Introduction
Global c limate change causes increasing water temperatures and decreasing oxygen levels, which leads to changing or destroying aquatic habitats. Additionally , the direct contact of the fish with the changed environment leads to physiological changes in the species , which can be studied using cell culture systems . In vitro models represent an essential tool in aquaculture-related research and a flexible system for investigating the effects of altered biotic and abiotic factors at the cellular level.
The maraena whitefish (Coregonus maraena ) is an ecologically important species in the region of the Baltic Sea. Due to extensive fishing, habitat fragmentation, and eutrophication, these populations have declined sharply. Today, this vulnerable salmonid species is on the IUCN Red List. The effects of rising water temperatures on the already vulnerable species are not yet been well studied. In studies conducted by our working group, we have succeeded in establishing a cell line (CMA-fin1) derived from maraena whitefish ( Grunow et al. 2021, Kaya et al. 2022) . The aim of the present work was to use t his in vitro model to examine the impact of increasing temperatures on growth and energetic parameters of CMA-fin1 cell line . Additionally, the use of cell lines helps to reduce the number of animals used in research in accordance with the 3Rs principle.
Materials and Methods
The proliferation of CMA-fin1 cells was analysed and compared at two different temperatures, at 20°C (control) and 25°C (high temperature). For this, all cells were cultivated in Leibovitz-15 Medium (L-15, Gibco ) with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. G rowth and vitality were measured after 6 days by trypan blue staining and image-based automated cell counting (EVE™ Plus, NanoEnTek) .
To conduct a comparative analysis (20°C vs. 25°C) of morphological cell changes, we visualized actin by Phalloidin-iFluor 488 Reagent staining . Additionally , mRNA abundance of selected temperature and stress- related genes was measured by quantitative reverse transcription PCR (RT-qPCR). To measure parameters related to mitochondrial function and glycolysis, we used the Seahorse XF Cell Mito Stress Test Kit and Glycolysis Stress Test Kit (Seahorse, Agilent Technologies) . The tests were performed after 24 h for cell attachment. The detection of lactate dehydrogenase (LDH) is a tool for determin ing the status of necrosis of cells. The enzyme activity was measured by Cytotoxicity Detection Kit (Roche Applied Science) in the supernatant of the CMA-fin1 cells
Results
CMA-fin1 cells had a stable, fast-growing phenotype at optimized conditions . We observed that increasing the temperature by 5 °C resulted in a higher proliferation and a higher amount of viable cells but a concomitant decrease in the size of these cells . The changes in doubling time had no significant impact on cell viability . Measuring mitochondrial function using Mito stress test, basal and maximum respiration and ATP production but also proton leak and non-mitochondrial respiration were decreased in cells cultured at 25°C compared to those grown at 20°C . CMA-fin1 cells did not perform glycolysis under basal conditions. However, cells grown at 25°C had a significantly lower glycolytic capacity and reserve than cells grown at 20°C. T he cell culture growth dynamics affected LDH release and were determined simultaneously in CMA-fin1 cells grow ing at 20°C and 25°C.
Discussion
In the context of global warming, the effects of rising water temperatures on fish populations are of great interest. The aquaculture sector is also growing and needs easy-to-use diagnostic tools in research and practice. Fish cell models provide a basis and an alternative to animal experiments. To date, in vitro models focus on fish health management, pathological, and transgenic studies (Goswami et al. 2022). Direct links between studies on climatic effects on fish (Crawshaw, 1977) and in vitro models such as the CMA-fin1 cell line have yet to be established. However, a good example of the correspondence between in vitro and in vivo studies is the brain-derived eelB cell line of the American eel, which showed morphological and functional properties of neuronal stem cells (Bloch et al. 2016). In our study, the fish cell line CMA- fin1, derived from maraena whitefish, showed different cell activities, expression and morphological changes as well as impaired mitochondrial and glycolytic metabolic potential at increasing ambient temperature. In total, t his in vitro model can be used for studies of environmental effects on Coregonus maraena to better understand t he cell physiology, like the here shown metabolic changes in more detail.
References
Crawshaw, L. I. (1977). Physiological and behavioral reactions of fishes to temperature change. J. Fish. Res. Board. Can., 34, 730–734.
Goswami , M.; Yashwanth, B. S.; Trudeau, V.; Lakra, W. S. (2022). Role and relevance of fish cell lines in advanced in vitro research. Molecular Biology Reports, 49, 2393-2411.
Grunow, B., Franz, G. P., & Tönißen, K. (2021). In vitro fish models for the analysis of ecotoxins and temperature increase in the context of global warming. Toxics , 9, 286.
Kaya, Y., Tönißen, K., Verleih, M., Rebl, H., & Grunow, B. (2022 ). Establishment of an in vitro model from the vulnerable fish species Coregonus maraena (maraena whitefish): Optimization of growth conditions and characterization of the cell line. Cell Biology International, 47, 548– 559.