Photoperiodic time measurement in Antarctic krill: - The effect of photoperiod on the seasonal development and the role of clock-genes -
Final Report Abstract
In the first project period, we identified critical molecular components of an endogenous circadian clock in Antarctic krill and showed evidence of its functional significance. Long-term simulative photoperiod experiments with live krill indicated the presence of an additional circannual timing mechanism which controls endogenous seasonal metabolic functions in krill. In the second funding period we aimed to 1) increase the number of known genes from krill through 454 pyrosequencing, 2) investigate the circadian adaptation to the polar environment, and 3) identify differential gene expression in krill throughout the whole photoperiodic cycle. Within the second project period we first started to prepare two cDNA normalized libraries from whole krill and krill heads sampled in different seasons and combined these with two datasets of krill transcriptome projects, already published. This led to the first knowledgebase ‘master’ transcriptome of Antarctic krill and will be particularly valuable for characterizing the molecular networks of krill that respond to environmental stressors and Zeitgebers in the Southern Ocean. Based on the transcriptomic information we produced the most complete oligonucleotide microarray platform (named krill 1.2) with a total of 57,358 probes and used this new platform to define the circadian transcriptome of krill of LD/DD entrained krill samples for the first time. Rhythmic analyses revealed that 1,546 transcripts could be considered as circadian transcripts. We successfully annotated 429 of these sequences including transcripts involved in clock control, metabolic processes, and visual phototransduction. Expression profiles of several transcripts were successfully validated by quantitative RT-PCR. The results significantly enhance our understanding of the physiological processes that are regulated by krill’s circadian clock. However, using individual SYBR® dye–based assays, to look at several targets for validation, became unmanageable and expensive. For the planned analyses proposed in the second work package of the project period, were expression pattern of circadian genes were planned to be measured during several 24 h cycles throughout the seasonal photoperiodic cycle, we decided to use an alternative array platform. Starting from the recently available krill ‘master’ transcriptome library and based on the results of the microarray results of LD and DD entrained krill samples, we have designed a 48-genes clock-oriented TaqMan low density array card starting from a selection of genes involved in light perception, phototransduction, carbohydrate metabolism and genes involved in moulting, sexual maturation and stress response. Due to this methodological change, we were not able to process the experimental samples as proposed, but instead developed a highly flexible low density array platform that will enable us in the future to analyse krill samples coming from the several 24-hours’ time series which have been sampled at different time points during an annual photoperiodic simulation in the laboratory more efficiently. Using new genes which have been achieved from the 454 sequencing and the microarray, we finally studied differential gene expression throughout the whole photoperiodic cycle. In correspondence with the findings of the previous funding period we now could demonstrate that enzyme activity and mRNA levels of metabolic key enzymes involved in the respiratory chain and the carbohydrate metabolism (e.g. cox and mdh) followed the simulated annual photoperiodic course in the laboratory, irrespective of the constant food availability in the experiment. Similar seasonal patterns were observed for animals kept under constant darkness suggesting that a seasonal metabolic activity cycle is regulated endogenously. Together these studies strongly indicate that the overt cycle of metabolic activity represent an endogenous adaptational seasonal rhythm that is controlled by an endogenous timing system in krill and that photoperiod seems to act as a main Zeitgeber, synchronizing the clock with the natural year. Future studies are necessary to understand the clock mechanism that seems to be involved in the modulation of krill’s seasonal cycles. Based on the results of the project in early 2013 a new Helmholtz Virtual Institute (HVI) named “PolarTime” was established to create a centre of excellence of international standing to study the principles, interactions and evolution of endogenous biological rhythms and clocks in polar pelagic organisms (www.polartime.org) and in which Krill act as a model organism.
Publications
- (2009) Effects of simulated light regimes on gene expression in Antarctic krill (Euphausia superba). J Exp Mar Biol Ecol 381: 57-64
Seear P, Tarling G, Teschke M, Meyer B, Thorne MAS, Clark, MS, Gaten E, Rosato E
- (2010) Seasonal variation in body composition, metabolic activity, feeding, and growth of adult krill Euphausia superba in the Lazarev Sea. Mar Ecol Prog Ser 398: 1-18
Meyer B, Auerswald L, Siegel V, Spahić S, Pape C, Fach BA, Teschke M, Lopata AL, Fuentes V
- (2011) A circadian clock in Antarctic krill: An endogenous timing system governs metabolic output rhythms in the Euphausid species Euphausia superba. PLoS ONE
Teschke M, Wendt S, Kawaguchi S, Kramer A, Meyer B
(See online at https://doi.org/10.1371/journal.pone.0026090) - (2015) Pyrosequencing and de novo assembly of Antarctic krill (Euphausia superba) transcriptome to study of the adaptability of krill to climate induced environmental changes. Mol Ecol Resour
Meyer B, Martini P, Biscontin A et al.
(See online at https://doi.org/10.1111/1755-0998.12408)
