Investigation of taxonomic relationship and effect of seasonal temperature changes based on protein profiles of fishes from Beyşehir, Suğla lakes and Dam Apa

Besides traditional methods based on morphological characters, electrophoretic methods such as SDS-PAGE are preferred by taxonomists to make the right decision in the species identification process. In addition, the effect of environmental factors, such as pH, salinity, heat, and temperature on protein profiles are essential in various studies. In this study, we aimed to determine the degree of relationship in some fish species, such as Squalius lepidus, Cyprinus carpio, Carassius gibelio, Pseudophoxinus anatolicus, Tinca tinca, Alburnus orontis, Scardinius erythrophthalmus, Capoeta capoeta, Vimba vimba, Sander lucioperca living in Beyşehir, Suğla lakes and Apa Dam by SDS-PAGE method, and to examine seasonal differences by evaluating the effect of hot/cold water on protein profiles in fish. Although there were common major protein bands in all fish species studied, the presence of species-specific minor protein bands led to the separation of the species. The same fish species distributed in different lakes and dams were different both in minor bands, and changes in protein profiles were observed consequently on the same fish species synthesizing different proteins in different seasons. The data obtained from this study can contribute to systematic classification studies of fish.


Introduction
The family Cyprinidae, represented by 200 genera and 2010 species, constitutes the largest family among freshwater fish (Nelson, 1994;He et al., 2008). Although Cyprinidae members are not found in South America, Australia, and Antarctica, they include many fish with cultural and economic importance . A total of 236 species and subspecies belonging to 26 families are found and it constitutes approximately 8% of all fish species in the inland waters of Türkiye (Kuru, 2004).
Mostly in taxonomic studies, the species identification process is based on morphological and anatomical characteristics (Theophilus & Rao, 1998;Yılmaz et al., 2005).
Some morphological features may subsequently change as a result of environmental conditions (Fowler, 1970;Ganai et al., 2014). During the definitive identification of a species, classical morphological characters can be misleading due to the existence of these changes over time (Menon, 1989;Ganai et al., 2014). For this reason, comparisons based on morphological characters are not sufficient for taxonomists to make the right decision for determining the species (Hua et al., 2019;Şalcıoğlu et al., 2020). Proteins are used as genetic markers that play an important role in determining taxonomic relationships (Crick, 1963;Nirenberg et al., 1963;Ochoa, 1963;Ganai et al., 2014). In previous systematic studies on fish species, successful identifications were made by electrophoresis of serum proteins, and these studies brought a new perspective to taxonomic evolution (Theophilus & Rao, 1998, Yilmaz et al., 2007. The aim of this study to evaluate the degree of relationship of some fish species distributed in Beyşehir, Suğla lakes and Apa Dam according to their total protein profiles by using the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) method and to determine the effects of seasonal changes on the protein profiles of fish. Table 1 indicates the different fish species obtained from Beyşehir, Suğla lakes and Apa Dam, their localities and the seasonal period in which they were obtained.

Protein Isolation
The protocol proposed by Hoffman & Penny (1973) was used, partially modified, for protein isolation from muscle tissue of fish. Five grams of muscle was taken from each fish and thoroughly crushed in a mortar. The shredded muscles were transferred to the falcon tube and 10 ml of distilled water was added. After vortexing, the samples were kept at +4°C overnight. The falcons were thoroughly vortexed the next day and centrifuged for 25 min at 5000 rpm at +4°C in a refrigerated centrifuge (Hettich Universal, Zentrifugen). The supernatant was transferred to a new falcon tube and stored at -20°C until use.

SDS-PAGE Electrophoresis
The SDS-PAGE method was carried out by modifying the method of Laemmli (1970). Before electrophoresis, protein The samples were run in electrophoresis at 36 mA until bromophenol blue, which was used as an indicator, reached the end of the running gel. When the electrophoresis process was completed, the gels were removed from the glass plates and placed in staining cuvettes containing the staining solution (50% methanol, 10% Acetic acid, 0.1% Coomassie Brilliant Blue G-250 M, water). After staining, the gels were washed with a washing solution (5% methanol, 7% acetic acid, water). Finally, images of all gels were taken using a gel imaging system (Vilber Lourmat, France).

Data Analysis
Scoring was done according to the absence (0) and presence (1) of protein bands. Similarities were calculated with the Bio1D++ computer program according to Nei's genetic similarity (Nei, 1978). To construct a dendrogram with the UPGMA (unweighted pair-group method and arithmetic averages) method, the degree of relationship and protein differences in summer/winter months were evaluated by cluster analysis.

Results
Total proteins belonging to fish species used in this study were isolated and SDS-PAGE electropherograms were taken.
( Figure 1)  When we evaluate the seasonal changes within the species, the synthesis of some proteins increased while others decreased.
Other types have minor changes.

Discussion
Many researchers have successfully differentiated fish species using serum protein profiles via SDS-PAGE, isoelectric focusing and two-dimensional electrophoresis methods. In this study, we evaluated protein identification SDS-page method to understand taxonomy of Cyprinid and other Order fishes. The most important finding for this study is that species-specific minor protein bands provide good differentiation between species. In addition, another important finding is that the minor band profile changes even in the same fish species depending on seasonal changes. In the electropherogram obtained from some studies revealed that protein bands are characteristic to the species (Yılmaz et al., 2005;Berrini et al., 2006;Ganai et al., 2014). Yılmaz et al. (2000) examined the serum proteins of 66 Capoeta trutta and 92 Capoeta umbla by SDS-PAGE method. It has been stated that there are taxonomically significant differences between the serum proteins of these two fish species. They reported that these differences were due to differentiation of the genes which synthesizes different proteins.
On the other hand, Yilmaz et al. (2007)  tinca, and C. carpio were separately located in distant clades, consistent with our results. However, contrary to our results, Alburnus alburnus was found to be closer to T. tinca and much farther from C. carpio (Imoto et al., 2013). Consistent with our results, in a phylogenetic tree based on cyt b gene, Alburnus escherichii and Squalius lepidus were closely located in a clade, while C. carpio and C. capoeta were located in the same clade, although not very closely (Durand et al., 2002).  (Tang et al., 2010(Tang et al., , 2011. Similarly, in another phylogenetic analysis based on mitochondrial 16S rRNA, contrary to the results of our study, it was shown that C. carpio and Carassius carassius species, and Leuciscus genus and Gobio genus are closely related each other (Li et al., 2008). Similar to current result, Imoto et al. (2013) found the Cobitis striata to be the most distant species from other Cyprinidae family members.
It is known that environmental conditions lead to changes in the amount and number of proteins, which are the expression products of the gene. It has been stated that Native-PAGE and SDS-PAGE methods are useful in separating the proteins of fish samples whose structure changes as a result of exposure to high pressure (Etienne et al., 2001). Muhammad et al. (2018) analyzed the liver proteins of three different fish species using the SDS-PAGE technique and the similarities and differences between the species were determined. In addition, according to the results of their studies, they suggested that the SDS-PAGE method could be used to examine the toxicological aspects of the species (Muhammad et al., 2018). Another group of researchers were able to distinguish processed fish samples using different staining methods of SDS-PAGE . Tokur & Kandemir (2008) Abolfathi et al. (2022) found the presence of proteins with molecular weights ranging from 7-224 kDa in a study in which the seasonal changes in skin-epidermal structure and mucosal immunity parameters of the skin of O. mykiss were examined by SDS-PAGE method. They also reported that there were noticeable differences between the number and size of protein bands in seasonal changes. In the study, they reported that small proteins with a molecular weight of less than 35 kDa were found in high proportion in the late summer and spring, while proteins with a larger molecular weight (> 35 kDa) were clearly observed in the winter season. Similarly, in our study, the synthesis of some proteins increased in the winter season, while others decreased in the summer season (for example: Figure 1 indicated by the arrow). This change was most prominently observed in S. lepidus species.

Conclusion
Although the electrophoretic results of the proteins are incompatible with DNA-based methods, in taxonomic studies, it allows successful differentiation at the level of species and higher characters, as well as the determination of seasonal changes in protein number and amount.
The presence of species-specific protein bands in this study led to the determination of the degree of relatedness. In addition, it was determined that protein profiles changed according to seasonal changes in the same fish species. It was observed that this change was quite evident in the protein profile of S. lepidus. In further studies, specific protein determination and quantification by methods such as Westernblot or typing by methods such as peptide mapping can be performed.