Protein synthesis in wild-caught Norway lobster (Nephrops norvegicus L.)

Journal of Experimental Marine Biology and Ecology 409 (2011) 208–214

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Protein synthesis in wild-caught Norway lobster (Nephrops norvegicus L.) E. Mente a, b,⁎, C.G. Carter c, R.S. (Katersky) Barnes d, I.T. Karapanagiotidis a a

School of Agricultural Sciences, Department of Ichthyology and Aquatic Environment, Fytoko Street, GR-38446 N. Ionia Magnesia's, Greece Institute of Biological and Environmental Sciences, School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, AB24 2TZ Aberdeen, UK c Institute of Marine and Antarctic Studies, University of Tasmania, Private Bag 49, Hobart, Tasmania 7001, Australia d National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia b

a r t i c l e

i n f o

Article history: Received 7 May 2011 Received in revised form 27 August 2011 Accepted 29 August 2011 Available online 23 September 2011 Keywords: Crustacean Lobster Nephrops Protein Synthesis Tissue

a b s t r a c t Although protein metabolism has been studied in fish and other crustaceans, this is the first study to measure protein synthesis rates for Norway lobster Nephrops norvegicus. This research aimed to assess the nutritional status of wild caught Nephrops by measuring tissue rates of protein synthesis and comparing these with rates from fed and starved Nephrops maintained in aquaria. Rates of protein synthesis were measured in the hepatopancreas, gill and tail muscle tissue. A time-course validated the use of a flooding-dose of 3H phenylalanine to measure protein synthesis in Nephrops and showed that the flooding-dose method is suitable for the study of protein turnover in Nephrops. The relationship between the measured rate of protein synthesis and capacity for protein synthesis (Cs) showed differences between tissues and between nutritional history. Measures of protein metabolism were generally higher in hepatopancreas, then gill and then tail muscle and generally lower in starved animals. Although individual variation meant there were few significant differences in tail muscle values between treatments, RNA concentration was higher in wild-caught than starved and suggested they were not starving. This was supported by hepatopancreas protein synthesis in wild-caught being intermediate between fed and starved, indicating relatively recent feeding had increased hepatopancreas protein synthesis due to increased RNA activity at constant RNA capacity. This study will add to our current but limited understanding of the effects of environmental variations on nutritional status and on protein metabolism and deposition in Nephrops species. Knowledge of the mechanisms enabling their survival is required to understand their ability to adapt to environmental variables. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The Norway lobster, Nephrops norvegicus (Linnaeus, 1758), is a commercially important benthic decapod crustacean with total catches of 72,130 t in 2009 (FAO, 2011) Little is known about relationships between nutrition, protein metabolism and growth in N. norvegicus, despite the number of studies addressing a wide range of issues, the effect of capture and aerial exposure, hypoxia, acid– base status and carbohydrate storage and release (Baden et al., 1994; Hagerman et al., 1990; Ridgway et al., 2006; Schmitt and Uglow, 1997; Spicer et al., 1990; Stentiford et al., 2001), the reproduction, larval and population biology (Aguzzi et al., 2004; Briggs et al., 2002; Dickey-Collas et al., 2000a, 2000b; dos Santos and Peliz, 2005; Farmer, 1975; Maynou and Sarda, 1997; Rotllant et al.,

⁎ Corresponding author at: School of Agricultural Sciences, Department of Ichthyology and Aquatic Environment, Fytoko Street, GR-38446 N. Ionia Magnesias, Greece. Tel.: + 30 2421093176; fax: + 30 2421093157. E-mail addresses: [email protected], [email protected] (E. Mente). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.08.025

2001, 2004; Sarda, 1995; Tuck et al., 1997, 2000), the culture and aquaculture potential (Anger and Puschel, 1986; Dickey-Collas et al., 2000b; Figueiredo and Vilela, 1972; Mente, 2010; Mente et al., 2009; Morais et al., 2001; Rosa et al., 2003; Rotllant et al., 2001). Furthermore, intriguing results from invertebrates suggest high growth rates may be achieved by relatively low rates of protein turnover, fast growing invertebrates may maximise protein growth by having low protein degradation (Carter et al., 2009; Fraser and Rogers, 2007; Mente et al., 2002). This is the case for fast growing cephalopods with short life-cycles (Moltschaniwskyj and Carter, 2010). Thus, a growing body of research promises to reveal important differences and similarities across diverse invertebrate species (Fraser and Rogers, 2007). Further research on growth and protein turnover strategies is needed for lobsters, including Nephrops species, and this requires validation of protein synthesis measurements. The current study aimed to validate the use of the flooding-dose method to measure protein synthesis rates with the Norway lobster in order to study the effects of nutritional status, starvation and feeding, on protein metabolism and then relate this to wild-caught animals. This would then provide an approach to investigate their ability to adapt and survive when food supply changes in relation to changes in response to environmental events.

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2. Material and methods

2.4. Statistical analysis

2.1. Animals

Means with their standard error (SEM) are presented throughout. One-way ANOVA and linear regression were used to analyse the relationship between incorporation time and both free pool and proteinbound phenylalanine-specific radioactivity for each tissue (Garlick et al., 1980). A sub-set of 12 wild-caught Nephrops that met criteria for a valid flooding-dose was used as the wild-caught treatment. One-way ANOVA was then used to compare mean values followed by Scheffe's multiple comparison for unequal sample size. Homogeneity of the groups was tested using the Levene's test. Significance was accepted at 5% or less. All statistical analyses were carried out using PASW Statistics, version 18. The statistical methodology used is detailed in Zar (1999).

Norway lobsters (N. norvegicus) were obtained by creels (baited traps) from the Pagasitikos Gulf in central Greece. Nephrops were transferred individually in traps to the University of Thessaly in aerated seawater containers, following which they were maintained in aquaria under constant environmental conditions (11 ± 1 °C, salinity 33 ppt and 12:12 h L:D). They were randomly distributed into 100 l tanks consisting of five numbered rectangular compartments each and each animal was placed in each compartment individually to avoid cannibalism (further details in Mente, 2010). They were allowed to acclimate to aquarium conditions for ten days prior to experimentation. All lobsters were of intermoult stage (Aiken, 1980). Four Nephrops were fed individually a natural diet (frozen mussel tissue Mytilus galloprovincialis) and four were starved, for a period of 6 months. At the end of the experiment seventeen wild-caught Nephrops were obtained by creels, transferred to the laboratory as described above and used to validate the measurement of protein synthesis. A sub-group with valid rates of protein synthesis was compared with the eight Nephrops kept in the laboratory, four starved and four fed. Only male N. norvegicus of a similar size (carapace length range of 30–45 mm) were used for the experiments. 2.2. Sampling All Nephrops used to measure rates of protein synthesis and tissue concentrations of protein and RNA were intermoult and sampled on the same day. Nephrops were sacrificed by decapitation. Samples of tail muscle, hepatopancreas and gills tissues were rapidly dissected out, weighed, individually wrapped in plastic bags and immediately frozen in liquid nitrogen and stored at −80 °C for further analysis. 2.3. Protein synthesis measurements Rates of protein synthesis were measured following a single injection of 3H-phenylalanine using the flooding-dose method (Garlick et al., 1980; Houlihan et al., 1995). Nephrops were injected via the first pair of pleopods into the haemolyph, by passing the needle through the anterior dorsal section of the first abdominal segment, without anaesthesia with 0.5 ml 100 g −1 injection solution, using a 0.5 ml micro-fine syringe (Mente et al., 2002). Following injection, lobsters were returned to separate aquaria containing aerated seawater. The precise time of the injection was noted. After injection three wildcaught nephrops were randomly sampled at each of 30, 45, 60, 90 and 120 min, the four fed and four starved nephrops were sampled at 68 and 92 min. Recovery of the lobsters from the injection procedure was 100%. The injection solution contained 150 mmol L-phenylalanine and L(2,6- 3H) phenylalanine (Amersham Pharmacia Biotech, NSW, Australia) in 0.2 μm filtered seawater at pH 7.4 with a measured specific activity of 1473 ± 252 dpm nmol −1 phenylalanine. The treatment of samples to measure protein-bound and free-pool phenylalaninespecific radioactivity was as described previously for crustaceans (Mente et al., 2002). Fractional rates of protein synthesis (ks: % d −1) were calculated as ks = 100 ∗ ((Sb/Sa) · (1440/t1)), where Sb is the protein-bound phenylalanine-specific radioactivity at time t1 (min) and Sa the free-pool phenylalanine-specific radioactivity (Garlick et al., 1980, 1983; McMillan and Houlihan, 1988). Protein concentration was measured using a modification of the Folin-phenol method (Lowry et al., 1951) and RNA concentration was measured using dual wavelength absorbance (Ashford et al., 1986). RNA was also expressed as the capacity for protein synthesis (Cs: mg RNA · g protein −1) and as RNA activity (kRNA: ks · g −1 RNA · d −1) (Sugden and Fuller, 1991).

3. Results 3.1. Validation of flooding dose The time course of incorporation of [ 3H]phenylalanine showed that the flooding-dose technique is suitable for the study of protein turnover in Nephrops (Fig. 1). The incorporation times for individual Nephrops were 30–120 min. Following injection, the free phenylalanine concentrations in the tail muscle free pool (nmol Phe g −1 wet mass) were elevated over the range of incorporation times (ANOVA, P b 0.05). Taking a tail-muscle free phenylalanine concentration of 270 nmol Phe g −1 wet mass in uninjected Nephrops (Mente, 2010), the results indicate a fourfold increase in free phenylalanine levels following injection. There were no significant differences between tail muscle freepool (Sa) phenylalanine-specific radioactivity (dpm nmol −1 phenylalanine) at different times (F4,10 = 0.264, P = 0.894) and free pools therefore remained flooded for 120 min. The free-pool Sa in the tail muscle reached a mean value of 614 ± 52 dpm nmol −1 Phe, 42% of the Sa of the injection solution. The mean free-pool specific activity at each time interval was significantly lower (ANOVA, P b 0.05) than the specific activity of the injection solution. Over this time there was a linear relationship between time and protein-bound (Sb) phenylalanine-specific radioactivity (dpm nmol−1 phenylalanine) described by Sb =0.0003 t+0.072 (R2 =0.216, F1,13 =4.852, P=0.046). Thus, the flooding-dose method was valid for incorporation times of between 30 and 120 min in tail muscle. The hepatopancreas samples from 45 min were removed from the analysis due to very low free-pool (Sa) phenylalanine-specific radioactivity. There were no significant differences between hepatopancreas free-pool (Sa) phenylalanine-specific radioactivity (dpm nmol−1 phenylalanine) at different times (F3,8 = 0.710, P= 0.578). Over this time there was a linear relationship between time and protein-bound (Sb) phenylalanine-specific radioactivity (dpm nmol−1 phenylalanine) described by Sb = 0.042 t −1.504 (R2 =0.389, F1,10 =8.00, P= 0.018). Thus, the flooding-dose method was valid for incorporation times of

Fig. 1. Time course for phenylalanine-specific radioactivity (dpm nmol−1 phenylalanine) in a, tail muscle free-pool and b, tail muscle protein of wild-caught Nephrops.

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between 30 and 120 min in the hepatopancreas, although the large variation in Sa at 120 min suggests this time is around the cut-off time for valid measurements. There were significant differences between gill free-pool (Sa) phenylalanine-specific radioactivity (dpm nmol−1 phenylalanine) at different times (F4,10 =4.41, P=0.026), the free-pool (Sa) phenylalaninespecific radioactivity was significantly higher at 30 min than at 120 min. There was also a negative linear relationship between time and freepool (Sa) phenylalanine-specific radioactivity described by Sa = −3.232 t+881 (R2 =0.267, F1,13 =4.732, P=0.049) showing that the free-pool was not constant over 120 min. Over this time there was a linear relationship between time and protein-bound (Sb) phenylalaninespecific radioactivity (dpm nmol−1 phenylalanine) described by Sb =0.004 t+0.319 (R2 =0.292, F1,13 =6.777, P=0.022). The wild-caught Nephrops sampled for protein synthesis at 120 min were not considered in further analyses. A conservative approach was adopted to reduce errors in protein synthesis estimates for hepatopancreas and gill tissue from wild caught animals. This decision was based on the considerably higher individual variation in protein-bound specific radioactivity for all tissues at 120 min than at other time, the significantly lower free-pool specific radioactivity at 120 min, and the numerically lower hepatopancreas specific radioactivity at 120 min. 3.2. Morphometric analysis Nephrops showed slow growth rates. By the end of the experiment, the specific growth rates of the fed Nephrops were 0.03% ± 0.01% d −1. The average rate of weight loss for the starved lobsters was − 0.02 ± 0.09% d −1. On the day protein synthesis was measured the final weights of the starved Nephrops were significantly (P = 0.01) lower than the weights of the fed and the wild-caught animals (Table 1). The claw muscle as a percentage of wet weight was higher, but not significantly different, in the fed compared with the wild-caught and starved Nephrops. Interestingly, the hepatopancreas size for starved Nephrops was intermediate between higher wildcaught and lower fed animals. 3.3. Indices of protein metabolism There were no significant differences among treatments for tail muscle protein content, capacity for protein synthesis (Cs, mg RNA g protein −1), fractional rate of protein synthesis (ks, % d −1) or RNA activity (Table 2). The RNA concentration was higher (P = 0.01) in the tail muscle of wild-caught than in starved animals. In the hepatopancreas fractional rates of protein synthesis were significantly higher in fed than in starved animals (Table 3). RNA activity was significantly higher in fed than in wild-caught or starved animals. There were no significant differences in indices of protein metabolism in the gill tissue, whilst there was a significant difference in protein content multiple comparisons did not reveal which treatments were different (Table 4).

Table 1 Morphometric data for nephrops that were wild-caught, fed or starved (mean ± SEM, n = 4). Tissue

Wild-caught Fed Starved F P

Weight

Tail muscle (%W)

Claw muscle (%W)

Hepatopancreas

(g)

Total length (cm)

60.5a ± 3.8 44.9ab ± 6.8 32.4b ± 4.0 7.72 0.011

44.9a ± 0.7 38.4b ± 1.6 37.4b ± 1.9 7.70 0.011

24.0 ± 1.3 22.3 ± 2.5 22.1 ± 1.4 0.32 0.732

5.5 ± 0.5 7.6 ± 2.1 2.7 ± 0.7 3.71 0.067

3.4a ± 0.2 1.9b ± 0.5 2.1ab ± 0.4 5.94 0.023

(%W)

Same letters, down a column, indicate no significant difference following one-way ANOVA and multiple comparison by Tukey HSD (% data were arcsin transformed).

Table 2 Protein metabolism measures in tail muscle of nephrops that were wild-caught, fed or starved (mean ± SEM). Tissue

Protein (mg g−1)

RNA (mg g−1)

Cs (mg RNA g protein−1)

ks (% d−1)

kRNA (ks · g−1 RNA)

Wildcaught Fed Starved F P

149.2 ± 5.6

0.418a ± 0.028

2.88 ± 0.26

0.35 ± 0.04

1.29 ± 0.14

2.29 ± 0.26 1.94 ± 0.24 2.50 0.111

0.35 ± 0.08 0.21 ± 0.04 2.26 0.143

1.39 ± 0.27 1.13 ± 0.19 0.34 0.718

166.1 ± 21.3 127.8 ± 8.3 2.42 0.117

ab

0.365 ± 0.014 0.248b (0.033) 6.11 0.01

Same letters, down a column, indicate no significant difference following one-way ANOVA and multiple comparison by Scheffe's. Cs, capacity for protein synthesis; ks, fractional rate of protein synthesis; kRNA, RNA activity.

Across all treatments fractional rates of protein synthesis in the tail muscle were correlated with the tail muscle RNA content (r = 0.671, P = 0.004, n = 20) and with the capacity for protein synthesis (r= 0.706, P = 0.002, n = 20), protein synthesis in the gill also correlated with the capacity for protein synthesis (r = 0.643, P = 0.003, n = 20). Protein synthesis in the hepatopancreas did not correlate with other measures of protein metabolism in the same tissue, it did correlate with gill protein synthesis (r = 0.881, P = 0.000, n = 20) and gill capacity for protein synthesis (r= 0.542, P = 0.037, n = 20). 3.4. Prediction of nutritional status of wild-caught Nephrops The relationship between the measured rate (ks) and capacity (Cs) for protein synthesis showed differences between tissues and nutritional history according to starvation, fed and wild-caught treatments (Fig. 2). The tail muscle values were lowest for both indices, and tended to have higher capacity and measured rates in the fed and wild-caught animals. The gill values were intermediate of the tissues; the measured rates and capacity tended to be higher in fed animals. For fed animals the capacity and the measured protein synthesis rates were highest in the hepatopancreas, in the starved and wildcaught hepatopancreas the capacity was also high but measured rates were low for both groups (P = 0.0017). 4. Discussion 4.1. Validation of the methodology The methodology, previously used to measure protein synthesis in a wide variety of mammals and fish (Carter and Houlihan, 2001; Garlick et al., 1980; Houlihan et al., 1995) can be successfully applied to measure protein synthesis in crustaceans (Fraser and Rogers, 2007; Mente et al., 2001, 2002, 2003). This study provided the first measurements of protein synthesis rates in the commercially exploited crustacean, N. norvegicus. In the present study, the three criteria Table 3 Protein metabolism measures in hepatopancreas of nephrops that were wild-caught, fed or starved (mean ± SEM). Tissue

Protein (mg g−1)

RNA (mg g−1)

Cs (mg RNA g protein−1)

ks (% d−1)

kRNA (ks · g−1 RNA)

Wild-caught Fed Starved F P

68.0 ± 3.8 61.4 ± 3.2 75.7 ± 3.2 1.62 0.226

3.00 ± 0.32 2.25 ± 0.36 3.12 ± 0.41 0.95 0.408

43.76 ± 3.48 36.64 ± 5.14 40.97 ± 4.73 0.60 0.558

2.28ab ± 0.37 7.55a ± 2.73 1.01b ± 0.42 5.87 0.017

0.56b ± 0.11 1.99a ± 0.55 0.29b ± 0.14 9.36 0.004

Same letters, down a column, indicate no significant difference following one-way ANOVA and multiple comparison by Scheffe's. Cs, capacity for protein synthesis; ks, fractional rate of protein synthesis; kRNA, RNA activity.

E. Mente et al. / Journal of Experimental Marine Biology and Ecology 409 (2011) 208–214 Table 4 Protein metabolism measures in gill tissue of nephrops that were wild-caught, fed or starved (mean ± SEM). Tissue

Protein (mg g−1)

RNA (mg g−1)

Cs (mg RNA g protein−1)

ks (% d−1)

kRNA (ks · g−1 RNA)

Wild-caught Fed Starve F P

80.7 ± 6.2 44.4 ± 6.2 54.1 ± 15.9 4.31 0.030

0.942 ± 0.101 0.618 ± 0.030 0.561 ± 0.123 3.33 0.060

11.91 ± 0.87 17.14 ± 4.16 12.49 ± 2.76 1.80 0.196

2.12 ± 0.23 3.91 ± 0.87 2.59 ± 0.81 3.32 0.062

1.83 ± 0.22 2.52 ± 0.57 2.12 ± 0.55 0.87 0.435

Same letters, down a column, indicate no significant difference following one-way ANOVA and multiple comparison by Scheffe's. Cs, capacity for protein synthesis; ks, fractional rate of protein synthesis; kRNA, RNA activity.

used to assess whether the rates of protein synthesis can be accurately measured in vivo by the flooding-dose (high-dose) phenylalanine method of Garlick et al. (1980) were validated. Free pools were flooded and free phenylalanine showed a fourfold increase following injection. This is similar to the fourfold increase found in shrimps L. vannamei and lobsters H. gammarus (Mente et al., 2001, 2002). The phenylalanine specific activity of the tail-muscle free pool was lower than and not equal to that of the injected solution. However, the level remained elevated and stable over the incorporation period for tail muscle for 120 min and for at least 90 min in the other two tissues. For all tissues the incorporation of phenylalanine into protein was linear over the incorporation time. Thus, the time course results for Nephrops from the current study validate the use of the protein synthesis values obtained. In future where measurements are to be made on all tissues we recommend an incubation time of less than 120 min.

4.2. Protein synthesis and protein metabolism Protein metabolism is fundamental to the growth of all animals and understanding differences and similarities between different taxa will reveal important principles in relation to their growth strategies (Carter and Houlihan, 2001; Fraser and Rogers, 2007; Mente et al., 2010; Moltschaniwskyj and Carter, 2010). Protein turnover can be divided into its constituent processes, protein synthesis, protein growth and protein degradation (Houlihan, 1991). At any particular time, protein growth (kg, protein growth as a percentage of the total protein mass) is the net balance between protein synthesis (ks) and protein degradation (kd), i.e. kg = ks − kd (Millward et al., 1975). Major influences on protein metabolism include life-cycle variable such as age, size, sex

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and reproductive state and environmental variables including those related to feeding and nutritional status. Over the life-cycle of well-fed southern dumpling squid Euprymna tasmanica protein metabolism was influenced in a predictable way by both age and weight, and also by individual variation (Carter et al., 2009; Moltschaniwskyj and Carter, 2010). Individuals with high growth rates had higher protein synthesis and higher efficiency of retaining synthesized protein whilst younger smaller animals generally had higher rates of protein synthesis, higher retention of synthesised protein and more RNA (Moltschaniwskyj and Carter, 2010). Over a 24 h daily-cycle, feed intake has a strong influence on protein metabolism and stimulates post-prandial peaks in protein synthesis (Fraser and Rogers, 2007; Katersky and Carter, 2010). The measures of protein metabolism in the present study reflect a combination of influences and variation due to size and sex were largely removed in order to investigate the nutritional status of wild-caught animals relative to starved and fed aquaria animals of a similar size and sex. Thus, a large difference between fed and starved aquaria animals highlights measures with potential to be used to assess the nutritional status of wild-caught Nephrops. Protein synthesis was almost 7.5 times higher in the hepatopancreas of fed compared to starved animals, for wild-caught animals protein synthesis was intermediate. Thus, hepatopancreas protein synthesis provided a strong indication of recent feeding and showed that the aquaria animals had fed more recently than the wild-caught animals (see below). It was also apparent that the increases in protein synthesis were due to increases in RNA activity and Nephrops did not modify hepatopancreas RNA content to change synthesis capacity directly. In molluscs the digestive gland has a role of enzyme production that requires protein synthesis to ramp up quickly in response to food (Carter et al., 2009; Semmens, 2002). The present study showed that measures of protein metabolism in the tail muscle were less sensitive to nutritional status than the hepatopancreas. In fed animals, rates of protein synthesis were about 1.5 times higher than in starved animals, and similar to wild-caught animals. Although there were no significant differences with starved animals, the similarity in tail muscle protein synthesis between fed and wild-caught animals supported the proposal that wild-caught animals had fed relatively recently and were growing. Muscle RNA and Cs values often have a positive correlation with growth rate (Carter et al., 1998) and are more indicative of longer term processes, such as growth, rather than recent post-prandial metabolism in which protein synthesis increases due to increased RNA activity rather than increased RNA (Katersky and Carter, 2010). Thus, the higher tail muscle RNA concentration of wild-caught compared to starved animals and the positive correlations between individual fractional rates of protein synthesis and tail muscle RNA content and with the capacity for protein synthesis support the case that the wild-caught animals were growing (Carter et al., 1993; McCarthy et al., 1994). Differences in gill measures of protein metabolism were less apparent and there were no significant differences between treatments. Gills of many taxa are relatively active and often have a high turnover of tissue with relatively high rates of protein synthesis and other measures of protein metabolism. This is likely to be related to their essential physiological role that is maintained even during starvation. Crustaceans achieve high protein growth rates by minimising protein degradation (Fraser and Rogers, 2007; Mente et al., 2002). The data in the present study are limited but showed that protein synthesis is influenced, particularly in the hepatopancreas, by recent feeding and also by the degree to which the Nephrops is fasted. Furthermore, the tail muscle has potential to show differences in growth rates between individuals. 4.3. Nutritional status and indices of protein metabolism

Fig. 2. The relationship between mean (±SE) capacity for protein synthesis (Cs, mg RNA g protein−1) and fractional rate of protein synthesis (ks,%d−1) in tail muscle (square), hepatopancreas (circle) and gill (triangle) for wild-caught (shaded), fed (closed/black) or starved (open/white) Nephrops.

Crustaceans' diversity in body form and mode of life, their physiological and adaptation strategies for survival, recovery from long periods of starvation, feeding habits, moulting, and adaptation to environmental variations have been the driving forces to promote

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research and increase considerably our knowledge on this group of animals. Crustaceans are excellent taxa to study species for physiological research since differences in biochemistry and nutritional physiology exist among them. In recent years, the need to understand and control growth mechanisms in crustaceans has been stimulated by increasing interest in the aquaculture of shrimps, lobsters and crabs. There is little information on the nutritional requirements and required diet formulation for groups of crustaceans, such as lobsters (Williams, 2007), crayfishes (Sáez-Royuela et al., 2007) and crabs (Rotllant et al., 2010). Commercial culture of Nephrops appears to be feasible although the slow growth rate showed by juveniles of that species and lack of detailed knowledge about their nutritional requirements and rearing conditions are constraints in assessing their potential for aquaculture (Mente, 2010; Rotllant et al., 2001). To understand the basis of Nephrops nutrition it is important to consider growth which is accomplished by moults which are cycles of laying down reserves and subsequent reutilisation of nutrients leading to a “discontinuity” in nutrient flux (Bell et al., 2006; Guillaume and Ceccaldi, 2011). Studies on growth rates and moulting of Nephrops follow the general decapod trend of increasing intermoult time and decreasing percent length increase with increasing age, body length or moult number (Aiken, 1980). However, little information is available on growth and nutritional status to investigate protein metabolism and growth of Nephrops. Environmental parameters such as temperature and food availability control growth rates and underlying processes driving growth, such as protein synthesis (Fraser and Rogers, 2007; Houlihan, 1991; Katersky and Carter, 2007). Evans et al. (1992) and Vogt et al. (1985) observed that extended starvation leads to decreased hepatosomatic ratios and loss of ability to metabolise food even if its availability is increased. The size and moisture content of the hepatopancreas in crustaceans could be used as an indicator to determine and evaluate the nutritional condition of the crustacean (Evans et al., 1992; Huner et al., 1990; McClain, 1995a, 1995b; Villagran, 1993). In the prawn (Palaemon serratus) 56 h of starvation caused changes in the structure of the hepatopancreas, enlarged mitochondria and rough endoplasmic reticulum in both R-cells and F-cells of the hepatopancreas (Papathanassiou and King, 1984). The hypoactivity of the cells due to fasting was reflected by changes in R-cells and disturbances in absorption mechanisms and protein synthesis. Changes in R-cells could be used as an indicator of the nutritional value of the diets offered in the (P. monodon) since they reacted to feeding on different diets (Vogt et al., 1985). Several digestive enzymes are produced in crustacean hepatopancreas that are secreted for food digestion, including protease like trypsin and chymotryosin, lipases and carbohydrate degrading enzymes (Sάnchez-Paz et al., 2006). During starvation these enzymes are produced or activated and finely regulated inside the cells; however the mechanisms is still unknown although some evidence for changes are becoming available (Sάnchez-Paz et al., 2006). Developmental stage influences crustacean's responses to starvation in relation to their loss in their dry weight due to the depletion of proteins, lipids and carbohydrates (Ritar et al., 2003). The early stages of starvation involve depletion of lipid:protein ratios typical of short-term food deprivation (Anger, 2001), protein, carbohydrate and lipid reserves in the crustaceans (Anger, 2001; Hazlett et al., 1975; Speck and Urich, 1969) due to utilisation of these reserves during nutritional deprivation. Starvation of 140 days decreased hepatopancreas mean weight from 5.2% weight to 2.6% of wet body in the lobster (H. americanus) whilst for feeding animals it was maintained at 5% BW (Stewart et al., 1967). Starvation increases whole body water content in juvenile red claw (Cherax quadricarinatus) (Gu et al., 1996). McClain (1995a, 1995b) showed that the water content of hepatopancreas in red swamp crayfish (P. clarkii) decreased when the feeding rate increased and crayfish were shown to catabolise tissue protein for metabolic requirements in starvation. The large hepatopancreas size, especially when it is related to low hepatopancreas moisture content, can be taken as an indicator of good condition

in crayfish (Lowery, 1988). In this study hepatopancreas weight was higher in the wild-caught Nephrops than the fed and the starved ones indicating a good condition of the wild animal. Although starvation forces the use of body reserves to maintain metabolic functions, the order and quantity of depletion to indicate the minimum requirements for survival is different in the strategies used by crustaceans (McLeod et al., 2004). Histological investigation of the digestive gland showed a lower density of lipid droplets in both day 14 and day 28 starved lobsters, with this depletion apparently causing structural damage to the digestive tubule in lobsters starved for 28 days (McLeod et al., 2004). Nephrops fed a mussel diet showed higher hepatopancreas lipid concentrations than the starved ones but lower hepatopancreas lipid concentrations than the one that were fed a pellet diet, indicating that diet and food consumption may explain the observed differences in hepatopancreas size (Mente, 2010). However, in the present study for fed animals the capacity for protein synthesis and the measured protein synthesis rates were high in the hepatopancreas, whereas in the starved and wild-caught animals the capacity was also high but protein synthesis was low for both groups. Furthermore, the RNA activity was higher in the fed group than the starved and the wild-caught. Many studies on fish have demonstrated that increasing feed (protein) intake is positively related to the capacity for protein synthesis and fractional rates of protein synthesis (Carter et al., 2009; McCarthy et al., 1994), and in RNA activity (Carter et al., 1993; Katersky and Carter, 2010; McCarthy et al., 1994). Thus, as discussed above, recent feeding has an effect on protein metabolism in crustaceans. Fractional rates of protein synthesis of Nephrops tissues measured were highest in the hepatopancreas, then gill and then tail muscle. Ranking of fractional rates of protein synthesis in invertebrate tissues is summarized, from highest to lowest, as digestive gland (hepatopancreas), gill, digestive tract, heart and muscle (Carter et al., 2009; Fraser and Rogers, 2007). The high fractional rates of protein synthesis in the digestive gland relative to other tissues supports hypothesis that Nephrops are constantly producing and storing digestive enzymes due to feeding and facilitate rapid nutrient assimilation for metabolism and growth. Rates of protein synthesis will be a function of the immediate nutritional or environmental history of the individual, but these will be modified by the physiological age of the individual. Using protein in the muscle tissue as an energy store would result in a dynamic system of increasing and decreasing mass depending on the nutritional state of the animal. Knowledge of the dynamics of muscle protein deposition and the relationship between fed and starved protein synthesis rates and muscle protein deposition is required. This study has demonstrated that there was a higher capacity for protein synthesis rates for wildcaught Nephrops but lower protein synthesis rates. The results in this study give some important insights into protein metabolism of this species. Data of this type can be used to provide knowledge on the biochemical pathways involved in protein turnover during changes that occur naturally and under periods of starvation and to understand their ability to adapt to environmental variations on a species specific case. Acknowledgements The authors thank Dr. C. Smith, N. Papadopoulou, Dr. I. McCarthy and Professor D. F. Houlihan for helpful comments. This research was cofunded by the European Social Fund and the National Greek Resources — EPEAEK II–PYTHAGORAS. [SS] References Aguzzi, J., Sarda, F., Allue, R., 2004. Seasonal dynamics in Nephrops norvegicus (Decapoda: Nephropidae) catches off the Catalan coasts (Western Mediterranean). Fish. Fish. Res. 69, 293–300.

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