Skeletal muscle protease activities in the early growth and development of wild Atlantic salmon (Salmo salar L.)

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Skeletal muscle protease activities in the early growth and development of wild Atlantic salmon (Salmo salar L.) Liudmila A. Lysenko⁎, Nadezda P. Kantserova, Elena I. Kaivarainen, Marina Yu. Krupnova, Nina N. Nemova Laboratory of Environmental Biochemistry, Institute of Biology, Karelian Research Centre of Russian Academy of Sciences, Pushkinskaya str., 11, 185910 Petrozavodsk, Russian Federation

A R T I C L E I N F O

A B S T R A C T

Keywords: Proteasome Calpain Cathepsin Skeletal muscle Fish growth Smoltification Salmo salar

Growth-related dynamics of intracellular protease activities in four year classes of the Atlantic salmon (Salmo salar L. 1758) parr and smolts inhabiting salmon rivers of northwestern Russia (the White Sea basin) were studied. Cathepsin B, cathepsin D, proteasome, and calpain activities in the skeletal muscles of salmon were assessed to investigate their relative contribution to the total protein degradation as well as to young fish growth process. It was confirmed that calpain activity dominates in salmon muscles while proteasome plays a minor role, in contrast to terrestrial vertebrates. Calpain and proteasome activities were maximal at the early postlarval stage (in parrs 0 +) and declined with age (parrs 1 + through 2 +) dropping to the lowest level in salmon smolts. Annual growth increments and proteolytic activities of calpains and proteasome in the muscles of salmon juveniles changed with age in an orchestrated manner, while lysosomal cathepsin activities increased with age. Comparing protease activities and growth increments in salmon parr and smolts we suggested that the partial suppression of the protein degradation could be a mechanism stimulating efficient growth in smoltifying salmon. Growth and smoltification-related dynamics of protease activities was quite similar in salmon populations from studied spawning rivers, such as Varzuga and Indera; however, some habitat-related differences were observed. Growth increments and protease activities varied in salmon parr 0 + (but not on later ages) inhabiting either main rivers or small tributaries apparently due to habitat difference on the resources for fish growth.

1. Introduction Fish growth is generally indeterminate, with most species continuing to grow in mass and length throughout their life. The fish growth process was extensively studied primarily on skeletal muscles constituting more than a half of the total body weight and containing mainly proteins (Weatherly and Gill, 1987; Houlihan et al., 1993; Mommsen, 2001; Bureau et al., 2006; Johnston et al., 2011). The proteins in the body are subjected to continuous breakdown and replacement. The rates of muscle protein synthesis and degradation are apparently counterbalanced to maintain indeterminate fish growth regularly interrupted by the periods of accelerated protein breakdown such as gonad maturation, spawning migration, food restriction, or starvation when protein degradation far outweighs protein synthesis (Nemova et al., 1980; Mommsen, 2004; Salem et al., 2007; Cleveland and Burr, 2011; Salmerón et al., 2013). Protein degradation during both physiological protein turnover and quality control of the newly synthesized proteins is a highly regulated and selective process carried out by the

⁎

proteolytic enzymes (Ciechanover, 2005). Up- or down-regulation of protease activities can significantly alter fish muscle growth and protein accumulation rates. The main protein-degrading pathways in the cells of all vertebrates are lysosome-autophagic (lysosomal autophagy), ubiquitin-proteasomal, and calpain proteolytic systems. A role of individual proteases as well as their relative contribution to the total protein degradation in the skeletal muscles has been partially characterized in fish, including salmonids (Salem et al., 2004; Hagen et al., 2008; Overturf and Gaylord, 2009; Seiliez et al., 2014; Nemova et al., 2016; Kantserova et al., 2017). Atlantic salmon (Salmo salar) represents a potential model to study and better understand the role proteases play in the fish growth process. Wild salmon growth and annual increments vary with fish age and depend on a range of environmental factors such as the length of the growing season, food availability, photoperiod, and water temperature (Metcalfe et al., 1988; L'Abee-Lund et al., 1989; Saltveit, 1990). Young salmonids of northern, low-production watercourses of the White Sea basin are characterized by a decreased growth rate during critical early growing stages and increased smoltification

Corresponding author. E-mail address: [email protected] (L.A. Lysenko).

http://dx.doi.org/10.1016/j.cbpb.2017.05.001 Received 6 December 2016; Received in revised form 2 May 2017; Accepted 3 May 2017 1096-4959/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Lysenko, L.A., Comparative Biochemistry and Physiology, Part B (2017), http://dx.doi.org/10.1016/j.cbpb.2017.05.001

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age comparing to their conspecifics from the lower-latitude watercourses of the North Sea, Baltic Sea, or Barents Sea basins (Saltveit, 1990; Erkinaro and Niemelä, 1995; Berglund, 1995; Siira et al., 2006; Otero et al., 2014). The mechanism(s) responsible for a reduced growth and development rate in salmonids of northern rivers especially at the earliest stages (0–4 months) has not to be resolved. The study of salmonid growth has not just a theoretical value but is of practical importance for the rapidly developing global aquaculture industry, particularly with respect to production and quality. The intent of this work was to evaluate the activities of the main proteolytic enzymes involved in the muscle protein degradation, namely lysosomal cathepsin B (catB) and cathepsin D (catD), proteasome, and calciumdependent proteases (calpains), in the skeletal muscles of wild S. salar juveniles of various ages collected in the different rivers and local habitats.

Table 1 Sampling sites and length-weight parameters of the Atlantic salmon of the Rivers Varzuga and Indera watercourses. Age group

n

Length, cm

Weight, g

Condition factor

River Varzuga, threshold Sobachii (66°40′69.9″N, 36°62′65.8″E); October 2014 Parr 0 +, 125 5 5.64 ± 0.23 1.38 ± 0.09 0.77 dah a a Parr 1 + 7 9.75 ± 0.15 6.96 ± 0.31 0.75 Parr 2 + 7 11.56 ± 0.69ab 9.95 ± 0.86ab 0.64 River Varzuga, threshold Sobachii (66°40′69.9″N, 36°62′65.8″E); August 2015 Parr 1 + 5 5.35 ± 0.06 1.28 ± 0.04 0.84 River Varzuga, brook Sobachii (66°40′76.8″N, 36°61′60.2″E); August 2015 Parr 1 + 5 5.0 ± 0.09 1.07 ± 0.08 0.86 River Varzuga, threshold Porokushka (66°43′38.9″N, 36°52′68.4″E); Parr 1 + 9 6.15 ± 0.07 2.21 ± 0.08 Parr 2 + 5 7.54 ± 0.12b 4.49 ± 0.27b Smolt 3 + 8 12.14 ± 0.17c 20.14 ± 0.13c

2. Materials and methods

June 2016 0.95 1.05 1.13

River Varzuga, mainstem (66°83′96.8″N, 35°95′02.6″E); July 2013 Parr 0 +, 51 12 4.00 ± 0.20 0.43 ± 0.03 dah

2.1. Ethical procedures All animal handling procedures were approved by the Ethics and Animal Care Committee of the Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences, following EU-established norms and procedures. Salmonid fish catch and study were approved by Barentsevo-Belomorskoye territorial department of the Federal Agency for Fisheries (resolution no. 51 2015 03 0119).

0.67

River Varzuga, tributary Pyatka (66°83′85.0″N, 35°94′83.3″E); July 2013 0.79d Parr 0 +, 51 11 4.30 ± 0.20 0.63 ± 0.03d dah River Indera, sea threshold (66°24′31.0″N, 37°14′27.8″E); June 2015 Parr 0 +, 20 5 2.73 ± 0.07 0.15 ± 0.01 dah a 1.30 ± 0.07a Parr 1 + 5 5.40 ± 0.07 Parr 2 + 6 9.77 ± 0.68ab 8.22 ± 1.82ab Smolt 2 +, 9 11.94 ± 0.80abc 13.39 ± 3.34abc female Smolt 2 +, 5 12.11 ± 0.92abc 14.9 ± 3.7аbc male Smolt 3 +, 5 14.12 ± 0.54abc 21.31 ± 3.2abce female Smolt 3 +, 6 14.14 ± 0.9abc 20.92 ± 3.4abcf male

2.2. The study area Wild Atlantic salmon (Salmo salar L. 1758) was collected on summer (late June through mid-August) and autumn (October) seasons 2014–2016, from the salmon spawning rivers of the White Sea basin, Kola Peninsula, Russia: the Varzuga River (sampling sites: threshold Sobachii, brook Sobachii, tributary Pyatka, threshold Porokushka) and the Indera River (sea threshold) (for site coordinates see Table 1). Study sites were in the same geographical zone and characterized by the similar annual temperatures and biodiversity, except the differences between the mainstem thresholds and a tributary/brook. Daily temperatures, stream velocities, the amount of organic drifting food resources, and the macrozoobenthic mass and biodiversity were significantly greater in the tributary/brook than in the mainstem of the River Varzuga (G2-test, p < 0.001; Baryshev, 2014).

0.74 0.83a 0.88a 0.79c 0.84a 0.76c 0.74c

Note: Values for growth characteristics are given as means ± SD, dah – days after hatching. Letters indicate significant differences: a in comparison to 0+; b in comparison to 1+; c in comparison to parr 2 +; d in comparison to mainstem/threshold salmon; e in comparison to smolt 2 +, female; f in comparison to smolt 2+, male.

2.4. Reagents and equipment Chemical reagents, protease inhibitors and substrates were purchased from Sigma-Aldrich (St. Louis, MO, USA) and of analytical grade. Technical facilities of the Equipment Sharing Centre of the Institute of Biology, KarRC of RAS were used, such as freezing chamber UF 240-86 Е (Snijders Scientific, The Netherlands); homogenizer Tissue Lyser LT (Qiagen, Germany); centrifuge Allegra 64R (Beckman Coulter, USA); spectrophotometer SP-2000 (OKB Spectrum, Russia), and microplate reader CLARIOstar (BMG LABTECH, Germany).

2.3. Sampling Salmon parrs were captured by electrofishing (Fa-2, Norway) and kept for 24 h in cages located downstream of the river to avoid electrofishing stress. Salmon smolts were captured during their natural seaward migration by a smolt trap located in the river 300 m from an estuary. Each fish was weighed (g) and fork length (cm) measurements were obtained. Mean body weights and fork lengths are presented in Table 1. Condition factors were calculated according to Fulton's formula: K = 100(W / L3), where K is condition factor, W is weight (g), and L is fork length (cm). Prior to sampling, fish were anesthetized by 1 mL/L phenoxyethanol and sacrificed by decapitation. Carcasses were immediately frozen in liquid nitrogen and stored at −80 °C until assayed. White (fast) muscle near dorsal fin was extracted and sampled. Skeletal muscle protease activities were determined for pooled parrs 0 + (8–10 fish); in the larger size groups – parrs 1+, 2+, and smolts – protease activities were measured in individual fish. Smolt status was evaluated using both physiological and morphological variables based on visual assessments. These results were consistent with each other, showing that wild S. salar from the Varzuga and the Indera rivers exhibited smolting-associated characteristics at 2 or 3 years old.

2.5. Extraction of intracellular proteases Samples (0.1 g each) were homogenized in 1:10 w/v 20 mM TrisHCl (pH 7.5) with 150 mM NaСl, 5 mM EDTA, 20 mM dithiothreitol, 1 mM АТP, 5 mМ MgCl2, 0.1% Triton X-100, and protease inhibitors (0.5 mg/mL leupeptin, 1 mg/mL pepstatin, 1 mg/mL aprotinin, and 1 mM PMSF). Homogenates were centrifuged at 20,600g for 30 min to obtain a pooled fraction of cytoplasmic and organelle proteins referred as enzyme-containing fraction. 2.6. Calpain activity assay Calcium-dependent proteolytic activity was quantified using a microplate assay and casein as a substrate (Enns and Belcastro, 2006). A reaction mixture with 500 mL total volume was composed 2

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Fig. 1. Weight, length, and skeletal muscle protease activities of salmon parr (age-groups 0+, 1 +, 2+) and smolts (3+) from the thresholds of the rivers Varzuga and Indera. Statistically significant differences (Mann-Whitney U test, p ≤ 0.05) are indicated in Supplement table 1.Weight, length, and skeletal muscle protease activities of salmon parr (agegroups 0+, 1+, 2+) and smolts (3+) from the thresholds of the rivers Varzuga and Indera. Statistically significant differences (Mann-Whitney U test, p ≤ 0.05) are indicated in Supplement table 1.

normalized for 1.0 mg protein in a sample.

of the following: 0.4% alkali-denatured casein, 20 mM dithiothreitol, 50 mM Tris-HCl (pH 7.5), 5.0 mM Ca2 + (as CaCl2) or 5.0 mM EDTA (negative control), and the enzyme-containing fraction. Following incubation at 28 °C for 30 min (heating/cooling dry block CH-100; BioSan, Latvia), remaining protein was quantified by Bradford assay (Bradford, 1976). Enzymatic activity was expressed in activity units (AU), defined as the amount of the enzyme that causes an increase of 0.1 in absorbance at 595 nm per hour. Specific calpain activity was

2.7. Proteasome activity assay The chymotrypsin-like activity (CLA) of the proteasome was determined in the enzyme-containing fraction using a fluorescence assay (Rodgers and Dean, 2003). Peptidase activity against a synthetic oligopeptide substrate was measured in a reaction mixture containing 3

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Fig. 2. Weight, length of salmon parr (age-groups 0+, 1+) and protease activities in the muscles of the Atlantic salmon from the thresholds and the small tributaries (tributary Pyatka or brook Sobachii) of the River Varzuga watercourse; indicated as “mainstem” and “tributary” groups. Vertical lines indicate SD. Statistically significant differences are indicated by asterisks; Mann-Whitney U test, p ≤ 0.05.

1 mM dithiothreitol, 5 mM MgCl2, 1 mM АТP, 30 μM Suc-LLVY-AMC as a substrate, and 20 mM Tris-HCl (pH 7.5) in the absence or presence of 5 μM MG132, a specific inhibitor of CLA. Following incubation at 37 °C for 30 min, proteasome activity was calculated as the difference in fluorescence intensity between the samples with and without inhibitor at excitation and emission wavelengths 380 nm and 440 nm, respectively. Specific CLA proteasome activity was normalized to total protein concentration in a sample and expressed as relative fluorescence fold change (FU).

2.8. Cathepsin B activity assay Cathepsin B (catB) peptidase activity in a pooled fraction of cytoplasmic and organelle proteins (obtained as described above; Section 2.5) was measured by the hydrolysis of 65 mM ethyl ether Dbenzoyl L-arginine hydrochloride (BAEE) in 200 mM acetate buffer (pH 5.0) at 37 °C (Lysenko et al., 2014). Enzymatic activity of partially purified catB was expressed using conventional activity units (AU) by reaction product absorbance at 525 nm per g tissue per 30 min.

4

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Table 2 Specific protease activities in the skeletal muscles of the Atlantic salmon parr (age-groups 0 +, 1+, 2 +) and smolts (2 +, 3+) of both sexes sampled at the sea threshold of the River Indera mainstem. n

CatD activity, AU

CatB activity, AU

Calpain activity, AU

Proteasome CL activity, FU

Parr 0+ 1+ 2+

5 5 6

5.5 ± 0.9 1.0 ± 0.3 0.2 ± 0.1ab

10.0 ± 2.6 9.1 ± 0.6 8.0 ± 1.0

203.4 ± 15.9 335.5 ± 82.04 286.2 ± 76.4

180.1 ± 2.11 115.1 ± 13.2а 108.7 ± 13.2а

Smolts 2+, female 2+, male 3+, female 3+, male

9 5 5 6

0.3 ± 0.2a 0.4 ± 0.1a 0.04 ± 0.02abcd 0.05 ± 0.03abce

5.2 ± 0.8a 6.1 ± 1.1 21.0 ± 2.7abcd 17.0 ± 2.6bce

79.8 92.9 76.4 47.2

± ± ± ±

30.7abc 32.5ab 6.8abc 12.8abcd

Note: Values for protease activities are given as means ± SD. Letters indicate significant differences: a in comparison to parr 0 +; 2 +; d in comparison to smolt 2+, female; e in comparison to smolt 2 +, male; Mann-Whitney U test, p ≤ 0.05.

b

81.1 70.2 61.5 63.9

± ± ± ±

8.2ab 20.5ab 9.4abc 10.6abc

in comparison to parr 1 +; c in comparison to parr

p < 0.05) and the condition factor was better in tributary parr (0.79 vs 0.67 in counterparts in mainstem) despite of the same place of origin for both fish groups hatched from the redds in the River Varzuga watercourse. Substantial difference in size of salmon parr and smolts was detected (Table 1). Both female and male salmon smolts of age 2+ were larger than parrs of the same age (Mann-Whitney U test, p < 0.05) with a critical raise in growth rate on a year preceding smoltification (pairwise comparison: parr 1+ vs smolts 2+, parr 2+ vs smolts 3+). Besides, female smolts 2+ and 3+ were distinct by their body weight, and males – by fork length (Table 1). The peak in smoltification towards larger fish (over ca. 11 cm in length) was observed in salmon parr 2+ (within Indera's salmon population) or 3+ (within salmon from both rivers).

2.9. Cathepsin D activity assay Cathepsin D (catD) proteolytic activity was assessed in an enzymecontaining fraction by the degradation of 1.0% bovine serum hemoglobin in 0.1 М acetate buffer (pH 3.6) at 37 °C based on a modified Аnson's technique (Lysenko et al., 2014). CatD activity was expressed in units (AU) of trichloroacetic acid-soluble product absorbance at 280 nm per g tissue per hour. 2.10. Determination of protein content Water-soluble protein concentration (mg of protein per g of wet weight) in an enzyme-containing fraction was determined according to Bradford technique (Bradford, 1976) using bovine serum albumin as a standard.

3.2. Protein turnover 2.11. Statistical analyses The activities of cytoplasmic proteases, including calpain and chymotrypsin-like proteasome activities, tended to be maximal in the skeletal muscles of the youngest fish (parr 0+) and progressively decreased with fish age (Fig. 1). Both calpain and proteasome activities substantially dropped in smolts comparing with parr of same age and particularly with parr of a previous year-class (parr 1+ vs smolts 2+ or parr 2+ vs smolts 3 +) (Mann-Whitney U test, p < 0.05; Fig. 1, Table 2). The one exception of that pattern was near equal calpain activity in salmon parr of different year-classes collected from Varzuga in autumn, apparently indicating the effect of growing season completion on growth regulation. The activities of the individual enzymes involved in lysosomal-autophagic protein degradation were affected differently by the factors “fish age” and “drainage” (Kruskal-Wallis test, p < 0.05; Fig. 1, Table 2). CatD activity in Varzuga's salmon increased with age (from 0+ to 3+) and reached the maximum in smolts in contrast to calpain and proteasome activity dynamics; whereas catD activity decreased with age in Indera's salmon parr of different yearclasses, thus partially matching calpain/proteasome dynamics regarding the rate of body mass accumulation. The detected level of catB activity was near equal in salmon parr 0+ through 2+ and substantially increased in smolts. Calpain and catB activities varied among two ecological subgroups of salmon parr 0+ inhabiting either a river mainstem or a small tributary (Mann-Whitney U test, p < 0.05; Fig. 2, threshold Sobachii vs tributary Pyatka); the habitat-related differences in growth rate and proteolysis in salmon parr 1+ (threshold Sobachii vs brook Sobachii) were not detected (Fig. 2).

Raw data were initially checked for normality of distribution and homogeneity of variances by Kolmogorov-Smirnov and Levene's tests, respectively. As variances were unequal and not distributed normally, multiple groups were compared with non-parametric Kruskal-Wallis test and two groups with Mann-Whitney U test. Values are throughout presented as mean ± SD. A p value < 0.05 was considered statistically significant in all analyses. 3. Results 3.1. Annual growth increments and physiological state of salmon The factors “fish age” and “developmental stage (parr or smolt)” significantly affected all studied performance traits (Table 1, Fig. 1). The morphometric indices of salmon parr of three year-classes (0 +, 1 +, and 2+) and smolts of two year classes (2 + and 3+) showed the age-related statistical difference (Kruskal-Wallis test, p < 0.05) with no differences between Varzuga's or Indera's juveniles of same age collected at same season. The growth increments per time unit decreased with increasing age or size; for instance, according to the observations in 2014, the body weight of salmon under-yearlings (0 +) differed more than five times from that of the one-year-old fish (1 +) and the body weight gain during the third year was substantially lower showing only a 1.4-fold increase. The individual growth rates during the first year of life showed a considerably higher spread in salmon under-yearlings than in individuals of the other year-classes but the mean values of their condition factor were rather similar with an exception of the brook juveniles (Table 1). Annual growth increments in salmon parr 0+ varied among the local habitats within the River Varzuga (Table 1, Fig. 2). The first-year mass accumulation was higher in tributary parr than that of main river parr (Mann-Whitney U test,

4. Discussion 4.1. Proteolytic enzymes of salmon skeletal muscles In this study, we detected multiple proteolytic activities in the 5

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protein-degrading capacity of fish muscles particularly concerns calpain and proteasome systems.

skeletal muscles of salmon, including proteasome, catheptic, and calpain activities that are responsible for ubiquitin-proteasome, lysosomal-autophagic, and Ca2 +-dependent protein degradation pathways, respectively. The relative importance of different proteases in the total degradation of muscle proteins is under debate (Nemova et al., 2016). Based on previous observations (Martin et al., 2002; Dobly et al., 2004; Seiliez et al., 2014) the non-proteasomal ATP-independent proteolytic pathways predominate in the skeletal muscles of fish, with calpains responsible for 30–34% of total protein hydrolysis and lysosomal system for 40%, while proteasome-dependent degradation accounts up to 4%. Calpains recognized to play a key role in muscle protein degradation in fish since a most of muscle proteins both sarcoplasmic or myofibrillar reported to be calpain substrates (Kołodziejska and Smorski, 1996; Verrez-Bagnis et al., 2002; Goll et al., 2003). Besides the contribution to myofibrillar protein turnover, calpains specifically regulate myogenesis promoting myocyte differentiation and hyperplastic muscle growth (Cottin et al., 1994) inherent to young fish (Bureau et al., 2006). Among numerous lysosomal proteases and peptidases, the principal role in protein degradation by autophagy in fish is assigned to catD (Yamashita and Konagaya, 1992). Our data correspond well with the previous observations on a low level of proteasome activity in fish muscles and a minor contribution of the proteasome to the myofibrillar protein turnover (Dobly et al., 2004; Seiliez et al., 2014), despite, we found that proteasome activity responds well to the size and physiological changes in salmon. The physiological relevance of proteasome activity in fish was demonstrated in other studies (Salem et al., 2007; Martin et al., 2002; Seiliez et al., 2008). Thus, proteasomes, cathepsins, and calpains are involved in fish muscle protein degradation; besides, their relative contribution is quite different to those in mammals. Multiple protease activities in fish skeletal muscles such as protein turnover, quality control, degradation, and the limited hydrolysis of specific muscle growth-related substrates suggest their critical importance for muscle growth and integrity. To better understand the patterns of salmon growth and development, the muscle proteases should be the studied comprehensively.

4.3. Stage-related changes in growth and protease activities Significant increase in growth increments associated with decrease in proteolytic activities in the skeletal muscles of smolts comparing with salmon parr is apparently the most important finding of this work. It indicates a possible contribution of proteases to the enhanced growth of individuals in a year preceding parr/smolt transformation. Generally, the probability of smoltification increased with increasing body length (Metcalfe et al., 1988; Berglund, 1995), and our finding that both female and male salmon smolts are larger than parrs are consistent with the observations on increased protein accumulation and salmon growth rate preceding smoltification (Dickhoff et al., 1997; Stefansson et al., 2008; Seear et al., 2010). Among proteolytic pathways, calpain and proteasome systems are substantially suppressed in salmon smolts. We suppose that revealed stage-specific and apparently orchestrated suppression of calpain and proteasome-dependent proteolysis in the skeletal muscles of smolting individuals could contribute to their enhanced growth during this physiological stage. 4.4. Habitat-related changes in growth and protease activities Environmental heterogeneity among local habitats promotes the differentiation of salmon under-yearlings on phenotypic subgroups distinguished by growth and metabolic rates (Erkinaro and Niemelä, 1995; Pavlov et al., 2007). Salmon parr 0+ of same place of origin settled riverside habitats with a different complex of factors affecting growth, such as food availability, and ambient water temperature, have a different potency for growth. According to our data on body weight increments and condition factor, under-yearlings inhabiting tributaries or brooks, grow better than mainstem counterparts and possess relatively high activities of muscle proteases, including calpain and catB, indicative for extensive protein turnover and muscle growth. Habitat-related advantage seems to lose in brook salmon of elder age groups (since 1+) presumably due to changing nutritional preferences. Our study considerably expanded the knowledge on the growth patterns and parr/smolt transformation of wild salmon inhabiting the subarctic rivers and on the factors affecting salmon growth and development. Based on our observations on multiple protease activities, such as calpains, proteasome, and cathepsins, in the skeletal muscles of the Atlantic salmon juveniles at the early (river) period of their life, we suggest that the level of protein degradation corresponds with their growth rate and varies with age to maintain indeterminate muscle growth. Linear growth and body mass accumulation rates in salmon parr depend on overall protein turnover in their muscles mostly relying on calpain and, to a lesser extent, proteasome activities. We assumed that revealed stage-specific suppression of the calpain and proteasomedependent protein degradation enable accelerated growth of salmon preceding smoltification. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpb.2017.05.001.

4.2. Age-related changes in growth and protease activities Age-related growth patterns and the dynamics of protease activities in the skeletal muscles of salmon juveniles of both studied sub-arctic rivers (Varzuga and Indera) was quite similar (with a few exceptions) indicating common mechanisms governing fish growth and muscle physiology. Since the skeletal muscles comprise a substantial proportion of fish body weight, the growth of the organism to larger extent relies upon increased muscle mass (Weatherly and Gill, 1987; Houlihan et al., 1993; Mommsen, 2001). Muscle growth in juvenile fish is determined by high rates of muscle protein synthesis, accumulation, and new muscle fiber formation exceeding the rates of protein turnover and degradation. However, fish growth absolutely depends on proteolytic machinery carrying out the protein quality control at the excess of newly synthesized proteins through the digestion of abnormal ones in lysosomes and proteasomes and contributing to hyperplastic muscle growth mediated by calpains. Gradual age-dependent decrease in total body mass accumulation rate in salmon is tightly connected with the decrease in both protein synthesis and degradation. Churova et al. (2015) described a positive correlation between the fish weight and protein synthesis measured by RNA/DNA ratio in juveniles 0+ and 1+ but not in other year-classes. Besides, the expression of myosin heavy chain (MyHC), a basic myofibrillar component susceptible to calpaindependent hydrolysis in fish (Verrez-Bagnis et al., 2002), decreased at age 2+, whereas the positive correlation of body weight and MyHC expression level maintained in 0 + through 2+ (Churova et al., 2015, 2017). Youngest fish group (0 +) possess maximal proteolytic activities of calpains and proteasome as well as maximal growth increments; in the following years, the proteolysis and growth indices decreased indicating their coordinate regulation. The age-dependent decrease in

Acknowledgments Authors deeply appreciate DSc. Alexey Veselov, PhD Denis Efremov, and PhD student Mikhail Ruchyov (IB KarRC RAS, Lab. for Fish and Water Invertebrate Ecology) for their assistance in collection and processing of field material. The authors express their sincere gratitude to the anonymous reviewers for their critical comments and guidance on improving the text of the manuscript. The research was financed by the Russian Scientific Foundation through research project 14-2400102 “Salmonids of the northwestern Russia: Ecological and biochemical mechanisms of the early development”. 6

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