Introduction
For over 100 million years, the stable day-night cycle has driven the evolution of circadian clocks across different organisms, allowing them to anticipate and adapt to daily and seasonal environmental changes. In finfish, the circadian system regulates essential processes like digestion, cell proliferation, stress responses, and reproduction (Vallone et al., 2007). On the molecular level, the circadian clock genes (clock, arntl, per, cry) form the foundation of cellular time perception by generating rhythmicity through positive and negative transcription-translation feedback loops. While studies in typical model organisms, especially zebrafish, have shown early onset of circadian gene expression (Dekens & Whitmore, 2008), much less is known about this process in temperate commercial species such as Eurasian perch, a freshwater fish of significant ecological and aquaculture importance. Understanding when and how this mechanism becomes functional during early development is crucial, as it may enable adjustment of hatchery protocols for domesticated stocks (Cahill, 2002). This study aims to explore the development of the circadian clock in early life stages (embryos and larvae) of Eurasian perch, using high-resolution sampling. Relatively long period of embryogenesis and larval metamorphosis in Eurasian perch compared to species like zebrafish provided us with a unique opportunity to gain insights into circadian regulation of closely spaced developmental stages. Our objectives were to: (1) map the expression of core circadian genes at key developmental stages, (2) determine the onset of rhythmicity and compare it with other investigated species, and (3) explore the biological and aquaculture implications of our findings.
Materials and methods
Eight families of Eurasian perch were created by controlled reproduction. Eggs were fertilized in vitro using cryopreserved semen (Judycka et al., 2022), and incubated in a recirculating aquaculture system (RAS) under a 14L:10D photoperiod, which simulates the natural light conditions during the spawning and early developmental stages of this species. The hatched larvae were reared in the same light conditions as embryos, until 36 days post hatching (DPH) following the standardized procedure (Palińska-Żarska et al., 2020).
Embryos and larvae were sampled continuously, every 4 hours, from the moment of fertilization to 3 DPH (11 days). From 3 DPH, larvae were sampled every 4 days, also in 4-hour intervals. The RNA from embryos and whole-larvae was extracted and analyzed for circadian clock gene expression (clockb, cry1b, cry2, per1b, arntl1a) using real-time qPCR. Rhythmicity of gene expression was analysed using Cosinor (Molcan, 2023). Gene expression correlations were assessed using Spearman’s correlation matrices in RStudio.
Results
During embryonic development, expression patterns of clock genes were random with no clear daily rhythm, indicating the circadian clock mechanism had not yet become functional. The Eurasian perch larvae displayed rhythmic expression of circadian genes starting at 3 DPH, coinciding with onset of exogenous feeding, with peaks and troughs in per1b, clockb, arnlt1a, and cry1b aligned with transitions between the day and night. The highest expression amplitude was recorded for cry1b, while arnlt1a had the lowest. Rhythmicity of the analysed genes persisted and became more consistent at the end of the larval period (around 28 DPH).
Discussion
This study provides the first detailed description of the development of circadian clock gene rhythmicity in Eurasian perch. The delayed onset of rhythmicity in Eurasian perch in comparison with other investigated teleosts (e.g., medaka, zebrafish, and Senegalese sole) may be linked to its slower development. This suggests that circadian clock development could be tied to the specific ecological needs and life history traits of each species. Prolonged embryogenesis of the Eurasian perch which takes place in a relatively safe microenvironment of the chorion, further protected by gelatinous matrix around the eggs might delay the need for a functional circadian clock. Therefore, the Eurasian perch might prioritize the development of other functions, such as visual and motor systems, before activating the circadian mechanism. The rhythmic expression of circadian clock genes began synchronously at 3 DPH. The timing of the onset of circadian clock gene rhythmicity coincides with the beginning of exogenous feeding. The presence of a functional circadian clock at this developmental stage may optimize feeding strategies by synchronizing larval activity with diel zooplankton migrations, thereby enhancing foraging efficiency and reducing predation risk. (Mikheev & Wanzenböck, 2010). As development progressed, these rhythms became more consistent in juveniles, likely reflecting the maturation of the nervous system and improved regulation of circadian processes.
This study not only enhances our understanding of circadian clock development in non-model species but also contributes to the broader knowledge of how circadian rhythms evolve in response to environmental factors. Our data shed light on how early life stages in this species are synchronized with natural light-dark cycles, which is critical for survival, growth, and feeding in the wild. In an ecological context, disruptions to these rhythms - such as through increasing levels of artificial light at night (ALAN) - could interfere with key biological processes, potentially affecting recruitment success and population dynamics. From an aquaculture perspective, the common practice of rearing Eurasian perch larvae under constant light conditions could profoundly alter the natural development of their circadian system, with potential consequences for growth, digestion, behaviour, stress resilience, and long-term welfare.
Acknowledgment
This research was a part of the project “Exploration of the development, role and functioning of circadian rhythm in early life stages of Perca fluviatilis” funded by the National Science Centre, Poland (project number UMO-2021/43/B/NZ9/03056).
References
Cahill, G. M. (2002). Clock mechanisms in zebrafish. Cell and Tissue Research, 309(1), 27–34. https://doi.org/10.1007/s00441-002-0570-7
Dekens, M. P. S., & Whitmore, D. (2008). Autonomous onset of the circadian clock in the zebrafish embryo. The EMBO Journal, 27(20), 2757–2765. https://doi.org/10.1038/emboj.2008.183
Judycka, S., Żarski, D., Dietrich, M. A., Karol, H., Hliwa, P., Błażejewski, M., & Ciereszko, A. (2022). Toward commercialization: Improvement of a semen cryopreservation protocol for European perch enables its implementation for commercial-scale fertilization. Aquaculture, 549, 737790. https://doi.org/10.1016/j.aquaculture.2021.737790
Mikheev, V. N., & Wanzenböck, J. (2010). Diet changes in prey size selectivity in larvae of perch Perca fluviatilis. Journal of Ichthyology, 50(11), 1014–1020. https://doi.org/10.1134/S0032945210110068
Molcan, L. (2023). Time distributed data analysis by Cosinor.Online application (p. 805960). bioRxiv. https://doi.org/10.1101/805960
Palińska-Żarska, K., Woźny, M., Kamaszewski, M., Szudrowicz, H., Brzuzan, P., & Żarski, D. (2020). Domestication process modifies digestion ability in larvae of Eurasian perch (Perca fluviatilis), a freshwater Teleostei. Scientific Reports, 10(1), 2211. https://doi.org/10.1002/dvdy.20998