

O

N

D

J F 2009

M

A

Month Fig. 3. Seasonal variations in rates of physiological components in a standard individual of 195 mg tissue dry weight. (a) ingestion (I), (b) feces production (F), (c) ammonia excretion (U), and (d) assimilation (A) and oxygen consumption (R). Vertical bars represent 95% confidence interval. Shaded areas indicate negative (−) and positive (+) scope for growth (SFG), respectively.

O:N

I (J d-1)

200

Feces production

25 20 15 10 5 0

A M 2008

J

J

A

S

O

Month

N

D

J F 2009

M

A

Fig. 4. Seasonal variations in rate of oxygen consumption:ammonia excretion by atomic equivalents (O:N ratio) in a standard individual of 195 mg tissue dry weight.

− 0.08 − 0.43 − 0.82⁎⁎ − 0.62⁎ − 0.62⁎ − 0.56 − 0.41 − 0.16 − 0.38 − 0.85⁎⁎⁎ − 0.58⁎ − 0.62⁎ − 0.53 − 0.39

0.24 − 0.32 0.15 − 0.81⁎⁎ − 0.38 − 0.60⁎ − 0.48 − 0.22 0.41

− 0.43 0.53 0.42 0.92⁎⁎⁎ 0.83⁎⁎ − 0.36 0.48 0.62 0.89⁎⁎⁎

PON

0.24 0.47 0.68⁎ 0.64⁎

POC

0.17 0.41 0.67⁎ 0.58⁎

− 0.18 0.20 0.53 0.09 − 0.46 0.04 − 0.77⁎⁎ − 0.23 − 0.36 − 0.23 − 0.17

PCHO PPr

− 0.38 0.12 0.42 − 0.19 − 0.51 0.45 − 0.30 0.07 0.03 − 0.09 − 0.17 0.18 0.09 − 0.21 0.24 0.22 − 0.25 0.28 − 0.21 − 0.23 − 0.25 − 0.41

POM SPM

0.03 − 0.10 − 0.20 0.08 0.09 − 0.13 0.29 0.16 0.13 0.06 0.08 − 0.03 0.12 0.34 − 0.19 0.05 0.31 − 0.08 − 0.28 − 0.34 − 0.16 − 0.44

Chl a T

− 0.14 0.18 0.89⁎⁎⁎

− 0.63⁎⁎ − 0.75⁎⁎ − 0.32 − 0.59 − 0.50 0.47 0.15 0.83⁎⁎ 0.66⁎ 0.88⁎⁎⁎

CHO Lipid

− 0.65⁎ − 0.83⁎⁎ − 0.58⁎ − 0.75⁎⁎

Prot

− 0.54 − 0.55 − 0.69⁎ − 0.69⁎

− 0.81⁎⁎ − 0.80⁎⁎ − 0.54 − 0.86⁎⁎⁎ − 0.61⁎ 0.72⁎⁎

DW SFG

0.10 − 0.24 − 0.81⁎⁎ − 0.30 0.39 0.03 − 0.86⁎⁎ − 0.55 0.01 − 0.91⁎⁎⁎ − 0.72⁎⁎

O:N A

0.93⁎⁎⁎ 0.52 − 0.09 0.62⁎ 0.82⁎⁎ 0.70⁎ 0.36

U R

0.13 0.46 0.74⁎⁎ I F R U A O:N SFG DW Prot Lipid CHO

F

− 0.56 − 0.51 0.06 − 0.43 − 0.50 0.48 − 0.13 0.29 0.11 0.29 0.15

PLip

C.-K. Kang et al. / Aquaculture 319 (2011) 168–177 Table 5 Pearson product-moment correlation coefficients between internal (physiological and biochemical) and external (environmental) variables. ⁎ 0.05 N P N 0.01; ⁎⁎ 0.01 N P N 0.001; ⁎⁎⁎ P b 0.001. n = 12 for all cases except for n = 10 for correlations with chlorophyll a. I, ingestion; F, feces production; R, oxygen consumption, U, ammonia excretion; A, assimilation rate; SFG, scope for growth; DW, tissue dry weight; Prot, protein; CHO, carbohydrate; T, temperature; Chl a, chlorophyll a; SPM, total suspended particulate matter; POM, suspended particulate organic matter; PPr, particulate protein; PCHO, particulate carbohydrate; PLip, particulate lipid; POC, particulate organic carbon; PON, particulate organic nitrogen.

174

Table 6 Multiple regression analysis for several physiological (dependent) variables versus subsets of environmental (independent) variables. ⁎ 0.05 N P N 0.01; ⁎⁎ 0.01 N P N 0.001; ⁎⁎⁎ P b 0.001. See Table 5 for abbreviations. Dependent variables

Independent variables

R2

n

F

I R SFG

PPr Temp POC Temp

0.345 0.861 0.727 0.838

9 9 9 9

5.74⁎ 56.66⁎⁎⁎ 24.92⁎⁎ 24.32⁎⁎

well related to body weight in a manner typical of marine invertebrates. As shown by Jiang et al. (2008b), the exponent b-values of the allometric equation (Y= aX b) between ingestion rate (Y) and DW (X) in the present study also indicate that body mass of the ascidians can influence their rate of food consumption. The exponent values of 0.161–1.365 for S. clava in the present study represented a slightly wider range than 0.3272–0.5938 found for the same species (Jiang et al., 2008b) and 0.310–0.820 for other suspension-feeding mollusks (Bayne and Newell, 1983; references therein). Considering that the latter values were obtained from experiments under controlled dietary conditions, the wider b-range in the present study could reflect feeding activity of the ascidians in natural seston concentrations and composition. The exponent b-values of regressions for the relationship between F, U, R, and DW of S. clava in the present study ranged from 0.428 to 2.543, from 0.508 to 2.890 and from 0.475 to 2.257, respectively. These ranges are somewhat wider than those reported for other ascidians and bivalves (Bayne and Newell, 1983; Jiang et al., 2008b; references therein). Further, different monthly relationships between physiological rates (i.e. I, F, U and R) and DW confirmed different physiological adjustments depending on the body size of the ascidians under different monthly environmental conditions, as shown for some bivalves (Bougrier et al., 1995; Newell and Bayne, 1980). Suspension-feeding animals can control their feeding activity (i.e. clearance, ingestion and absorption), depending on the concentration and composition of seston as available food (Bayne and Newell, 1983; Iglesias et al., 1996; Navarro and Iglesias, 1993). Seston concentrations and quality, which represent the biopolymeric fraction (proteins, carbohydrates and lipids) of SPM as food available to suspension feeders (Danovaro and Fabiano, 1997; Navarro et al., 1993), varied irregularly and greatly within a daily cycle (as indicated by SD values in Table 1), as well as an annual cycle, in the present study. However, seston (SPM and POM) and chlorophyll a concentrations at the study site were very low compared to other coastal bay and estuarine systems (Berg and Newell, 1986; Danovaro and Fabiano, 1997; Navarro et al., 1993). Such a low concentration of seston probably reflects high feeding activity by the huge stocks of cultured suspension feeders in the study bay system (cf. Huang et al., 2008; Kang et al., 2009). Along with nonseasonality in seston concentrations and the low seston concentrations, C:N ratios of 7.0–9.3 indicate that most of the seston at the study site is derived from autochthonous marine production rather than significant loads from adjacent rivers or the land. However, high C:Chl a values (149–1703) indicate that a large quantity of the seston is composed of nonliving detritus such as phytodetritus (Berg and Newell, 1986). In the present study, nonsignificant relationships between ambient food environments and monthly ingestion rates of a standard animal were found to be because of low seston concentrations. Moreover, this result may reflect the ability of S. clava and other ascidians to regulate food intake and to retain complete particles down to a size of 2 to 3 μm (Bone et al., 2003; Jørgensen et al., 1984; Riisgård and Larsen, 2000). It is obvious that the mucus net of ascidians can effectively retain small particles (1 to 5 μm) at low seston concentrations (Armsworthy et al., 2001; Jørgensen et al., 1984; Petersen et al., 1995). Recently, Jiang et al. (2008c) also observed that S. clava retains small particles (1.5–5 μm) efficiently.

C.-K. Kang et al. / Aquaculture 319 (2011) 168–177

Kang et al. (2009) compared tissue isotope composition between another ascidian species Halocynthia roretzi and the cocultured oyster Crassostrea gigas. These authors demonstrated that while a 13Cdepletion in ascidian tissues reflects the use of dietary components of fine (pico-/nano-sized) particles, a marked 13C-enrichment in oyster tissues resulted from their strong selectivity of diatoms (N20 μm). In the present study, monthly isotopic measurements of the ascidian S. clava also showed that their tissue δ 13C had a seasonal pattern and values similar to those of fine (b20 μm) particles, indicating efficient utilization of small particles particularly during the autumn–winter period when the seston concentrations are low (Fig. 5). Petersen et al. (1995) showed that such a particle capture mechanism by net trapping enables ascidians to maintain a high growth rate at low food concentrations. Indeed, a high ingestion rate of the ascidians was observed during autumn and winter in the present study. Accordingly, the ingestion rate of a standard animal was not correlated with water temperatures. Rather, a negative correlation was found between seasonal fluctuations in the ingestion rate and DW (also biochemical components) of a standard animal, suggesting that seasonal food consumption of the ascidians could be related to reproductive activity. Microscopic observation of gonads confirmed that active gonads peaked in April and spawning occurred in May–June (unpubl. data). As a result, the low ingestion rate of the ascidians during spring and summer seems to be associated with feeding activity occurring at minimum rates during spawning. On the other hand, Jiang et al. (2008b) observed the maximum feeding activity of S. clava at temperatures of 20–24 °C, and significantly reduced activity at 28 °C. In the present study, higher water temperature (exceeding 26 °C) may be responsible for some of the low ingestion rates during summer. While energy losses associated with feces production represented 16–33% of the energy ingested by a standard animal of S. clava, ammonia excretion accounted for less than 6% of the ingested energy. An ammonia excretion percentage of consumption in S. clava falls within the range observed for suspension- and deposit-feeding mollusks, and the feces production percentage is at the low end of the range (Bayne and Newell, 1983; Navarro and Thompson, 1996; references therein). In the present study, positive significant correlations were found for the ingestion rate versus feces production and ammonia excretion of a standard animal. Jiang et al. (2008b) pointed out that feces production and nitrogen excretion are influenced by food availability and the feeding rate of the ascidian prior to the excretion measurement. They also showed that these excretion components increased with rising temperatures, which were in the

δ15N (o/oo)

14 13

J7

12

J6 A J1

11

M A

S O

N

D 10

CPOM

9

FPOM 8 7 -24

-23

-22

-21

-20

-19

-18

-17

δ13C (o/oo) Fig. 5. Plot of δ13C and δ15N determined for Styela clava, fine (N 20 μm, FPOM) and coarse (≤ 20 μm, CPOM) particulate organic matter (unpubl. data). Abbreviations indicate the sampling month of S. clava. J1, January; J6, June; J7, July. Considering the documented trophic enrichment (expressed by dashed line) of about + 0.5‰ and 2.5‰, respectively, for δ13C and δ15N of herbivores, the ascidian diets can be presumed from their isotopic values (Vander Zanden and Rasmussen, 2001).

175

range of 12–24 °C. However, our results showed that the seasonal cycles in excretion (both nitrogen and feces) are not simply temperature dependent. As previously shown for other bivalves (Navarro and Thompson, 1996; references therein), other factors such as biochemical energy storage cycles are likely to influence the physiological responses of the ascidian (see also later discussion). Oxygen consumption of S. clava in the present study displayed a remarkable seasonal change that follows the fluctuation in water temperatures, where the rates peaked during summer when temperatures were highest (Fig. 3). Accordingly, respiratory energy losses accounted for 40–50% of the energy ingested by a standard animal of S. clava during the low-temperature (b20 °C) period of autumn–winter, but exceeded the ingested energy during summer. A positive relationship between oxygen consumption and temperature was observed in the ascidian S. clava (Jiang et al., 2008a) and also some bivalve species (Bricelj et al., 1987; MacDonald and Thompson, 1986). In relation to thermal acclimation of mollusks, while some species (Chlamys opercularis, Crepidula fornicate, and Mytilus edulis) can adjust their rate of oxygen consumption in response to long-term (i.e. seasonal) changes in environmental temperatures (Bayne and Newell, 1983; McLusky, 1973; Newell and Kofoed, 1977; Widdows, 1978), other species (Donax vittatus and Ostrea edulis) cannot adjust their metabolic rate, which increases rapidly with temperature (Ansell and Sivadas, 1973; Newell et al., 1977). Similar to the latter case, the ascidian S. clava exhibited little evidence of adjustment of its metabolic cost to seasonal changes in temperature. Recently, Jiang et al. (2008b) also found from a laboratory experiment that, at high temperatures (above 24 °C), the rate of oxygen consumption of S. clava increased with increasing temperature. In many cases of suspension feeders, the seasonal variation of oxygen consumption has been also related to food availability and gametogenesis, together with temperature (Babarro et al., 2000; Hawkins et al., 1985; Iglesia and Navarro, 1991; Navarro and Thompson, 1996; Newell and Bayne, 1980; Rueda and Smaal, 2004). In the present study, the oxygen consumption rate decreased during the low-temperature period of winter when the gonads are mature, suggesting no close link between oxygen consumption and the gametogenic cycle of S. clava. Moreover, although considerable energetic costs of feeding (associated with water transport, filtration, digestion, and absorption) in S. clava were expected as in other suspension-feeding mollusks (Bayne and Newell, 1983; Hawkins et al., 1985) and ascidians (Petersen et al., 1995), no correlation between oxygen consumption and food availability (also ingestion rate of food) was observed. Increased filtration (ingestion) rates during the warm conditions in the summer is needed as a physiological adjustment through which suspension-feeding animals can compensate for increased metabolic energy costs during that time (Bayne and Newell, 1983; Newell and Kofoed, 1977). As shown in a bivalve species, Donax vittatus (Ansell and Sivadas, 1973), metabolic costs of S. clava increased with temperature during the summer. However, although increased oxygen consumption in September may reflect the energetic costs of feeding, the increase in ingestion rate was undetectable in July and August. Such an increased oxygen consumption rate and a lowered ingestion rate in S. clava during the warm conditions may be indicators of thermal stress (Jiang et al., 2008b; Newell and Branch, 1980). The SFG measurement can be used to integrate compensatory adjustments of animals in response to changing environmental conditions and thus is a useful index to estimate the effect of environmental stressors on the overall performance of animals (Bayne and Newell, 1983; Hawkins et al., 1985). In the present study, a lack of synchronous adjustment of energy loss and gain (i.e. increased metabolic energy cost and lowered ingestion rate) in S. clava during the warm conditions led to negative SFG during summer. In contrast, the decreased oxygen consumption rate and the elevated ingestion rate during autumn–winter, when water temperatures were low, resulted in positive SFG during that time. Obviously, the inverse

176

C.-K. Kang et al. / Aquaculture 319 (2011) 168–177

relationship between oxygen consumption and SFG demonstrates the influence of respiratory losses on the growth of S. clava. The seasonal pattern of SFG in S. clava is inconsistent with that in most bivalve species which have higher SFG values during spring–summer and lower values during autumn–winter (Hawkins et al., 1985; Navarro and Thompson, 1996; Rueda and Smaal, 2004). Such a clear seasonal pattern in the SFG of S. clava was well reflected in its seasonal growth pattern. Styela clava showed weight gain during autumn–winter, followed by a maximum in April, and a loss during spring–summer. In addition, monthly changes in the gross biochemical composition of the ascidians gave an insight into the seasonal patterns in storage utilization of energy reserves in flesh tissues, showing rapid exhaustion of energy reserves during spring– summer and subsequent accumulation of reserves during autumn– winter. Weight loss between April and July was largely explained by a concurrent decline in the absolute values of protein. Considering that proteins constitute the major organic component of gametes, weight loss during that time could be related to the spawning. Moreover, high (N90) O:N ratios from April to June suggest considerable contributions of carbohydrates and lipids to catabolic substrates (Bayne, 1973; Bayne and Newell, 1983; Hawkins et al., 1985). A relatively low ingestion rate and thus negative SFG during that time must indicate predominant catabolism of prestored nutrient reserves compared to ingested nutrients. Negative SFG caused by thermal stress resulted in successive mobilizations of reserve materials during summer, with the absolute values of the biochemical components being minimal. An increase in the dry flesh weight, which paralleled the progressive recovery and accumulation of nutrient reserves in the ascidians, is clearly related to a positive SFG during autumn–winter. Along with this phenomenon, low (b16) O:N ratios compared to those in spring suggest a higher use of proteins from the ingested nutrients as an energy source (Bayne, 1973; Bayne and Newell, 1983; Hawkins et al., 1985). On the other hand, microscopic observation of gonads showed that gametogenesis overlapped with the accumulation of nutrient reserves at low temperatures during late autumn–winter (unpubl. data) and thus it is characterized as an opportunistic species (see Hawkins et al., 1985; Navarro et al., 1989). Our results indicate that seasonal patterns in storage utilization of energy reserves of the ascidians are well linked to physiological adjustments as their adaptive responses concerning energy balance to seasonal variation of local environmental conditions within an annual cycle. Our results indicate that fast growth in the ascidian flesh tissues happens during the autumn–winter culturing period when their SFG is positive. Accordingly, seeding of larvae in spring and the transplantation of seed ascidians to grow-out areas in early summer are expected to be appropriate to harvest marketable-sized ascidians in the next spring when their growth is depressed. Negative SFG and thereby an abrupt decline in their flesh weight during the spring– summer period clearly indicates that the prolonged culture activity to spring-summer is inappropriate. However, since harvesting in early spring may result in a lack of mature ascidians and consequently a seed shortage, it may be necessary to secure husbandry ground for seed collection. Finally, our results suggest a need of reformation in the ascidian-culturing cycle based on their seasonal cycles of storage and utilization of energy reserves as well as physiological adjustments.

5. Conclusion Changes in the physiology and somatic growth of S. clava are largely influenced by seasonal variations in temperature. As shown by the inverse relationship between SFG of the ascidians and POC, the ability of ascidians to effectively retain small particles seems to account for the reduced importance of food availability in explaining their physiological adjustments in this particular environment with low seston concentra-

tions. Seasonal variations in ingestion and respiration of the ascidians were mismatched, resulting in an energy imbalance (i.e. increased metabolic energy cost and lowered ingestion rate; and vice versa). This mismatched activity resulted in negative SFG values during spring– summer, followed by a rapid exhaustion of energy reserves and flesh weight loss. Weight loss during that time is also related to spring spawning. During autumn–winter, the ascidians showed a positive SFG as a result of a decreased oxygen consumption rate and an elevated ingestion rate, showing accumulation of nutrient reserves and weight gain. Gametogenesis initiates concurrently with the accumulation of nutrient reserves. Jiang et al. (2008b) concluded that considering SFG values obtained from the experimental temperatures (12–28 °C), a temperature range of 16–20 °C is suitable for rearing S. clava to achieve optimum development. However, the ascidians showed a positive SFG, even in cold conditions below 12 °C in the present study. In this respect, the culturing period during autumn–winter may be recommended to maximize ascidian production in long-line suspended culture under the natural environmental conditions occurring in the study area. Acknowledgements The authors are grateful to Miran Park and Jung Hyun Kwak for their support in the collection of field samples and environmental data. We also thank two anonymous reviewers for their valuable comments. This research was financed by the project “Environmental Research of Aquaculture Farms” of the Ministry for Food, Agriculture, Forestry, and Fisheries (MFAFF) of Korea. References Ansell, A.D., Sivadas, P., 1973. Some effects of temperature and starvation on the bivalve Donax vittatus (da Costa) in experimental laboratory population. J. Exp. Mar. Biol. Ecol. 13, 229–262. Armsworthy, S.L., MacDonald, B.A., Ward, J.E., 2001. Feeding activity, absorption efficiency and suspension feeding process in the ascidian, Halosynthia pyriformis (Stolidobranchia: Ascidiacea): response to variations in diet quantity and quality. J. Exp. Mar. Biol. Ecol. 260, 41–69. Babarro, J.M.F., Fernández-Reiriz, M.J., Labarta, U., 2000. Metabolism of the mussel Mytilus galloprovincialis from two origins in the Ría de Arousa (northwest Spain). J. Mar. Biol. Assoc. U. K. 80, 865−872–865. Bayne, B.L., 1973. Physiological changes in Mytilus edulis L. induced by temperature and nutritive stress. J. Mar. Biol. Ass. U.K. 53, 39–58. Bayne, B.L., Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A., Livingstone, D.R., Lowe, D.M., Moore, M.N., Stebbing, A.R.D., Widdows, J., 1985. The effects of stress and pollution on marine animals (Praeger special studies). Praeger Scientific, Westport, CT. Bayne, B.L., Newell, R.C., 1983. Physiological energetics of marine mollusca. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca, vol. 4. Academic press, New York, pp. 407–515. Berg, J.A., Newell, R.I.G., 1986. Temporal and spatial variations in the composition of seston available to the suspension feeder Crassostrea virginica. Estuar. Coast. Shelf Sci. 23, 375–386. Bligh, E.G., Dyer, W.F., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Bone, Q., Carré, C., Chang, P., 2003. Tunicate feeding filters. J. Mar. Biol. Assoc. U. K. 83, 907–919. Bougrier, S., Collet, B., Geairon, P., Geffard, O., Héral, M., Deslous-Paoli, J.M., 1998. Respiratory time activity of the Japanese oyster Crassostrea gigas (Thunberg). J. Exp. Mar. Biol. Ecol. 219, 205–216. Bougrier, S., Geairon, P., Deslous-Paoli, J.M., Bacher, C., Jonquières, G., 1995. Allometric relationships and effects of temperature on clearance and oxygen consumption rates of Crassostrea gigas (Thunberg). Aquaculture 134, 143–154. Bourque, D., Davidson, J., MacNair, N.G., Arsenault, G., LeBlanc, A.R., Landry, T., Miron, G., 2007. Reproduction and early life history of the invasive ascidian Styela clava Herdman in Prince Edward Island. Canada. J. Exp. Mar. Biol. 342, 78–84. Bricelj, V.M., Epp, J., Malouf, R.E., 1987. Comparative physiology of young and old cohorts of the bay scallop, Argopecten irradians irradians (Lamarck): mortality, growth and oxygen consumption. J. Exp. Mar. Biol. Ecol. 112, 73–91. Danovaro, R., Fabiano, M., 1997. Seasonal changes in quality and quantity of food available for benthic suspension-feeders in the Golfo Marconi (north-western Mediterranean). Estuar. Coast. Shelf Sci. 44, 723–736. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebecs, P.A., Smith, F., 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28, 350–356. Elliot, J.M., Davison, W., 1975. Energy equivalents of oxygen consumption in animal energetics. Oecologia (Berl.) 19, 195–201. Farías, A., García-Esquivel, Z., Viana, M.T., 2003. Physiological energetics of the green abalone, Haliotis fulgens, fed on a balanced diet. J. Exp. Mar. Biol. Ecol. 289, 263–276.

C.-K. Kang et al. / Aquaculture 319 (2011) 168–177 Gnaiger, E., 1983. Calculation of energetic and biochemical equivalents of respiratory oxygen consumption. In: Gnaiger, E., Forstner, H. (Eds.), Polarographic Oxygen Sensors. Appendix C, Springer-Verlag, Belin, pp. 337–345. Hawkins, A.J.S., Salkeld, P.N., Bayne, B.L., Gnaiger, E., Lowe, D.M., 1985. Feeding and resource allocation in the mussel Mytilus edulis: evidence for time-averaged optimization. Mar. Ecol. Prog. Ser. 20, 273–287. Holm-Hassen, O., Lorenzen, C.J., Holms, R.W., Strickland, J.D.H., 1965. Fluorometric determination of chrolophyll. J. Cons. Perm. Int. Explor. Mer. 30, 3–15. Huang, C.H., Lin, H.J., Huang, T.C., Su, H.M., Hung, J.J., 2008. Responses of phytoplankton and periphyton to system-scale removal of oyster-culture racks from a eutrophic tropical lagoon. Mar. Ecol. Prog. Ser. 358, 1–12. Iglesia, J.I.P., Navarro, E., 1991. Energetics of growth and reproduction in cockle (Cerastoderma edule): seasonal and age-dependent variations. Mar. Biol. 111, 359–368. Iglesias, J.I.P., Urrutia, M.B., Navarro, E., Alvarez-Jorna, P., Larretxea, X., Bougrier, S., Héral, M., 1996. Variability of feeding processes in the cockle Cerastoderma edule (L.) in response to changes in seston concentration and composition. J. Exp. Mar. Biol. Ecol. 197, 121–143. Jiang, A.L., Guo, J.L., Cai, W.G., Wang, C.H., 2008a. Oxygen consumption of the ascidian Styela clava in relation to body mass, temperature and salinity. Aquacul. Res. 39, 1562–1568. Jiang, A.L., Lin, J., Wang, C.H., 2008b. Physiological energetics of the ascidian Styela clava in relation to body size and temperature. Comp. Biochem. Physiol. A 149, 129–136. Jiang, A.L., Yu, Z., Cai, W.G., Wang, C.H., 2008c. Feeding selectivity of the marine ascidian Styela clava. Aquacul. Res. 39, 1190–1197. Jørgensen, C.B., Kiørboe, T., Møhlenberg, F., Riisgård, H.U., 1984. Ciliary and mucus-net filter feeding, with special reference to fluid mechanical characteristics. Mar. Ecol. Prog. Ser. 15, 283–292. Kang, C.K., Choy, E.J., Hur, Y.B., Meyong, J.I., 2009. Isotopic evidence of particle sizedependent food partitioning in cocultured sea squirt Halocynthia roretzi and Pacific oyster Crassotrea gigas. Aquat. Biol. 6, 289–302. Kim, D.S., Cho, K.D., Park, C.K., 2001. The oceanic environmental property in the Jindong Bay of the red-tide appearance area. J. Environ. Sci. 10, 159−166–159− (Korean with English abstract). Kim, D.S., Kim, S.U., 2003. Mechanism of oxygen-deficient water formation in Jindong Bay. The Sea-J. Korean Soc. Oceanogr. 8, 177–186 (Korean with English abstract). Lee, P.Y., Kang, C.K., Choi, W.J., Lee, W.C., Yang, H.S., 2001. Temporal and spatial variations of particulate organic matter in the southeastern coastal bays of Korea. J. Korean Fish. Soc. 34, 57–69 (Korean with English abstract). Lowry, O.M., Roseborough, N.I., Farrand, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 263–275. MacDonald, B.A., Thompson, R.J., 1986. Influence of temperature and food availability on the ecological energetics of the giant scallop Placopecten magellanicus. III. Physiological ecology, the gametogenic cycle and scope for growth. Mar. Biol. 93, 37–48. Marsh, J.B., Weinstein, D.B., 1966. Simple charring method for determination of lipid. J. Lipid Res. 7, 574–576.

177

McLusky, D.S., 1973. The effect of temperature on the oxygen consumption and filtration rate of Chlamys (Aequipectan) opercularis (L.) (Bivalvia). Ophelia 10, 141–154. Navarro, E., Iglesias, J.I.P., 1993. Infaunal filter-feeding bivalves and the physiological response to short-term fluctuations in food availability and composition. In: Dame, R.F. (Ed.), Bivalve Filter feeders in Estuarines and Coastal Ecosystem Processes. Springer, Heidelberg, pp. 25–56. Navarro, E., Iglesias, J.I.P., Larranaga, A., 1989. Interannual variation in the reproductive cycle and biochemical composition of the cockle Cerastoderma edule from Mundaca Estuary (Biscay, North Spain). Mar. Biol. 101, 503–511. Navarro, J.M., Clasing, E., Urrutia, G., Asencio, G., Stead, R., Herrera, C., 1993. Biochemical composition and nutritive value of suspended particulate matter over a tidal flat of southern Chile. Estuar. Coast. Shelf Sci. 37, 59–73. Navarro, J.M., Thompson, R.J., 1996. Physiological energetics of the horse mussel Modiolus modiolus in a cold ocean environment. Mar. Ecol. Prog. Ser. 138, 135–148. Newell, R.C., Branch, G.M., 1980. The effects of temperature on the maintenance of metabolic energy balance in marine invertebrates. Adv. Mar. Biol. 17, 329–396. Newell, R.C., Johnson, L.G., Kofoed, L.H., 1977. Adjustment of the components of energy balance in response to temperature change in Ostrea edulis. Oecologia 30, 97–110. Newell, R.C., Kofoed, L.H., 1977. Adjustment of the components of energy balance in the gastropod Crepidula fornicate in response to thermal acclimation. Mar. Biol. 44, 275–286. Newell, R.I., Bayne, B.L., 1980. Seasonal changes in the physiology, reproductive condition and carbohydrate content of the cockle Cardium (= Cerastoderma) edule (Bivalve: Cardiidae). Mar. Biol. 56, 11–19. Petersen, J.K., Schou, O., Thor, P., 1995. Growth and energetics in the ascidian Ciona intestinalis. Mar. Ecol. Prog. Ser. 120, 175–184. Riisgård, H.U., Larsen, P.S., 2000. Comparative ecophysiology of active zoobenthic filter feeding, essence of current knowledge. J. Sea Res. 44, 169–193. Rueda, J.L., Smaal, A.C., 2004. Variation of the physiological energetics of the bivalve Spisula subtruncata (da Casta, 1778) with an annual cycle. J. Exp. Mar. Biol. Ecol. 301, 141–157. Sokal, R.R., Rholf, F.J., 1995. Biometry. The Principles and Practice of Statistics in Biological Research, third ed. W.H. Freeman and Company, New York. Thompson, B., MacNair, N., 2004. An Overview of the Clubbed Tunicate (Styela clava) in Prince Edward Island. PEI Department of Agriculture, Fisheries, Aquaculture and Forestry Technical Report, 234, p. 29. Vander Zanden, M.J., Rasmussen, J.B., 2001. Variation in δ15N and δ13C trophic fractionation: implications for aquatic food web studies. Limnol. Oceanogr. 46, 2061–2066. Widdows, J., 1978. Combined effects of body size, food concentration and season on the physiology of Mytilus edulis. J. Mar. Biol. Assoc. U. K. 53, 109–124. Zhang, J.H., Fang, J.G., 2000. Study on the oxygen consumption rates of some common species of ascidian. J. Fish. Sci. China 3, 16–19. Zhang, J.H., Fang, J.G., Dong, S.L., 2000. Study on the ammonia excretion rate of four species ascidian. Mar. Fish. Res. 31–36.