The challenge of studying fish chromosomes has long been compounded by the fact that karyotypes can only be reliably obtained from living specimens or fresh tissues. While chromosome structures have been documented for nearly 4,000 fish species out of over 37,000 known species, many marine species remain unstudied due to their biological characteristics. The Mediterranean horse mackerel, Trachurus mediterraneus, and the black scorpionfish, Scorpaena porcus, were selected for this study as they represent two distinct families, Carangidae and Scorpaenidae, respectively. Both species inhabit the coasts of Türkiye, specifically the Black Sea region, and are of significant ecological and economic interest. The ability to analyze their chromosomes without the need for long-term live culture opens new avenues for understanding their evolutionary history and taxonomic relationships.

The methodology employed in this study relies on a short-term culture protocol that can be applied to fish samples collected within four hours of death. Kidney tissues, which have been identified as the optimal source for cytogenetic analysis in fish, were removed from the specimens and placed in Falcon tubes containing the PB-MAX™ culture solution. The tissues were incubated at 4°C for approximately three to four hours, a process that allows for the proliferation of cells without the need for complex in vivo maintenance. Following incubation, the cells were treated with colchicine to arrest them in metaphase, followed by hypotonic treatment and fixation. The resulting cell suspensions were spread onto slides and stained using Giemsa, C-banding, and Ag-NOR banding techniques to visualize chromosomal structures and specific genetic markers.

The results of the study provided updated cytogenetic data for both species, revealing distinct karyotypes that differ from previous reports. For the Mediterranean horse mackerel, the diploid chromosome number was determined to be 48, with a karyotype consisting of 6 metacentric, 16 submetacentric, 18 subtelocentric, and 8 acrocentric chromosomes. This results in a fundamental number of arms (NF) of 70. In contrast, the black scorpionfish exhibited a diploid number of 42, with a karyotype of 4 metacentric, 4 submetacentric, 10 subtelocentric, and 24 telocentric chromosomes, yielding an NF of 50. These findings align with the general hypothesis that teleost fishes often possess a diploid number of 48, but they also highlight specific variations that may be attributed to evolutionary rearrangements such as inversions and fusions.

The study also examined the distribution of constitutive heterochromatin and nucleolar organizer regions (NORs) in both species. In the Mediterranean horse mackerel, C-positive regions were identified in the centromeric positions of two subtelocentric chromosomes, while NOR-positive regions were found on six chromosomes, including four small acrocentric chromosomes and two subtelocentric chromosomes. For the black scorpionfish, C-positive regions were located in the centromeres of two telocentric and two subtelocentric chromosomes, and NOR-positive regions were detected on the short arms of two submetacentric chromosomes. These banding patterns provide insights into the structural organization of the genomes and may serve as markers for further molecular cytogenetic studies, such as fluorescence in situ hybridization (FISH), which can detect microstructural rearrangements and gene loci.

The effectiveness of the PB-MAX™ method was demonstrated by the high success rate in obtaining quality metaphases. For the Mediterranean horse mackerel, an average of 59 metaphase sites were identified per preparation, with an average of 48 high-quality chromosome sites, resulting in an 81% success rate for karyotyping. The black scorpionfish showed a similar success rate, with 77 chromosome sites identified per preparation and 49% of high-quality metaphases. These results are comparable to or exceed those obtained using traditional direct methods or long-term culture techniques, which often yield fewer usable chromosomes. The method’s ability to produce high-quality spreads from postmortem samples is particularly advantageous for species that are difficult to keep alive, such as deep-sea fish or those that are sensitive to handling and environmental changes.

The karyotypic variations observed in this study contribute to the broader understanding of karyoevolution in marine fish. The presence of metacentric and submetacentric chromosomes in the Mediterranean horse mackerel, which is generally characterized by a high number of acrocentric chromosomes, suggests that chromosomal rearrangements have played a role in its evolution. Similarly, the reduction in chromosome number in the black scorpionfish, from the typical 48 to 42, may be the result of Robertsonian fusions or pericentromeric inversions. These evolutionary changes can influence genetic diversity and adaptation, and further studies using molecular techniques will be necessary to elucidate the specific mechanisms involved. The study also notes that the differences in karyotypes between populations of the same species may be due to geographical isolation or local adaptation, highlighting the importance of studying multiple populations to capture the full extent of genetic variation.

The implications of this research extend beyond the two species studied, as the method has the potential to be applied to a wide range of marine fish families. The study reports successful outcomes in 90% of 12 different sea fish families and 20 species, indicating that the technique is robust and versatile. This is particularly significant for the study of economically important species, such as those in the Carangidae and Scorpaenidae families, which are key targets for aquaculture and fisheries management. By providing a reliable and efficient method for chromosome analysis, this research paves the way for more detailed studies of fish genetics, evolution, and conservation. The ability to work with postmortem samples also reduces the ethical and logistical challenges associated with keeping live fish in laboratories, making cytogenetic studies more accessible to a broader range of researchers.

Future research will likely focus on applying molecular cytogenetic techniques, such as multi-color FISH and comparative genomic hybridization, to the chromosomes obtained using this method. These techniques can provide higher-resolution maps of the genome and help identify specific genes and regulatory regions involved in fish development, reproduction, and adaptation. The study also suggests that collaborative international efforts could lead to a more comprehensive understanding of the karyotypes of marine fish species worldwide. By standardizing methods and sharing data, researchers can build a global database of fish cytogenetics that will aid in the classification and conservation of these diverse and important vertebrates. The success of this short-term in vitro method marks a significant step forward in marine fish cytogenetics, offering a practical and efficient tool for studying the genetic diversity of the ocean’s inhabitants.

Subject of Research: Cytogenetic analysis of marine fish using short-term in vitro culture methods

Article Title: The next level in marine fish cytogenetics via the latest developed short-term in vitro method: A novel karyoevolutive consideration of two marine fish species ( Actinopterygii )

Article References: Saygun, S., & Gündoğan, M. (2025). The next level in marine fish cytogenetics via the latest developed short-term in vitro method: A novel karyoevolutive consideration of two marine fish species (Actinopterygii). Acta Ichthyologica et Piscatoria, 55, 297-308. https://doi.org/10.3897/aiep.55.158826

Image Credits: AI Generated

DOI: 10.3897/aiep.55.158826

Keywords: marine fish, cytogenetics, karyotype, in vitro culture, PB-MAX, chromosome analysis, Trachurus mediterraneus, Scorpaena porcus, fish evolution, molecular biology, next, level