Interactions of season , sex and size on nutrient composition of freshwater crayfish ( Astacus leptodactylus Eschscholtz , 1823 ) from Lake Eğirdir

Astacus leptodactylus is naturally and widely distributed in lakes, ponds and rivers throughout Turkey (Harlioğlu & Harlioğlu, 2004). On recent years, there has been a gradual decrease in the production of crayfish in Turkey from 5000 tons in 1984 to 532 tons in 2015 (Turkish Statistical Onstitute, 2017). The greatest cause of this decline is crayfish plague fungus (Aphanomyces astaci) seen after 1985 (Baran et al., 1987; Rahe & Soylu, 1989; Harlioğlu, 2008). There is no crayfish culture in Turkey and all production is obtained from wild harvests (Harlioğlu et al., 2012). Therefore, culture of crayfish is needed to replenish native stocks of A. leptodactylus.


Introduction
Astacus leptodactylus is naturally and widely distributed in lakes, ponds and rivers throughout Turkey (Harlioğlu & Harlioğlu, 2004).On recent years, there has been a gradual decrease in the production of crayfish in Turkey from 5000 tons in 1984 to 532 tons in 2015 (Turkish Statistical Onstitute, 2017).The greatest cause of this decline is crayfish plague fungus (Aphanomyces astaci) seen after 1985 (Baran et al., 1987;Rahe & Soylu, 1989;Harlioğlu, 2008).There is no crayfish culture in Turkey and all production is obtained from wild harvests (Harlioğlu et al., 2012).Therefore, culture of crayfish is needed to replenish native stocks of A. leptodactylus.
The knowledge of nutritional requirements in decapods is crucial to successful culture (Moreno-Reyes et al., 2015).According to Dempson et al. (2004), the proximate body composition (moisture, lipids, protein and ash) is a good indicator of the nutritional status of an organism.There is not a study about interactions of season, sex and size on nutrient composition of crayfish.This investigation provides basic information about nutritional requirements of the species in recovering natural populations.

Sample collection
Crayfish were supplied by the Egirdir Lake, Osparta, Turkey.They were collected by trapping from their natural habitat at 4 seasons and size range (7.1-9.9 cm).Total of 335 crayfish were caught in autumn (Dctober), winter (January), spring (April) and summer (July) (2014)(2015).The crayfish were separated by sex and weighed on an electronic scale (0.001 g) in the laboratory (Table 1).Total lenght was measured from the rostrum to the end of the telson.Crayfish were anaesthetized by placing them at freezer (-80 ± 1 °C) for 30 min.The abdominal muscle were removed and stored in a freezer at −80 °C until they were used for the biochemical analyses.

Proximate composition
Moisture contents were detected with an automatic moisture analyzer (AND MX-50).The crude protein contents according to Kjeldahl method (Nx6,25) (Association of Dfficial Analytical Chemists, 2000), crude lipid contents by Bligh & Dyer (1959)'s method and crude ash contents according to ADAC (Association of Dfficial Analytical Chemists, 2006) were done.

Fatty acid analysis
The operating conditions of the GC-MS (GC Clarus 500 with autosampler (Perkin Elmer, USA): Column was SGE (60 m x 0.25 mm OD.BPX5.0.25 μm USA), the oven temperature was maintained at 60 °C for 10 min and increased to 220 °C at a rate of 4 °C/min.The oven temperature was maintained at 220 °C for 10 min, then increased to 250 °C at a rate of 4 °C/min and maintained at 250 °C for 10 min, the Carrier gas was Helium (1.5 mL/min), and Onjector temperature was 240 °C, Split ratio was 1:100, Mass spectra was 70 eV, and Mass range was 35-425 m/z.Fatty acids were identified by comparing the retention times of FAME (catalogue number 18919; Supelco) with the standard 37-component FAME mixture.

Statistical analysis
The data obtained were analyzed in terms of specifications elaborated by analysis of factorial design ANDVA using the SPSS 13.0 computer program (SPSS Onc., Chicago, USA) techniques.On the factorial analysis; 4 levels of season factors (autumn, winter, spring, and summer), sex factor (male, female) and size factor (7.1-9.9 cm) were available.Tukey test was used to determine the differences between the average levels of the factor.

Proximate compositions
Binary or triple interaction was not obtained when crude protein, crude ash and moisture analysis results were evaluated in tail meat of A. leptodactylus.Alone the season significantly affect these nutrient compositions (p < 0.05).The highest protein contents were found in spring and summer (p < 0.05).The highest ash content was determined in the autumn while the lowest ash content was found in the winter (p < 0.05).The highest moisture contents were determined in the spring and the winter while the lowest moisture content was found in the summer (p < 0.05) (Table 2).Any an interaction was not found for crude fat contents of tail meat of A. leptodactylus.Crude fat means in tail meat of A. leptodactylus were given in the Table 3.Also, the sex factor was a significant affect over crude ash contents (p < 0.05) (Table 4).

Crude protein contents
On this study, effect of season only was determined on crude protein contents in tail meat of A. leptodactylus.The highest crude protein content (15.90%) was detected in summer samples and similar result was obtained in spring, as well.The lowest protein content was detected in the winter and similar result was recorded in the autumn (Table 2).Similarly to the present study, Berber et al. (2014) reported that the highest protein contents were assigned in both sexes in tail meat of A.leptodactylus in summer.But, Berber et al. (2014) did not research differences between sexes.Silva-Castiglioni et al. ( 2007) indicated that total muscle proteins were high during summer in both sexes of Parastacus varicosus and there was no difference between the sexes during the year.Onanlı & Çoban (2007) and Bilgin et al., (2008) did not determine difference between sexes in terms of protein contents in tail meat of A.leptodactylus.The protein contents in tail meat of A. leptodactylus were determined as 15.98% male and 16.39% female in (Onanlı & Çoban 2007), 14.61% in (Harlioğlu et al., 2012), 16.32% in (Çoban et al., 2012), in 15.23-16.66% in (Duman et al., 2012), male (15.68%) and female (17.59%) in (Berber et al., 2014), 15.41% in (Dksuz & Mazlum 2016).The reason of differences in protein contents in these studies can be habitat different, environmental factors and nutrient availability (Winberg, 1956).A. leptodactylus feeds as active from spring to early autumn (Alpbaz, 1993) therefore the protein contents in their muscles are high in summer (nutrient-rich in high temperature).The protein content in their muscles loses during starvation period in winter (Dall, 1981;Rosa & Nunes 2003).A. leptodactylus completes its gonad development in the autumn and mates in winter (Alpbaz, 1993).For this reason, the protein, fat and energy are transferred to the when crayfish reaches maturity (Güner & Mazlum, 2010).Also, Abdu et al. (2002) determined that there is an increase in the protein composition in the gonad as a result of transfer of energy from hepatopancreas.

Crude lipid contents
On the present study, no interaction was observed in crude lipid contents of tail meat of A. leptodactylus (Table 3).Similarly to the present study, Onanlı & Çoban (2007) and Silva-Castiglioni et al. (2007) did not determine difference between sexes.Also, Buckup et al. (2008) did not found seasonal differences in lipids contents in muscle tissue of P.defossus.Contrastly, Berber et al. (2014) determined seasonal differences in tail meat lipid contents of A. leptodactylus.Silva-Castiglioni et al. (2007) indicated the highest lipid contents were during winter in both sexes of P.varicosus.Buckup et al. (2008) determined differences between sexes in terms of total lipids content.
There are different reports in terms of crude lipid contents tail meat of A. leptodactylus; 0.57% (Harlioğlu et al., 2012), 1.25% (Dksuz & Mazlum 2016).Bilgin et al. (2008) reported the highest lipid content was 1.91% and the lowest lipid content was 1.09% in different size female crayfish.These differences may be caused by environmental factors, age, species and nutrient in environment.

∑MUFA contents
On the present study, season/sex interaction effected to ∑MUFA.The high ∑MUFA contents in female were found in the spring (19.65%) and summer (19.64%), the lowest ∑MUFA content was in the autumn (17.99%).High ∑MUFA contents in male were found in the autumn (18.35%) and summer (18.46%) and the lowest ∑MUFA content was found in the winter (16.99%).Also, ∑MUFA contents in males were found lower than female in winter, spring and summer (Table 7).Similarly, Stanek et al. (2013) indicated that ∑MUFA contents were higher in female (29.26%) than male (26.61%) in tail muscle of O. limosus from Lake Goplo (Polond).Berber et al. (2014) reported that ∑MUFA reached maximum levels in April and May while stayed as normal in the other months.On contrast, Stanek et al. (2011) reported that ∑ MUFA contents were similar between spring and summer in tail muscle of O. limosus from Lake Goplo (Polond).The reason of this difference may be that two season only were researched this study.Harlioğlu et al. (2012) detected ∑MUFA content as 28.17% in tail muscle of A. leptodacylus.Dksuz & Mazlum (2016) indicated as 24.60% ∑MUFA content in tail muscle of A. leptodacylus.Contrary to our results, ∑MUFA contents were high in previous studies.
∑PUFA, ∑SFA, ARA, EPA and DHA contents On this study, only seasonal differences were determined for ∑SFA, ∑PUFA, ARA, EPA and DHA.The highest ∑PUFA content (34.91%) was in the winter while the lowest content (30.63%) was in autumn.Also, ∑PUFA contents were higher than ∑MUFA, ∑SFA and ∑HUFA.Similarly the present study, Berber et al. (2014) defined that ∑PUFA content in the muscle was higher than ∑HUFA and ∑SFA but ∑PUFA content detected higher in female than that of male in tail meat of A. leptodactylus (Table 5).Similarly, Stanek et al. (2013) indicated no differences between sexes in ∑PUFA contents, however ∑PUFA contents were found similar between spring and summer.Harlioğlu et al. (2012) and Dksuz & Mazlum (2016) determined higher ∑PUFA (45.97%, 53.52% respectively) than ∑SFA and ∑MUFA.Also, they detected higher ∑PUFA contents than that of the present study in tail muscle of A. leptodacylus.
The highest ∑SFA content (22.14%) was found in the autumn in this study (Table 5).Unlike for the present study, Stanek et al. (2011) indicated that there were no difference in SFA contents between spring and summer and SFA contents were difference in between female (22.27%) and male (23.56%) in tail muscle of O. limosus.The cause of this situation may be species and habitat differences.Similarly, Dksuz & Mazlum (2016) determined SFA content as 21.32% in tail muscle A. leptodactylus caught from Egirdir Lake.Harlioğlu et al. (2012) reported SFA content as 25.56% in tail muscle of A. leptodactylus caught from Lake Keban Dam.
On the present study, the highest ARA content (13.42%) was found in the winter and the lowest content (9.70%) was determined in the summer.The highest EPA content (14.27%) was found in the summer and the lowest level (10.11%) was determined in the autumn (Table 5).The high DHA's were observed in the spring (5.34%) and summer (5.40%).The lowest DHA content was determined in the autumn (% 4.43) (Table 5).Similarly, Bottino et al. (1980) reported that ARA content in shrimps decrease in the summer when the water temperature rises.Berber et al. (2014) determined seasonal differences in EPA contents in the female, in DHA contents in the male.Berber et al. (2014) signed that the highest EPA content in the female in November, the lowest in June, the highest DHA content in the male in January -March, the lowest contents DHA content in November-May.They explained this situation with reproductive and gonadal development period of the female, indicated that reproductive was affected directly by DHA content.Whereas, in the present study, both of EPA and DHA contents was low in tail meat of both of male and female in reproductive season.Unlike the present study, Stanek et al. (2011) did not determine differences in point of EPA, ARA, in tail muscle of O. limosus between spring and summer.Bilgin et al. (2008) did not observe differences sexes in DHA contents while Stanek et al. (2013) observed differences sexes.

LOA and α-LNA contents
On current study, differences among seasonal and sex were determined on C18:2n6 linoleic acid and C18:3n3 linolenic acid.The highest linoleic acid content (3.87%) and linolenic acid content (1.14%) were found in the autumn and linoleic acid contents were higher than linolenic acid contents.Also, linoleic and linolenic acid contents were higher in female than male.Similarly, Castell (1983) and Chanmugam et al. (1983) reported that linoleic acid contents were higher than linolenic acid contents in freshwater crustaceans.Dn the contrary, Berber et al. (2014) did not determine differences among seasons in linoleic acid and linolenic acid contents.Stanek et al. (2013) and Bilgin et al. (2008) were not observed differences sexes (Table 6).

Conclusion
The protein content in tail muscle of A. leptodactylus decreases by gonadal development in autumn but increases by feeding in summer.This decreasing continues in food deprivation (winter).However, the lipid contents didn't not show seasonal difference.Ot may be that this species do not prefer fatty foods.DHA, EPA and n-3 contents in tail muscle of A. leptodactylus increase by feeding in summer while these contents decrease by gonadal development in autumn.Therefore, EPA, n-3 and DHA contents are important for A. leptodactylus.According to results of study, the female brood stocks in culture are feed diets with high protein, EPA, DHA and n-3 contents.On addition, nutritional contents in terms of protein, EPA, DHA and n-3 of this species are higher in summer.
Means within a row having different superscripts are significantly different (P ˂ 0.05).

Table 1 .
Mean lenght and weight of samples.

Table 2 .
Seasonal variation of some nutrient composition in tail meat of A. leptodactylus (mean ± S.E.).

Table 4 .
Effect of sex on crude ash in tail meat of A. leptodactylus (mean ± S.E.).
aMeans within a column having different superscripts are significantly different (P ˂ 0.05).

Table 5 .
Effect of season on some fatty acids in tail meat of A. leptodactylus (mean ± S.E.)

Table 7 .
Variation of total ∑ MUFA contents in the tail of A. leptodactylus according to season and sex interaction (mean ± S.E.).
AbThe big letters in the line, different among seasons in same sex; small letters in column, different among sexes in season.

Table 6 .
Effect of sex on some fatty acids in the tail of A. leptodactylus (mean ± S.E.).