In this study, the chromosome numbers and karyotypes of two marine fish species, representing two families, living off the south coast of the Black Sea were successfully determined using the short-term culture method (PB-MAX™ application). Although the culture incubation periods, depending on postmortem timing, are non-identical for each species, the kidney tissues of the fish samples were treated in Falcon tubes filled with PB-MAX™ for 3–4 h. Subsequently, hypotonic and fixation procedures were applied consecutively, and then spreading was done by dropping the cell suspension onto steamed, clean slides. Giemsa staining, C-banding, and Ag-NOR banding were applied to the preparations. We determined that the Mediterranean horse mackerel, Trachurus mediterraneus (Steindachner, 1868), 2n = 48, has 6 metacentric, 16 submetacentric, 18 subtelocentric, and 6 acrocentric chromosomes (NF–70); while the black scorpionfish, Scorpaena porcus Linnaeus, 1758, 2n = 42, has 4 metacentric, 4 submetacentric, 10 subtelocentric, and 24 telocentric chromosomes (NF–50). Trachurus mediterraneus represents the family Carangidae, while Scorpaena porcus belongs to the family Scorpaenidae. In conclusion, while the number and location of NOR and C+ chromosomes in both species differed from previous studies, in the presently reported study we also observed similarities in the 2n counts. The differences in karyotypes were also observed, in comparison to other studies. This study, using image analysis software, updated information on the chromosomal structures of both species. Thus, the in vitro method used has proven to eliminate the problem of failing to obtain high-quality chromosomes in marine fish, making it undeniably the most efficient and economical short-term culture method. Furthermore, it can be predicted that enabling the exposure of chromosomes from even post-mortem fish samples will provide advantages and time savings, as well as facilitate the easy, rapid, and high-quality chromosomes required for further molecular cytogenetic studies. This study aims to pave the way for chromosome research on sea fish to be conducted in a shorter timeframe with practical approaches. It also seeks to revise the karyotypes of the two marine fish revealed by this short-term cell culture.

chromosome, marine fishes, Scorpaena porcus, short-term culture, Trachurus mediterraneus

In many vertebrate groups, the study of chromosome morphology, number, structure, and genome size, along with the analysis of mitochondrial and nuclear gene sequences, has helped to resolve challenges in understanding fish biology, systematics, and evolution. However, fish, the most diverse vertebrate group, have traditionally been classified based on higher taxa, with much less cytogenetic information compared to morphology and paleontology. This is partly because karyotypes can only be obtained from living specimens, tissues, or cells, making it difficult to study the karyotypes of fish that are hard to collect and keep alive (e.g., deep-water, pelagic, fast-swimming, long-range migrant sea fishes). Today, researchers have published information on the chromosome structures of nearly 4000 fish species (Arai 2011) out of 37 408 fish species (Fricke et al. 2025). It has been reported that approximately 552 marine and brackish water fish species inhabit the coasts of Türkiye (Bilecenoglu 2024), as well as 427 freshwater fish species in inland waters of Türkiye (Cicek et al. 2023). Since the first known academic study in Türkiye conducted by Gul (unpublished1), the chromosomes of 103 fish species have been examined (Saygun 2021). Although the number of scientists working in fish cytogenetics is decreasing, chromosome analyses are performed on dozens of fish species each year, providing insights into fish taxonomy. The Mediterranean horse mackerel, Trachurus mediterraneus (Steindachner, 1868), one of the three horse mackerel species in Turkish waters (Bektas and Belduz 2008), represents the family Carangidae, which includes 153 species found in the eastern Atlantic from the Bay of Biscay to the Mediterranean, including Mauritania (Froese and Pauly 2025; Fricke et al. 2025). Karyological studies have been conducted on 34 species (Arai 2011; Jacobina et al. 2016) within the family Carangidae, which contains 14 valid species in the genus Trachurus worldwide (Froese and Pauly 2025). Additionally, chromosome morphology data are still available for three of additional species: Trachurus mediterraneus, Trachurus japonicus (Temminck et Schlegel, 1844), and Trachurus trachurus (Linnaeus, 1758). The second family represented in our study—the Scorpaenidae (order Perciformes)—includes 387 species (Fricke et al. 2025). As a result of cytogenetic studies (Arai 2011), chromosome information is available for 36 species out of the valid 63 species belonging to the family Scorpaenidae (see Froese and Pauly 2025). The black scorpionfish, Scorpaena porcus Linnaeus, 1758, is one of the six valid scorpaenid species living in Turkish marine waters (Yedier and Bostanci 2022), as studied in this research.

Advances in genetics during the last quarter of the twentieth century led to significant developments in fish karyology and cytogenetics. The widespread use of DNA gene markers (MM, DAPI, CMA3, etc.) and probes in fluorescent staining greatly improved in the 1980s, replacing traditional sequential staining methods. Pinkel et al. (1986) pioneered the era of FISH staining with probes they designed for the 45S rDNA locus. Today, many variations of the technique exist, providing valuable tools for collecting important data, especially in medical genetics and fish evolution. The use of monoFISH (mFISH) staining has since diversified and expanded. Currently, in medical cytogenetics and zoology, molecular cytogenetic techniques such as dual-color FISH (D-FISH), M-FISH (multicolor spectral karyotyping), and other chromosome mapping methods are used to detect interspecies and interpopulation structural and numerical chromosome rearrangements, sex chromosome variations, polymorphisms in active or inactive heterochromatin and NOR regions, and numerical differences. These methods can also provide deeper insights into gene regulation systems that influence heredity (Rossi 2021). To better understand the results of the widely used D-FISH method in fish cytogenetics, some of its applications in Carangiformes and Scorpaeniformes include: mapping 18S rDNA and 5S rDNA to examine the relationship between body shape and genetic makeup in carangid species of the genus Selene: Selene brownii (Cuvier, 1816), Selene setapinnis (Mitchill, 1815), and Selene vomer (Linnaeus, 1758), found along the coasts of Rio Grande do Norte State in Northeastern Brazil. It was found that morphological differences emerged between individuals carrying these two loci together on a chromosome during speciation (Jacobina et al. 2013). The co-occurrence of these loci on a chromosome pair also contributed to body shape polymorphism in the related Caranx lugubris Poey, 1860 (Carangiformes) populations from two nearby regions in Saint Pedro and Saint Paulo Archipelago (SPSPA) in the central Atlantic (Jacobina et al. 2014). Four different carangid species—Elagatis bipinnulata (Quoy et Gaimard, 1825), Seriola rivoliana Valenciennes, 1833, Gnathanodon speciosus (Forsskål, 1775), and Trachinotus carolinus (Linnaeus, 1766)—are migratory reef species with wide distributions, sharing the same chromosome number (2n = 48) and very similar karyotypes. However, differences among species exist in chromosomes containing NOR regions where the 5S rDNA loci are located. This influences the migration ability of these populations and helps explain their widespread presence across the Atlantic, Pacific, and Indian Oceans (Soares et al. 2021). Another study showcasing microevolution within populations found that two gene loci coexist on a chromosome pair in populations of the invasive lionfish Pterois volitans (Linnaeus, 1758) (Scorpaeniformes), distributed along the coasts of the Indian Ocean (Java), Costa Rica, and Venezuela, with this pattern differing from original Indian Ocean populations (Nirchio et al. 2014b). Additionally, Nirchio et al. (2016b) found, through classical and molecular cytogenetic analyses, that Scorpaena plumieri Bloch, 1789 sampled in the Caribbean exhibited two distinct cytotypes.

The D-FISH technique, which can detect even microstructural rearrangements in chromosomes, has also been applied to other fish species: Detection of karyotype differentiation in scombriform species: Ruvettus pretiosus Cocco, 1833 and Promethichthys prometheus (Cuvier, 1832), that are geographically distributed in distant regions and have the same chromosome number (2n = 48) (Sousa Santos et al. 2024); determination of the relationship between karyotype differentiation and the effect of post-reproductive barrier formation in the family Epinephelus striatus (Caribbean Sea), Epinephelus coioides (Hamilton, 1822) and Epinephelus tauvina (Forsskål, 1775) (Indo-Pacific Region) of the Epinephelidae (Amorim et al. 2024); observation of geographical karyotype differentiation in 19 studied coral butterfly fish species (Chaetodontidae) (Molina et al. 2024). The same molecular cytogenetic methods were used to determine the karyotype diversities among three different species: Kyphosus sectatrix (Linnaeus, 1758), Pempheris schomburgkii Müller et Troschel, 1848, and Amblycirrhitus pinos (Mowbray, 1927), distributed in the Atlantic and Stygnobrotula latebricola Böhlke, 1957 spread across the Pacific (Molina et al. 2025).

In this study, we evaluated a short-term in vitro method that can be used even with dead fish and was developed to address challenges in directly determining chromosome structures in marine fish that cannot be kept alive for long periods. We employed sequential staining techniques such as conventional NOR and C-banding to update the cytogenetic data of two species from different fish groups, Trachurus mediterraneus and Scorpaena porcus. Additionally, we aimed to demonstrate the effectiveness of this method in producing high-quality chromosomes through molecular cytogenetic techniques to identify previously undetected genetic markers involved in the evolution of these two species, which we plan to explore further in future research.

This study was based on two randomly obtained fish samples by professional and amateur fishermen at two sites (41°03.51′N, 037°30.50′E and 41°02.45′N, 37°31.42′E) in the Fatsa Bay of Ordu Province (Fig. 1). Based on the decision (Number: 82678388 and Date: 26 October 2021) issued by the Animal Experiments Ethics Committee of the Ordu University, this research is not subject to AEEC permission and therefore does not require an ethical approval document. For this purpose, random sampling was carried out between February and November 2023. The chromosome structures of seven Mediterranean horse mackerel (3 male, 4 female) and four black scorpion fish samples (2 male and 2 female individuals) were determined. In the study, the species were determined in the light of the taxonomic information provided by Aksiray (1987), Bat et al. (2008), Bilecenoglu et al. (2011), and Nelson et al. (2016). While the fish samples were fresh for at most four hours during the postmortem period, the tissues obtained were transported to the Fatsa Faculty of Marine Sciences biochemistry laboratory without delay and placed in a culture environment.

Figure 1. 

Random sampling sites of Trachurus mediterraneus and Scorpaena porcus in the Black Sea off the coasts of Türkiye.

As suggested by Araya-Jaime et al. (2021), better results were obtained from the kidney. The first samples were taken from the spleen and gills, but then preparations were made from the kidney, followed by all other samples. According to Kligerman and Bloom (1977), the slides were prepared using the air-drying techniques specified by Blanco et al. (2012). Without losing the sterility of the fish samples, kidney tissues were removed by wiping, especially the outer surfaces, with alcohol. Approximately 1 g of tissue sample was added to Falcon tubes that had been taken out of the freezer the day before, thawed, and contained at least 5 mL of culture solution, then left for incubation in the refrigerator at +4°C for at least 2.5 h. After the tissues in the Falcon tubes were transferred to petri dishes and thoroughly crushed and disintegrated with a scalpel, forceps, or scissors, they were returned to the Falcon tubes using a Pasteur pipette. After adding 0.01% colchicine solution to the tubes, they were homogenized in a vortex mixer and left for incubation. Following incubation, centrifugation was performed at 1000 RPM, and the supernatant was discarded.

The hypotonic solution prepared from 0.075M KCl was added to the cell pellet and incubated for at least 2 h. 1–2 drops of cold Carnoy’s fixative were added to the cells in the hypotonic solution, and they were allowed to sit for 5 min. Then, the volume was adjusted to 12–13 mL with cold fixative. After centrifugation at 1000 RPM, the supernatant was carefully discarded with a Pasteur pipette. Fixative was added to the cell pellet, centrifuged again at 1000 RPM, and the supernatant was discarded. This process was repeated several times. Finally, a few milliliters of fixative were added to the cells, and homogenization was performed; then, the spread was made by dropping onto clean slides exposed to steam in a bain marie and left to dry in the air. The preparations were first stained in 5% Giemsa (pH 6.8–7.1) solution for at least 10–15 min, rinsed with distilled water, and air-dried.

According to Sumner’s (1972) C-band method modified by Artoni et al. (2001), the preparations were adapted with minor changes using the specified methods. These regulations are briefly as follows: Slide preparations were washed with ethanol and Carnoy’s fixative to remove immersion and Giemsa stain. The dried slides were rinsed with distilled water and placed in 0.2N HCl solution for 10–15 min at room temperature. The HCl-soaked slides were then washed with distilled water. The washed slides were placed in a freshly filtered 5% Ba(OH)₂ solution (filtered through #1 Whatman filter paper) for 15–20 s. They were then dipped in 0.2N HCl for a few seconds, then washed with distilled water and allowed to dry. The dried slides were placed in a bain-marie at 60°C in 2 × SSC for 1 h. The slides were gently washed with distilled water and allowed to air dry. Dried slides were stained with 5% Giemsa (pH 6.8–7.1, phosphate-buffered) for 10–15 min, rinsed with distilled water, and allowed to dry.

In the study, various adaptations of the techniques of Howell and Black (1980), which were successfully applied in fish by Kavalco and Pazza (2004), were carried out. To summarize, C-band or Giemsa slide preparations were first stained with ethanol and fixative. Two drops of 1% gelatin (1 g gelatin and 0.25 mL formic acid) and four drops of 25% silver nitrate (0.25 g · L^–1) were placed on each slide. A 40 × 22 mm cover slip was placed on the slide, and the preparation was placed in the microwave. After 5–10 s, the slide was removed and rinsed in tap water to remove excess stain and the cover slip. Finally, the slide preparations were stained with Giemsa solution (5%, pH 6.8–7.1) for 10–30 s.

The chromosome preparations were observed with the Leica DM500 (Leica, Germany) and photographed digitally with the Leica ICC50 (Leica, Germany) digital camera. At least 20 metaphase sites were selected from the most suitable ones for each sample from the digital photographs taken under the microscope (Maneechot et al. 2015), AKAS Multispecies© (v.3.5.1.0; Argenit Smart Information, Technology Limited Company, İstanbul, Türkiye) (Unal Karakus et al. 2024), and the microscope-specific LAS EZ© (v.3.4.0; Leica, Germany) image analysis software programs, and diploid (2n) chromosome numbers were determined and arranged. According to Levan et al.’s (1964) nomenclature, homologous chromosomes were measured in the centromeric plane, extracting karyotypes, and preparing karyogamy with the special image processing program AKAS Multispecies© and Adobe Photoshop CC (v.19.1.5). As stated in the same study (Levan et al. 1964), the number of chromosome arms (NF, the number of fundamental) is contained from biarmed chromosomes (m, metacentric and sm, submetacentric chromosomes or m-sm, meta-submetacentric chromosomes) and uniarmed chromosomes (st, subtelocentric or st-a, subtelo-acrocentric and a, acrocentric, or t, telocentric chromosome).

In the study, the species with the highest number of samples examined, Mediterranean horse mackerel, Trachurus mediterraneus, had no chromosome plates found in four out of seven samples after 3–4 h of PB-MAX™ incubation, while perfect metaphases were found after 3 h. Five preparations were made for each of the seven specimens. The average metaphase count in these preparations was 59, and the mean number of high-quality chromosome sites was determined to be 48. This corresponded to an average success rate of 81% for the best karyotyping, and an average number of high-quality metaphases per slide of approximately 10.

As shown in Fig. 2, as a result of the counts, the 2n = 48 cytotype was by far the highest at 49% among the 14 cytotypes detected, which determined the diploid number for this species. It was determined that the 2n diploid number of Trachurus mediterraneus from the preparations obtained from horse mackerel fish was 48 and that it had 6 metacentric, 16 submetacentric, 18 subtelocentric, and 8 acrocentric chromosomes in the karyotype (Fig. 3B). According to this karyotype, the NF arm number was found to be 70. The diagrammatic classification of homologous chromosomes in n numbers is given in the karyogamy in Fig. 2.

Figure 2. 

Idiogram (A) and frequency distribution (B) of detected cytotypes according to the karyotype of Trachurus mediterraneus.

Figure 3. 

Metaphase (A) and karyotype (B) of Trachurus mediterraneus, the bar is 5μ.

The constitutive heterochromatin and Ag-NOR banding results of the preparations from Trachurus mediterraneus samples obtained in the study are shown in Fig. 4A, B. According to the results, the C+ regions were identified in the centromeric position of two subtelocentric chromosomes in T. mediterraneus (Fig. 4A). However, examinations of silver nitrate-stained preparations of this species revealed NOR+ regions on six chromosomes. These were located in the pericentromeric positions of four small acrocentric chromosomes and at the centromeres of two subtelocentric chromosomes (Fig. 4B).

Figure 4. 

Chromosome regions (Arrows) with constitutive heterochromatin (A–C) and NORs (B–D) in Trachurus mediterraneus (A–B) and Scorpaena porcus (C–D), the bar is 5μ.

Scorpion fish (Scorpaena porcus) had the highest rate of success (3 out of 4 samples). These were the samples taken from the fishermen, and the kidney samples from these specimens had a 4-h short-term culture application with PB-MAX™. An average of 77 chromosome sites was identified from five preparations prepared from each of the four specimens obtained from S. porcus. Of these, 49% were of high quality, and the number of metaphases that could be karyotyped was 33 (49%), and the average number of metaphases that could be karyotyped was calculated as 63% (21 metaphases). The average number of high-quality metaphases per slide corresponded to approximately 7 metaphases. As can be seen from the graph in Fig. 5, which was created based on the chromosome counts, it was determined that 2n = 42, the highest frequency among the seven cytotypes, corresponds to a diploid number of chromosomes.

Figure 5. 

Idiogram (A) and frequency distribution (B) of detected cytotypes according to karyotype in Scorpaena porcus.

According to results obtained from Scorpaena porcus samples in the study, it was determined that the diploid number was 2n = 42 from the metaphases found (Fig. 6A), and the karyotype consisted of 4 metacentric, 4 submetacentric, 10 subtelocentric, and 24 telocentric chromosomes. As shown in Fig. 6B, the arm number (NF) from the detected karyotype was found to be 50. The diagrammatic classification of homologous chromosomes in n numbers is given in the idiogram in Fig. 5.

Figure 6. 

Metaphase (A) and karyotype (B) of Scorpaena porcus, the bar is 5μ.

As a result of the banding of constitutive heterochromatin regions in the study, C+ regions were identified in 4 centromeric cases in two telocentric and two subtelocentric chromosomes in Scorpaena porcus (Fig. 4C). When the Ag(NO)₃ staining results were evaluated in the study, two NOR+ regions were detected in the telomeric state on short arms of two submetacentric chromosomes in Scorpaena porcus (Fig. 4D). However, no sex chromosomes from S. porcus and T. mediterraneus were detected in the preparations examined after staining.

Essentially, to facilitate the study of fish species (e.g., Holoshestes sp. and Odontostilbe sp.) too small to be directly cultured, Fenocchio et al. (1991) incubated kidney tissues from live fish samples under anesthesia with TC 199 culture medium in a CO₂ culture oven for 6–24 h as a short-term culture. Although their results showed satisfactory success in terms of fish cytogenetics, our study found this method impractical due to both technical and time constraints. Netto et al. (2007) reported that incubating fish samples known to be in the postmortem period for at least 20 min and up to 2.5 h in RPMI 1640 culture medium (PB-MAX™ was developed from this medium) for up to 12 h would achieve good cytogenetic performance. While there is a difference in the number of metaphases between the studies conducted and this study, there are no species for which obtaining at least 10 quality metaphases for karyotyping was impossible. This method proved more advantageous than traditional in vivo methods, which often cannot produce chromosomes from marine fish—the main purpose and hypothesis of the study, is not always possible, despite many attempts on numerous samples with little success. A disadvantage of this approach is that the freshness of the fish sample and the time elapsed since death must be precisely known (no more than 4 h). In this study, where a minimum of 3 h and a maximum of 4 h was deemed suitable for PB-MAX™ treatment in terms of working time, positive results were observed for these two species.

An evaluation of the data from the most comprehensive experimental direct method developed by Blanco et al. (2012) showed that preparations from a hypotonic fixative-containing cell solution, which could be stored at –20°C for up to 7 days, yielded approximately 64% of high-quality metaphases per slide. While the results from a freshwater fish (Hoplias malabaricus (Bloch, 1794)) in this study were adequate for direct studies, they were less successful than those in the current in vitro study. However, the results are significantly lower than those reported by Rey et al. (2015), where more than 150 high-quality chromosomes were obtained from a single drop of cell solution in one preparation using rapid (5 to 7 days) cell culture. The most developed long-term culture method is applied to marine fish species (Notothenioidae), where chromosome retrieval by direct methods was impossible.

The studies show that members of this genus have 2n = 48 chromosomes, as seen in Table 1. This finding aligns with the cytotaxonomic results reported by Vasil′ev (1978) and Caputo et al. (1996) for this species regarding chromosome number. The idea that the teleosts typically have2n = 48 acrocentric chromosomes, a common ancestral trait (plesiomorphic character) proposed by Ohno (1970, 1974) and widely accepted today, is also observed in species within the Carangidae family. While T. trachurus and T. mediterraneus, members of the genus Trachurus, possess 48 chromosomes, the presence of a few meta- and submetacentric chromosomes causes deviations from this rule. Nevertheless, it is generally believed that these exceptions do not violate the overall pattern (Caputo et al. 1996). In this study, T. mediterraneus with 48 chromosomes was found to have 32 uniarmed (st+a) chromosomes as reported by Caputo et al. (1996), though our analysis shows 26 st+a chromosomes, indicating distinctive karyological features in phylogenetic comparison with T. japonicus and T. trachurus. Differences were also noted in the number of biarmed chromosomes; this study identified 11 pairs of m+sm, whereas Vasil′ev (1978) and Jacobina et al. (2016) reported 5 pairs, and Caputo et al. (1996) found 8 pairs, as shown in Table 1. However, Jacobina et al. (2016) reported a karyotype of biarmed (m-sm) and uniarmed (st-a) chromosomes in T. mediterraneus, a result that fully coincides with that reported by Vasil′ev (1978). This phylogenetic study (Jacobina et al. 2016) did not mention the C+ and NOR+ regions carried by the chromosomes. However, they also reported that T. japonicus and T. mediterraneus have the most dynamic karyotypes, and the more restricted distribution of these two species within the Carangidae family suggests that orogenic movements during continental formation may have been more intense among habitable zones.

Cytogenetic studies on Trachurus mediterraneus and T. trachurus revealed that NORs were located in the interstitial region on the long arm of the first pair of chromosomes (Fig. 7). Conversely, constitutive heterochromatin regions (C+ regions) were reported at centromeric and telomeric positions on seven chromosome pairs (Caputo et al. 1996). In our study, C+ region signals were observed at the centromere of two subtelocentric chromosomes. Although the position is consistent, the number of chromosomes with C+ regions differs. NOR+ regions were found in the pericentromeric area on the short arm of four small acrocentric chromosomes and at the centromere of two subtelocentric chromosomes in our study. The hypothesis that chromosomal rearrangements in T. mediterraneus result from paracentric inversion of the terminal NOR region in the metacentric chromosome pair of T. trachurus—transforming it into a pericentromeric biarmed chromosome, as reported by Caputo et al. (1996)—aligns with the NOR phenotype in our study, despite observing polymorphic variation in NOR chromosome numbers.

Figure 7. 

Graphical representation of C+ (Red dots) and NOR+ (Blue dots) results of Trachurus mediterraneus compared with other studies.

In this study, the fact that the biarmed chromosome number differs from the 48 NF values obtained in other species of the family Carangidae, which includes Trachurus mediterraneus (NF–70) examined, seems to move away from the hypothesis of a plesiomorphic character of bony fishes in general terms. For example, Murofushi and Yosida (1979) reported that Kaiwarinus equula (Temminck et Schlegel, 1844) and Caranx sexfasciatus Quoy et Gaimard, 1825 studied from the southeastern coast of Japan had 2st+46a (NF–48) chromosomes, and Alectis ciliaris (Bloch, 1787) had 48a chromosomes. Rodrigues et al. (2007) reported that Selene vomer from the south coast of Brazil had 2st+46a (NF–48) chromosomes. Chai et al. (2009) reported that the karyotype of Seriola lalandi Valenciennes, 1833 from farms on the coast of South Australia had 4m+6st+38a (NF–52) chromosomes, and Jacobina et al. (2013, 2014) found 48a (NF–48) in Selene brownii, 2m+46a (NF–50) in Selene setapinnis, and 2st+46a (NF–48) in Selene vomer, living off the Brazilian coast of the Atlantic Ocean. 6sm+42a (NF–48) chromosome was found in Caranx lugubris from the Faroe Islands in the middle of the Atlantic Ocean, and most recently, in a study conducted by Soares et al. (2021) on Western Atlantic Carangids, 2st+46a (NF–48) chromosomes were found in Elagatis bipinnulata and 2st+46a (NF–48) in Gnathanodon speciosus. Similarly, in Seriola dumerili (Risso, 1810), one of the most important and economically valuable carangid species, karyotype differentiation was observed using classical methods (Vitturi et al. 1986), and polymorphism in diploid number (2n = 47–48) due to Robertsonian fusions was confirmed. In contrast, for this species, Sola et al. (1997) reported that mFISH staining and 18S rDNA FISH signals overlapped with AgNOR and CMA₃-stained regions, demonstrating karyotype differentiation, but showed no signs of Robertsonian translocation.

As seen in Table 2, chromosome numbers and karyological data of 10species belonging to the genus Scorpaena have been reported. The chromosomes of the examined members of this genus vary between 34 and 48. While the 2n number is 42 in five studies conducted on S. porcus, as in this study, differences are also seen between the karyotypes in other research results.

All studied members of Scorpaenidae are karyologically close to 2n = 48, which is the widely accepted diploid number for fish. This supports Ohno’s (1970) hypothesis of single-ancestor evolution for teleosts (Caputo et al. 2003). The first karyological studies for Scorpaena porcus were conducted by Cataudella et al. (1973), Sola and Cataudella (1978), and Sola et al. (1978, 1981). They reported that this species had a karyotype with 6 metacentric, 10 subtelocentric, and 26 acrocentric chromosomes (2n = 42) as shown in Table 2. Although the model diploid chromosome number was not determined for scorpaeniforms, the reason for the decrease from 2n = 48 to 42 in S. porcus compared to other scorpaenids was stated by Cano et al. (1982) and Thode et al. (1985) as the result of the decrease from pericentromeric inversions and random fusions in the biarmed chromosomes (m+sm). This was confirmed by the reduction to 2n = 34 in Scorpaena notata Rafinesque, 1810, determined by Caputo et al. (1998) and Galetti et al. (2000). The chromosome type and arm number observed in S. porcus in our study closely resemble those reported by Caputo et al. (1998). However, while all studies conducted on S. porcus up to 1998 identified 13 pairs of acrocentric chromosomes, our study identified 24 telocentric chromosomes instead of acrocentric ones. As can be seen from Table 2, it is notable that no telocentric chromosomes are visible in any of the karyotypes of the studied Scorpaena species. The variation in the chromosome arm number (NF), resulting from differences in submetacentric and acrocentric chromosome counts, is likely due to variations in the techniques or technologies used to analyze and assess chromosome morphology.

In the studies conducted by Thode et al. (1985) and Caputo et al. (1998) on scorpaeniform species, it was noted that there were C+ regions in the centromere of nearly all chromosomes in S. porcus, and NOR+ regions were found in one pair of chromosomes (Fig. 8). It has been reported that the numerical differences between S. porcus, S. notata, and other species are reflected in NOR variations caused by duplications and deletions. Caputo et al. (1998) used fluorescence staining with GC-specific chromomycin A₃ (CMA₃) and AT-specific 49,6-diamidino-2-phenylindole (DAPI), which revealed ectopic telomeric sequences in some chromosomes of S. notata; tandem fusions were shown to significantly contribute to the reduction in both chromosome number and number of fundamental (NF) observed in this species. This reduction appears in biarmed chromosomes in S. porcus as 5 (m), 8 (m+sm), and 10 (m+sm) (Table 2). In contrast, Sofradžija (1984) found 16 m-sm chromosomes in the Adriatic S. porcus populations examined. The hypothesis explaining the karyological situation in S. porcus studied here is that the chromosome number in scorpaenids (Scorpaena onaria Jordan et Snyder, 1900; Scorpaena miostoma Günther, 1877) from the Indo-Pacific Far East region is 48, and the variation in NFs, such as biarmed chromosomes between 6 and 8, results from Robertsonian fusions (Nishikawa et al. 1977; Murofushi et al. 1987; Yokoyama et al. 1992). These chromosomes may have been lost during evolution or may remain inert in the genome as microchromosomal fragments. In our study, NOR+ carrying chromosomes were seen on uniarmed chromosomes (two telocentric or acrocentric), as in all other studies.

Figure 8. 

Graphical illustration of C+ (Red dots) and NOR+ (Blue dots) results of Scorpaena porcus compared with other studies.

According to Table 2, Corrêa and Galetti (1997) reported that the karyotypes of Scorpaena brasiliensis Cuvier, 1829 and Scorpaena isthmensis Meek et Hildebrand, 1928, sampled from the Brazilian coast via short-term culture, were 2m+12sm+22sta (2n = 44, NF–60) and 6m+8sm+26sta (2n = 40, NF–56), respectively, along with direct chromosome preparation methods. Nirchio et al. (2014a) stated that the karyotypes of the same species sampled from the Venezuelan coast were 2m+44sta (2n = 46, NF–48) for S. brasiliensis and 8m+10st+20a (2n = 38, NF–56) for S. isthmensis. The differences between these studies likely reflect an increase in biarmed chromosomes for S. brasiliensis due to centric fission or pericentric inversions, as suggested by Galetti et al. (2000) for scorpaenids, and a decrease in biarmed chromosomes for S. isthmensis due to Robertsonian fusions. Our results suggest that the increase in biarmed chromosomes in S. porcus may have been caused by similar rearrangements. Nirchio et al. (2016b) observed the formation of two distinct cytotypes in Scorpaena plumieri from the Caribbean. NOR phenotypes were identified in the short arm of the 11th pair of st-a in cytotype 1 (48st-a) and in the terminal region of the 4th pair of st-a in cytotype 2 (2m+48st-a), as shown in Table 2. In dFISH analyses, they reported that the 18S and 5S rDNA regions appeared interstitially in two different pairs of medium-sized chromosomes in both cytotypes. Based on the C-banding results, they hypothesized that the formation of these cytotypes was due to a large subtelocentric chromosome pair, one of which was pericentric and the other paracentric, leading to a metacentric chromosome inversion.

In the species of the Indo-Pacific Scorpaena species, C+ regions were reported in one pair of chromosomes of Scorpaena izensis Jordan et Starks, 1904, Scorpaena neglecta Temminck et Schlegel, 1843 and NOR regions were found in the short arm of a pair of metacentric chromosomes of these species (Yokoyama et al. 1992). In studies conducted at two different times, NOR+ regions were detected on the short arm of a biarmed chromosome (subtelocentric/acrocentric) in S. porcus (see Thode et al. 1985; Caputo et al. 1998). As illustrated in Fig. 8, while C+ positive regions were found in the paracentromeric regions of all chromosomes of S. porcus sampled from the coasts of Málaga in the Mediterranean, it was recorded that they appeared on both sides of the centromere of the 2nd pair of chromosomes and that they had a pair of chromosomes carrying NOR (Thode et al. 1985). Caputo et al. (1998) obtained very similar results in S. porcus samples from Senigallia (Italy), a different population from the previous study. Inversions and fissions observed in single-armed chromosomes (mostly subtelocentrics), which are thought to lead to the formation of biarmed chromosomes in scorpaeniforms (Thode et al. 1985; Corrêa and Galetti 1997; Caputo et al. 1998; Galetti et al. 2000; Nirchio et al. 2014a, 2016b), can be considered as a result of pericentromeric inversions in the centromeric C+ regions of a pair of subtelocentric chromosomes, forming a pair of submetacentric chromosomes, as we found for S. porcus. Considering that these changes in karyology take millions of years to occur, according to the results of phylogenetic studies (Nirchio et al. 2016b; Rossi 2021). Since no other results have been found in the Black Sea populations, more detailed studies, such as molecular cytogenetic studies, should be conducted to elucidate the cause of karyotype differences among populations.

This study retested the in vitro method proposed by Araya-Jaime et al. (2021), which is the most recent and shortest-term cell culture method with successful results. Successful outcomes were observed in 90% of 12 different sea fish families and 20 species included in this study, suggesting that definitive and optimal metaphases can be achieved in future research on marine fish, regardless of species. Additionally, this study provided new cytogenetic data for both species, likely paving the way for further studies in marine fish cytogenetics. In addition to the superior properties of this newly developed short-term culture for cytogenetic studies, it is also necessary to know the exact post-mortem period of fish samples, which do not exceed 4 h, as a handicap. Based on cytogenetic examinations and supported by the other studies, it was observed that both species showed less or more variation according to the karyotype and chromosomal features, NOR+ and C+ positive regions in this study. These determined variations may be attributed to research conditions, research techniques, and chromosome image analysis methods (such as the latest developed AKAS Multispecies© software). Future collaborative international studies could conduct detailed chromosome mapping of other scorpaenid and carangid species living in Turkish marine waters and karyotype differences between populations of the same/separate species living in other seas and/or oceans, using molecular cytogenetic techniques such as mFISH, D-FISH, M-FISH, GISH, and others.

We would like to thank Prof. Dr. Roberto Ferreira Artoni and Asst. Prof. Dr. Cristian Araya-Jaime for their feedback on the study’s method and evaluation. We are pleased to thank Dr. Nazan Gillie from the University of Wisconsin–Madison (USA) for reading the manuscript and improving the language. This work was supported by the Ordu University Scientific Research Projects Coordination Office [Grant number AR-2304] and TUBITAK [Grant number 123Z621].