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Introduction
Crangon crangon (Linnaeus, 1758) is a common crustacean in European seas (Holthuis 1980; Jeffery & Revill 2002; Oh et al. 1999; Revill & Holst 2004) and has a large and stable population in the Gulf of Gdansk (Baltic Sea) (Żmudziński & Ostrowski 1990; Łapińska & Szaniawska 2006). The stability of this species in this region allows a number of processes to be followed. Certain regularities can thus be established for this population and extrapolated to other populations from different latitudes. The body of shrimps is rich in highly nutritional substances, e.g. protein, unsaturated fatty acids and mineral compounds (Heu et al. 2003; Revill & Holst 2004; Sachindra et al. 2005; Kurita 2006). Biological activity of protein-containing tissues and organs of marine animals depends on its amino acid composition (Yoshinaka et al. 1989; Sivakumar et al. 1997). It is assumed that the amino acids occurring in aquatic invertebrates are involved in various processes and can affect the body development or the condition of individuals (Hulmes 2008; Kavitha & Thampan 2008). One of them is hydroxyproline, an amino acid typical of collagen (Ignat'eva et al. 2007). Hydroxyproline is a characteristic constituent of collagen, accounting for 13 to 14% (Neuman & Logan 1949) or 12.8 to 14.7% (Ignat'eva et al. 2007) of the amino acids forming this protein. Knowing the hydroxyproline content, we can estimate the amount of collagen in a given material, although we have to be aware of the fact that the method of converting the hydroxyproline level into the quantity of collagen may vary slightly depending on the group of animals we are dealing with (Ignat'eva et al. 2007). Siddiqi et al. (2001) assumed that hydroxyproline constitutes 12.5% of collagen. In the case of the shrimps Penaeus monodon and Penaeus vannamei, Sriket et al. (2007) used the conversion factor of 11.42 suggested by Sato et al. (1986). Mizuta et al. (1998) estimated the amount of collagen in the Antarctic krill Euphausa superba based on the fact that hydroxyproline accounts for 12.7% of that protein. Recent research has shown that hydroxyproline, and hence collagen, is present in different parts of the invertebrate body and participates in a range of life processes (Hulmes 2008; Kavitha & Thampan 2008). For example, collagen is involved in the regeneration of animals and in the modulation of stress during swimming (Féral 1988; Sivakumar & Chandrakasan 1999). Collagen synthesis has been shown to intensify wherever the body has been damaged (Féral 1988). Hydroxyproline significantly affects the temperature of collagen denaturation, which is important in the context of thermal stability of this protein (Muyonga et al. 2004). Water temperature affects the level of hydroxyproline and thus collagen content in animals (Furukawa et al. 1973; Shuster et al. 1975; Kavitha & Thampan 2008). This applies in particular to species exposed to changing ambient temperatures (Brauer et al. 2003). The level of hydroxyproline is higher in organisms living in warm waters (Brauer et al. 2003). A number of studies indicate the influence of the sex of animals, water salinity and dietary composition on hydroxyproline and collagen levels in invertebrates (Sato et al. 1978; Vasanthakumar et al. 1980; Touhata et al. 2000; Brauer et al. 2003).
The objective of this work was to define the level of hydroxyproline in C. crangon from the Gulf of Gdansk (southern Baltic). Knowing the quantity of hydroxyproline, one can determine the level of collagen and explain whether and how this protein participates in various life processes of this shrimp. This research should indicate 1) whether the hydroxyproline content is only associated with the growth and ageing of shrimps, or whether it may also be involved in other processes such as reproduction; 2) whether, in view of the evident sexual dimorphism in C. crangon, collagen synthesis is correlated with the sex of animals; 3) whether, despite the stable, uniform population in the Baltic, the hydroxyproline content is determined by the distribution of individual animals; 4) whether changes in the levels of this protein are due to environmental factors such as water temperature and food availability.
Materials and methods
Fieldwork
The material for analysis was collected in 2008 at six sites in the Gulf of Gdansk (southern Baltic) divided into two profiles – Gdynia and Sopot (Table 1). The animals were captured using a drag net (30 x 60 cm; mesh size 2.5 cm). On each occasion, salinity (S = PSU) and temperature (T = °C) of water were measured using a MULTI 340i probe (WTW, Germany).
Study area, depth, geographical coordinates, and temperature and salinity of water in the following months of 2008
Sampling sites
Depth
Geographical coordinates
Water temperature
Salinity
m
λ,ϕ
°C
PSU
IV
V
VI
VII
VIII
IV
V
VI
VII
VIII
Gdynia profile
G1
10
1854°°36.427.9'E 'N
6.2
13.6
11.2
18.0
18.0
6.2
6.0
6.6
7.1
7.0
G2
14
1854°°40.029.0'E 'N
6.1
10.1
11.3
17.9
17.5
6.3
6.7
6.7
7.0
7.0
G3
17
18°41.4'E 54°29.4'N
5.7
8.9
11.5
16.3
16.9
6.5
6.8
6.6
7.0
7.0
Sopot profile
S1
8
1854°°36.126.4'E 'N
7.1
14.9
11.2
18.1
18.3
6.0
5.3
6.5
7.0
7.0
S2
10
1854°°37.927.5'E 'N
6.9
13.8
11.2
17.6
17.6
5.9
5.9
6.5
7.0
7.0
S3
15
18°39.7'E 54°28.6'N
6.5
11.2
11.3
16.9
17.0
6.3
6.5
6.6
7.1
7.0
Laboratory work
The animals were deep-frozen (T = –80°C) and subsequently stored in this state until required for analysis. After thawing, the total length of animals was measured from the tip of the rostrum to the end of the telson, with an accuracy of 0.1 mm. They were also weighed on an electronic scale (XS 205 Dual Range – Mettler Toledo, Poland), with an accuracy of 0.001 g. The animals were sexed and ovigerous females were included in a separate group. They were then divided into three length classes, i.e. 21.0–30.0 mm, 31.0–40.0mm and 41.0–50.0 mm. Preparations were made from the tissues as well as from the thoracic and abdominal muscles of each shrimp. In the ovigerous females, the thoracic tissues were separated from the eggs.
The prepared tissues and muscles were placed in a desiccator (KBC-65G; Wamed, Poland) and dried at 105°C to constant mass. The hydroxyproline levels in male and female Crangon crangon from the three length classes were measured using Stegemann's technique (1958) modified by Czarnowski & Krechniak (1989). The analysis is based on the spectrophotometric measurement of pink color, which is the effect of the condensation reaction of products resulting from the oxidation of hydroxyproline (Hyp) with p-dimethylaminobenzaldehyde (PABA). Samples of 100 mg (n = 3 replicates) were hydrolyzed with 6N HCl at 120ºC for 2.5 h. To determine the actual content of hydroxyproline in the material, 200 μl of hydroxyproline standard (trans-4-hydroxy-L-proline ≥ 99%, Sigma-Aldrich) were added to one of the vials. The product of hydrolysis was neutralized with 6N NaOH to pH = 7.0, and then centrifuged at 2800 rpm. Chloramine T was added to a suitably diluted supernatant in the amount of 1 ml, then 1 ml of HClO4 was added after 20 min, and 1.5 ml of p-dimethylaminobenzaldehyde after another 5 min. The solutions were heated over a water bath at 60ºC for 18 min. After the test-tubes were cooled, the absorbance of the solution at a wavelength of 555 nm was measured using a UV–1202 spectrophotometer (SHIMADZU Europe GmbH). Quartz cuvettes with a 10 mm optical path length were used.
Calculation of hydroxyproline
A calibration curve of hydroxyproline (Hyp) was plotted, on the basis of which the hydroxyproline content in a dry mass sample of 100 mg was calculated for each type of material. For this purpose, solutions of hydroxyproline (10 μg ml–1) of known concentration were prepared: 0.1, 0.2, 0.3, 0.4, 0.5 μg ml–1 along with a blank test. Three replicates of each concentration were prepared. Knowing the absorbance of the material in each length class, it was possible to read the hydroxyproline concentration per 100 mg from the plot.
Values were expressed as a mean together with the standard deviation (mean ± SD) for a specified number (n) of length classes. The relationship between the studied parameters was determined using the linear equation y = ax+ b, at a significance level of 5%. Differences between the groups of results were tested with the Mann-Whitney U-test at a level of 5%.
Results
Fieldwork
Water temperature and salinity
The near-bottom water temperatures in the two profiles (Gdynia and Sopot) differed only slightly and changed throughout the year (Table 1). The lowest mean water temperatures were recorded in April (6.8 ± 0.3°C in Gdynia and 6.3 ± 0.2°C in Sopot), and increased in the following months. The highest temperature at all sites was recorded (18°C) in July and August.
The mean salinity remained at the same level at all sites throughout the study period (6.6 ± 0.4 PSU; Table 1).
Laboratory work
A total of 2886 individuals of C. crangon were collected during the whole study period – 1302 males and 1291 females. Of the total number of females, 293 were ovigerous from May to August 2008. The largest numbers of C. crangon were recorded at the shallowest sites in both profiles (Table 2). All animals captured were used to determine the hydroxyproline levels.
The number of C. crangon males, non-ovigerous females and ovigerous females in length classes (mm)
Sampling sites
Number of individuals in length classes (mm)
Males
Non-ovigerous females
Ovigerous females
21.0–30.0
31.0–40.0
41.0–50.0
21.0–30.0
31.0–40.0
41.0–50.0
21.0–30.0
31.0–40.0
41.0–50.0
Gdynia profile
G1
193
95
1
74
109
28
1
24
3
G2
117
20
0
84
24
3
6
36
2
G3
51
15
0
64
52
3
1
25
2
Sopot profile
S1
246
115
0
103
173
19
1
69
20
S2
201
55
0
125
130
13
3
40
9
S3
106
24
0
81
88
4
1
45
5
Total number
1302
1291
293
Sex and size of individuals
The mean hydroxyproline levels in males were 1.81 ± 0.61 μg 100 mg–1 d.w. and in non-ovigerous females – 1.71 ± 0.64 μg 100 mg–1 d.w. Ovigerous females have significantly higher levels of hydroxyproline in their tissues (2.12 ± 0.65 μg 100 mg–1 d.w.) compared to other groups, which applies to both profiles of the study area and all the depths (Fig. 1).
Figure 1
Gdynia and Sopot profiles: hydroxyproline levels (μg 100 mg–1 d.w.) in C. crangon males, non-ovigerous females and ovigerous females. Mean value from triplicate determinations
In both the Gdynia and Sopot profiles, the hydroxyproline content increased with the individual body length of males and non-ovigerous females (Fig. 2). Mean values were significantly lower in younger specimens compared to the older ones. In ovigerous females from both profiles, the hydroxyproline levels decreased with the length of individuals (Fig. 2).
Figure 2
Gdynia and Sopot profiles: hydroxyproline content (μg 100 mg–1 d.w.) in C. crangon in different length classes. Mean value from triplicate determinations
Study area
Mean hydroxyproline levels in all the groups of the examined shrimps were similar throughout the study area (Table 3, Fig. 3). The differences were not statistically significant (p > 0.05). The average hydroxyproline level did not exceed 2.0 μg 100 mg–1 d.w. in both males and non-ovigerous females (Fig. 3). Ovigerous females from the Gdynia profile had on average 15% lower content of hydroxyproline in their tissues compared to individuals collected in the Sopot profile (Table 3).
Figure 3
Gdynia and Sopot profiles. Hydroxyproline levels (μg 100 mg–1 d.w.) in C. crangon males, non-ovigerous females and ovigerous females collected from different depths. Mean value from triplicate determinations
Hydroxyproline levels in males, non-ovigerous females and ovigerous females at two profiles
Study area
Males
Non- ovigerous females
Ovigerous females
Gdynia profile
1.80 ± 0.64
1.74 ± 0.66
1.88 ± 0.65
Sopot profile
1.83 ± 0.59
1.74 ± 0.63
2.20 ± 0.63
Values are given as means ± SD (Standard Deviation) from triplicate determinations and expressed as μg Hyp x 100 mg–1 d.w. (Dry Weight)
Depth
Males collected at the shallowest sites in both profiles of the study area (site G1 and S1) had the lowest hydroxyproline levels (Fig. 3). The highest values were determined at the deeper sites: G2 (14 m) and S3 (15 m) (Fig. 3). The mean hydroxyproline levels in non-ovigerous females varied with depth. The lowest values were observed at site G2 (14 m) and S3 (15 m). The highest values were determined at site G3 (17 m) and S1 (8 m) (Fig. 3). The average content of hydroxyproline in ovigerous females was at a similar level at all depths in the Gdynia profile (Fig. 3). There was a slight decrease in hydroxyproline levels with depth in the Sopot profile (Fig. 3).
Hydroxyproline levels from April to August 2008
Gdynia profile
A 30% increase in the mean hydroxyproline level was observed in males from April to May (Fig. 4). In the following months, the hydroxyproline content declined. In non-ovigerous females, the lowest value of the analyzed amino acid was observed in May, and the highest one in June (Fig. 4). Ovigerous females appeared in May and then reached the highest mean hydroxyproline levels in their body (Fig. 4). In the following months, the amino acid level decreased.
Figure 4
Gdynia and Sopot profiles. Hydroxyproline levels (μg 100 mg–1 d.w.) in C. crangon males, non-ovigerous females and ovigerous females collected from April to August 2008. Mean value from triplicate determinations
Sopot profile
From April to June, mean hydroxyproline levels in males slightly increased and then decreased in subsequent months (Fig. 4). Low mean hydroxyproline
content in non-ovigerous female tissues was observed in May and August, and the highest content was found in July (1.91 ± 0.56 μg 100 mg–1 d.w.). In ovigerous females, the mean hydroxyproline level increased by 34% from May to June and then decreased to 1.78 ± 0.17 μg 100 mg–1 d.w. in August (Fig. 4).
Discussion
The level of hydroxyproline was determined in the shrimp C. crangon from the coastal waters of the Gulf of Gdansk, where the species has established a permanent population (Żmudziński & Ostrowski 1990; Łapińska & Szaniawska 2006). Our studies show that C. crangon uses this amino acid in various life processes. The development and growth of individuals are significantly related to the chemical composition of a protein that builds their tissues. The age and size of an organism is a factor strongly correlated with the hydroxyproline level in tissues. Usually, the rate of collagen synthesis in cells is inversely proportional to age and is associated with growth hormones and the presence of ascorbic acid, which in turn has a significant effect on the enzyme catalyzing the production of hydroxyproline (Touhata et al. 2000; Kavitha & Thampan 2008). The hydroxyproline content in males and non-ovigerous females of C. crangon from the Gulf of Gdansk was found to increase with body length. Changes in the hydroxyproline level in the length class of 21 to 40 mm of these shrimps likely result mainly from collagen synthesis during their growth. On the other hand, the clear decrease in the hydroxyproline content in males from the largest length class (41.0–50.0 mm) is probably the effect of senescence. Morales et al. (2000) observed a higher level of collagen in adult compared to immature cephalopods Todaropsis eblanae, which could indicate intensive collagen production during sexual maturation, growth and development of the animal. Muyonga et al. (2004) also recorded higher hydroxyproline levels in adult compared to immature Nile perch (Lates niloticus). Akel (1981) observed an increase in the level of collagen formed with age and size of the shrimp Pandalus jordani, however, it contained very limited amounts of hydroxyproline. In the present work, it was found that hydroxyproline levels in ovigerous females of C. crangon decreased with body length. It has to be emphasized, however, that ovigerous females were not present in the length classes smaller than 31.0–40.0 mm, since they do not become sexually mature until they reach the age of two, and the length of 30–55 mm (Lloyd & Yonge 1947; Tiews 1970; Oh & Hartnoll 2004).
It was once believed that changes in the content of hydroxyproline, and hence of collagen in aquatic
invertebrates, were primarily associated with the age and size of animals (Kavitha & Thampan 2008). It appears, however, that collagen probably participates in a variety of processes, including maturation and egg production. Morales et al. (2000) demonstrated a significant effect of the sex on collagen levels in the cephalopods T. eblanae and Eledone cirrhosa, in which these levels were higher in females than in males. Touhata et al. (2000) pointed out that during the reproductive period, sex hormones increase the rate of collagen synthesis in the body. In the case of C. crangon from the Gulf of Gdansk, this may indicate a much higher level of hydroxyproline in ovigerous females than in males and non-ovigerous females. In some fish species, collagen levels decrease in both males and females during the reproductive period, although in this context it is very important to know whether the fish die after spawning (Toyohara et al. 1997).
Since the population of C. crangon in the Gulf of Gdansk is homogeneous, notwithstanding sexual dimorphism and differences resulting from reproductive processes, this region has no significant effect on hydroxyproline levels. However, there is a correlation between the hydroxyproline content and the depth at which the shrimps live. Hydroxyproline levels, both in males and non-ovigerous females, increased with depth in the Gdynia profile. In the Sopot profile, the hydroxyproline content in males increased with depth, and in non-ovigerous females – decreased with depth. Presumably, the food conditions affected these amino acid levels to a greater extent than water temperature.
The hydroxyproline content in C. crangon from the Gulf of Gdansk depends on the season, as these shrimps migrate between the coastal zone and the deeper waters of the Gulf. These movements are governed by the availability of food, temperature and oxygenation of water, as well as the reproductive period (Łapińska & Szaniawska 2006). Water temperature is often a factor responsible for changes in a population and affects the life cycles of organisms. In aquatic organisms, a high level of hydroxyproline is directly responsible for the lower susceptibility of collagen to denaturization (Brauer et al. 2003). In both males and females of C. crangon from the Gulf of Gdansk, high levels of hydroxyproline were found at the beginning of the season, in April and May. The lowest water temperature was observed in April 2008. This is the time when shrimps prepare for reproduction, while the abundant food available promotes individual development in the population, increases in the body size and the storage of substances in the tissues to be used in the coming months. The dietary composition of these animals may be of great importance. Research by Sato et al. (1978) has shown that a diet poor in ascorbic acid leads to a decrease in the level of hydroxyproline, and hence, of collagen. Ovigerous females do not appear before May, but then they have the highest concentrations of hydroxyproline in their tissues. This significantly affects the total amount of hydroxyproline in this species at that time. It decreases at the end of the season in August, which does not correspond with the measured water temperature – the highest in the entire study period.
It may be concluded that hydroxyproline and thus collagen participates in different life processes of C. crangon. Male individuals use this amino acid mainly in the body growth, whereas females – both in the body growth and the reproductive period. Our studies show that the hydroxyproline level in C. crangon is correlated with the sex and the size of individuals. The hydroxyproline content increased with body length in both males and females (except for ovigerous females). Moreover, the hydroxyproline levels in C. crangon are seasonally dependent. The highest hydroxyproline levels are found in spring, due to the maturation processes in specimens and food availability. Despite numerous reports in the world literature on the presence and properties of hydroxyproline and thus collagen in invertebrates, there are no reports on the amino acid content of collagen in Baltic invertebrates, especially representatives of the macrozoobenthos. These are the first studies that determine the level of hydroxyproline in C. crangon from the southern Baltic, depending on biotic and abiotic factors.
