Chlorophyll-a (chl-a) measurements were made according to the spectrophotometric method (Parsons et al., 1984). For the determination of chl-a content, 1 liter of water was sampled monthly from the specified stations. The sample was then filtered through GFC filters with a pore size of 1 µm. The filter papers were folded and placed in 15 mL centrifuge tubes and 10 mL 90% acetone solution were added to the centrifuge tubes. They were kept in the refrigerator at 4 ^oC for 24 hours. Then the samples were brought to room temperature and their absorbances were determined at 750, 664, 647, and 630 nm wavelengths by means of a spectrophotometer (SHIMADZU UV 2100).

Lipid extraction was performed on the separated muscle tissue samples. The weight of each sample was determined with a precision of 0.001 mg of wet weight (WW). The wet weight of each sample was about 1-2 g. Hexane/isopropanol (3/2) was used for lipid extraction as suggested by Hara and Radin (1978). For fatty acid transmethylation, 20 g methanolic sulfuric acid were mixed with 1 liter of distilled water to prepare a 2% methanolic sulfuric acid solution. Five mL of this solution were added to a test tube and completely mixed by vortex. The mixture was left to methylate in an oven at 55 ^oC for 15 hours. At the end of this period, 5 mL of 5% NaCl were added to it and mixed thoroughly. Afterwards, 5 mL of hexane were added to the fatty acid methyl esters formed in the tubes, and the tubes were mixed (Christie, 1992). After waiting three hours at room temperature, the hexane phase formed was taken from the top, 5 mL of 2% KHCO₃ solution were added to the tubes and the sample was dried in a nitrogen evaporator (Allsheng WD-12). A weight measurement was taken on a precision scale to determine the dry lipid content after the evaporation process, and the average total lipid content (%) per individual was calculated. Then, the dry lipid layer was hydrated with the addition of 1 mL hexane, and then vortexed. The samples were transferred to 2 mL capped autosampler vials and analyzed in a gas chromatograph/mass spectrometer (GC/MS) system.

The fatty acids were analyzed with a GC/MS system (Agilent 5975 C). A Macherey-Nagel (Germany) capillary column (100 m x 0.25 mm, 0.25 μm) was used for the analysis. The column temperature was kept at 120-220 °C; whereas the injection temperature was 240 °C and the detector temperature was 280 °C throughout the analysis. The column temperature program was set from 120 to 220 °C. The temperature ramp was set at 5 °C / min up to 200 °C and at 4 °C / min from 200 to 220 °C. It was held at 220 °C for 8 min and the total time was 52 min. Helium (0.5 ml/min) was used as carrier gas. The fatty acid methyl esters (FAMEs) of the samples were identified initially based on the retention time of each fatty acid by using the analytical standard of FAMEs (Supelco Component FAME Mix). After analysis, wsearch32 mass spectrometry software (Wsearch 2008; version 1.6 2005, Sidney, Australia) was used to confirm the peak identities of each fatty acid.

The effect of location difference was observed to be significant, albeit only slightly, in the change in the fatty acid composition of G. rufa (ANOSIM-R=0.27). The season factor was partially significant in the fatty acid composition of the fish at both stations. However, seasonal variation was more prominent at the Ilıcalar station (ANOSIM-R=0.21) than at the Garip station (ANOSIM-R=0.07). The ANOVA test (TUKEY HSD) results showed that monthly and seasonal changes were significant for total polyunsaturated fatty acids (∑PUFA), 18:0, 22:6ω3 (docosahexaenoic acid, DHA), 20:1ω9 and 20:4ω6 (arachidonic Acid, ARA) at the Ilıcalar station (Tables 2 and 3) and total monounsaturated fatty acids (∑MUFA), 14:0, 20:5ω3 (eicosapentaenoic acid, EPA), 18:1ω9 and 18:0 at the Garip station (Tables 2 and 3). The pair-wise test results, PERMANOVA, revealed that the difference in FA composition was significant between spring and winter at the Ilıcalar station, and between spring and autumn in the Garip station (P _perm =0.002). The SIMPER results revealed that the average highest similarity was in winter at the Garip and Ilıcalar stations with close values (88, 89%, respectively). The gender difference in fatty acid composition was not significant at the stations because gender formed barely-separated groups.

Figure 2 shows a two-dimensional configuration plot of the PCO analysis of a resemblance matrix of fatty acids in G. rufa collected from different locations (Garip and Ilıcalar). The fish samples from the Ilıcalar station were characterized by 18:1ω9, EPA, DHA and 18:0 fatty acids; whereas those from the Garip station were characterized by 18:1ω9 and 22:1ω9. The stations formed separate but overlapping groups for fatty acid composition (ANOSIM-R=0.27), and differences between the stations were not significant.

The major SFA in all factor groups were 16:0 and 18:0 (Table 2). Similarly, these two fatty acids were reported by Guler et al. (2008) as the major fatty acids in Cyprinus carpio, ranging from 14.6 to 16.6% in all seasons. Misir et al. (2013) reported that 16:0 was the prominent SFA contributing to approximately 60% of ∑SFA, followed by 18:0 for Chalcalburnus tarichi. 18:1ω9 was the main MUFA in all muscle tissues of nine freshwater fish species collected from the Tigris River (Turkey). In addition, 18:1ω9 was recorded as the predominant fatty acid in Cyrinus carpio for all seasons (15.1-20.3%) (Guler et al., 2008). The SIMPER results also showed that the FAs which contributed the most to the similarity between the Garip and Ilıcalar stations were 18:1ω9 (28%), 16:0 (22%) and DHA (9%), respectively. It was determined that 18:1ω9 was the most significant contributor to FAs for all seasons and genders at both stations (Table 3).

Oleic acid, 18:1ω9 is the subrate for two important desaturases, Δ12 and Δ15, which are only available from primary producers. These enzymes enable the conversion of 18:1ω9 to 18:2ω6 (linoleic acid, LNA) and 18:3ω3 (linolenic acid, LA). Animals obtain these two essential fatty acids from their diet rather than replacing other fatty acids (Dalsgaard et al., 2003). It is known that G. rufa prefers phytoplankton in its diet especially, Bacillariophyceae members from Chrysophyta (Demirci et al., 2016). Yalcin-Ozdilek and Ekmekci (2006) reported that Chrysophyta members were abundant in all seasons in the diet of G. rufa in the Asi River, Turkey. Moreover, 18:1ω9 is used as a characteristic fatty acid marker for Cryptophyceae, together with Dinophyceae and Chlorophyta (Dalsgaard et al., 2003). The percentage of abundance of other types of food in the diet of G. rufa varies depending on the season (Yalcin-Ozdilek and Ekmekci, 2006).

Guler et al. (2008) reported that the PUFA content in Cyprinus carpio fillets differed in each season and was 39% in spring, 43% in summer and 36% in autumn. They indicated that DHA was the major PUFA for Cyrinus carpio in summer and winter. DHA plays an important role in adaptation processes. When fish are exposed to low water temperatures, PUFA content increases (Lavens et al., 1999). Similarly, the highest ∑PUFA content in G. rufa (34%; 35%, respectively) was observed to be found in the lowest temperatures detected during the sampling period, which were in winter (8.03 °C) at the Garip station and in spring (12.63 °C) at the Ilıcalar station. The PUFA content was higher in the fish samples from the Garip station (Table 2) than from the Ilıcalar station (Table 3). This is thought to be related to temperature because the temperature of the Ilıcalar station was higher than the Garip station during the sampling period (Table 4).

The fatty acid marker for diatoms (Bacillariophyceae) is EPA and for dinoflagellates (Dinophyceae) is DHA (Viso and Marty, 1993). EPA and DHA contents vary among/within species depending on environmental factors such as diet and habitat, as well as whether the fish are wild or farmed (Tocher, 2010). The fact that 18:1ω9, EPA and DHA are the most abundant fatty acids may indicate that G. rufa prefers the members of Cryptophyceae, Dinophyceae, and Chlophyta as food sources in the Murat River. Moreover, the 16:1ω7/16:0 ratio has been used to infer a diatom-dominated food chain base (Auel et al., 2002). While 16:1ω7 is found in cyanobacteria, dinoflagellates, and a specific isomer, the trans one, is found in bacteria, 16:1ω7 is most prevalently associated with diatoms (Parrish, 2013). The 16:1ω7/16:0 ratio varied from 0.02 to 0.08% (summer-autumn) at the Ilıcalar station and 0.01-0.03% (autumn-winter) at the Garip station. These results showed that G. rufa at consumed more diatoms the Ilıcalar station than those at the Garip station during the sampling period. However, 18:1ω9, EPA and DHA were the most abundant dietary fatty acids in G. rufa. Yalcin-Ozdilek and Ekmekci (2006) showed that G. rufa’s main diets primarily comprised diatoms found in the Chrysophyta. However, the fatty acid composition detected in the present study indicated there were more dinoflagellates than diatoms in G. rufa’s diet. Freshwater fish cannot synthesize certain fatty acids, especially C₁₈ PUFA, such as 18:2ω6, 18:3ω6, although they can directly ingest many LC-PUFAs such as ARA, DHA, EPA from their prey (Tocher, 2010). Dietary EPA, DHA, and ARA improve reproductive success and increase the quality of broodstock eggs (Mazorra et al., 2003). They are critical to the general health of organisms and most consumers synthesize them inefficiently from their precursors (e.g., 18:3ω3 or ALA and 18:2ω6 or LNA). Therefore, EPA, DHA, and ARA are considered essential dietary FAs in aquatic ecosystems (Dalsgaard et al., 2003; Parrish, 2009). 18:3ω3 is also higher in freshwater herbivorous Cypriniformes, including G. rufa. 18:3ω3 was one of the highest PUFA at both stations during the sampling period (Tables 2 and 3). It is stated in many studies that terrestrial plants abundantly synthesize 18:3ω3 and 18:2ω6, which are also used as dietary marker in the fatty acid composition of aquatic organisms (Parrish, 2013). ARA was more prominent in the fish from the Ilıcalar station than those from the Garip station for PUFA in the PCO analysis (Figure 2). Freshwater fish have relatively high contents of 18:2ω6 and ARA, which are indicative of freshwater algae and terrestrial dietary sources (Parzanini et al., 2020). These fatty acids were present in significant percentages in the fatty acid composition of G. rufa. From the fatty acid values, it was deduced that G. rufa also preferred diets of terrestrial origin.

C₁₃, C₁₅, C₁₆ and C₁₇ SFA and MUFA and their isomers, as well as 18:1ω6 characterize bacterial fatty acids (Dalsgaard et al., 2003; Parrish, 2013). Therefore, the presence of these fatty acids in fish tissue may indicate a bacterial diet. The results of the present study showed that G. rufa fed on bacteria (Tables 2 and 3). However, terrestrial markers outnumbered both bacterial and zooplanktonic markers at both stations during the sampling period (Tables 2 and 3).

The C₂₀ and C₂₂ group zooplankton fatty acids were in very small pergentages in G. rufa, which were found in a smaller amount in the fish samples from the Garip station than in those from the Ilıcalar station. These fatty acids characterize copepod species and are used in the analysis of marine fish to reveal nutritional relationships (Iverson et al., 2009). These fatty acids were observed in G. rufa in winter as well as other seasons at the Ilıcalar station, and not in winter at the Garip station. G. rufa preferred zooplankton as food in winter months, because the Chl-a content was found to be lower at the Ilıcalar station (1.19 µg/L) than at the Garip station (2.55 µg/L) in winter. The highest Chl-a content was detected in summer at both stations. It was higher at the Garip station (5.81 µg/L) (Table 2) than the Ilıcalar station (4.18 µg/L) in summer (Table 3). In the periods when Chl-a was abundant (Table 4), G. rufa tended toward an herbivorous diet, which is their main diet.

The w3/w6 ratio is greater in herbivorous freshwater fish (Parzanini et al., 2020). The w3/w6 ratio varied between 3-5 at the Ilıcalar station (Table 3) and 4-7 at the Garip station (Table 2). It reached the highest value in August at 6.60 at the Garip station (Table 2). The Ilıcalar station included zooplanktonic FAs at higher levels than the Garip station. Phytoplankton were probably more abundant than zooplankton at the Garip station, and G. rufa preferred phytoplankton, which is the primary food source in all seasons at the Garip station.

The results of seasonal changes in dietary fatty acid levels showed that the fatty acid composition of G. rufa changed according to the change in nutritional content and variety depending on the season. There are already many approaches and studies showing that fatty acids give clues about the food consumed. All these approaches can provide valuable information about consumer nutrients and food ecology in complex aquatic ecosystems. Each of these approaches is evaluated and used in studies according to the content of the study (Dalsgaard et al., 2003). FAs are known indicators of specific food sources, the results can indicate the diet of consumers and are a potentially powerful trophic measures which reflect what is included in an individual’s diet over a period of several weeks (Kirsch, 1998). From this point of view, it would not be wrong to say that G. rufa predominantly feeds on plants, but can also feed omnivorously. It can be stated that G. rufa has a very wide food preference from bacteria to zooplankton. Demirci et al. (2016) emphasized similar results in their study on the nutritional characteristics of G. rufa from organisms in stomach-intestinal contents. G. rufa can feed on various plankton species, although they prefer phytoplankton (Demirci et al., 2016). It was also found in the present study that the results of dietary fatty acids indicated the same results.