ALBERT

Description: Antarctic krill (Euphausia superba) are high latitude pelagic organisms which play a key ecological role in the ecosystem of the Southern Ocean. To synchronize their daily and seasonal life-traits with their highly rhythmic environment, krill rely on the implementation of rhythmic strategies which might be regulated by a circadian clock. A recent analysis of krill circadian transcriptome revealed that their clock might be characterized by an endogenous free-running period of about 12–15 h. Using krill exposed to simulated light/dark cycles (LD) and constant darkness (DD), we investigated the circadian regulation of krill diel vertical migration (DVM) and oxygen consumption, together with daily patterns of clock gene expression in brain and eyestalk tissue. In LD, we found clear 24 h rhythms of DVM and oxygen consumption, suggesting a synchronization with photoperiod. In DD, the DVM rhythm shifted to a 12 h period, while the peak of oxygen consumption displayed a temporal advance during the subjective light phase. This suggested that in free-running conditions the periodicity of these clock-regulated output functions might reflect the shortening of the endogenous period observed at the transcriptional level. Moreover, differences in the expression patterns of clock gene in brain and eyestalk, in LD and DD, suggested the presence in krill of a multiple oscillator system. Evidence of short periodicities in krill behavior and physiology further supports the hypothesis that a short endogenous period might represent a circadian adaption to cope with extreme seasonal photoperiodic variability at high latitude.

Description: Antarctic krill (Euphausia superba), a key species in the Southern Ocean, reduce their metabolism as an energy saving mechanism in response to the harsh environmental conditions during the Antarctic winter. Although the adaptive significance of this seasonal metabolic shift seems obvious, the driving factors are still unclear. In particular, it is debated whether the seasonal metabolic cycle is driven by changes in food availability, or if an endogenous timing system entrained by photoperiod might be involved. In this study, we used different long-term photoperiodic simulations to examine the influence of light regime and endogenous rhythmicity on the regulation of krill seasonal metabolic cycle. Krill showed a seasonal cycle of growth characterized by null-to-negative growth rates during autumn-winter and positive growth rates during spring-summer, which was manifested also in constant darkness, indicating strong endogenous regulation. Similar endogenous cycles were observed for the activity of the key-metabolic enzyme malate dehydrogenase (MDH) and for the expression levels of a selection of metabolic-related genes, with higher values in spring-summer and lower values in autumn-winter. On the other side, a seasonal cycle of oxygen consumption was observed only when krill were exposed to simulated seasonal changes in photoperiod, indicating that light-related cues might play a major role in the regulation of krill oxygen consumption. The influence of light-regime on oxygen consumption was minimal during winter, when light-phase duration was below 8 h, and it was maximal during summer, when light-phase duration was above 16 h. Significant upregulation of the krill clock genes clk, cry2, and tim1, as well as of the circadian-related opsins rh1a and rrh, was observed after light-phase duration had started to decrease in early autumn, suggesting the presence of a signaling cascade linking specific seasonal changes in the Antarctic light regime with clock gene activity and the regulation of krill metabolic dormancy over the winter.

Description: Antarctic krill (Euphausia superba), hereafter krill, are key players in the ecosystem of the Southern Ocean. They are distributed all around Antarctica, and they are exceptionally abundant, representing the main link between primary producers and the higher trophic levels in the Antarctic marine food web. Due to their high ecological relevance, krill have been extensively studied in the field and in the laboratory, and it is known that their life-cycle is shaped by fundamental daily and seasonal rhythmic events. Actual knowledge about the external and internal factors involved in the regulation of rhythmic functions in krill is still quite limited but pivotal, especially in the context of future environmental changes driven by climate change. One hypothesis is that the daily and seasonal rhythmic functions in krill might be regulated through the activity of so-called “endogenous” clocks. Endogenous clocks are molecular function units, which promote rhythmic oscillations in transcription, physiology and behavior at the daily and seasonal levels. Endogenous clocks can be entrained (i.e. synchronized) by rhythmic environmental cues, like the day/night cycle (i.e. photoperiod = day length) at the daily level, and the seasonal photoperiodic cycle at the seasonal level. The implications of endogenous rhythmicity (i.e. rhythmicity promoted by endogenous clocks) in the regulation of rhythmic biological functions are well documented among terrestrial species, but studies dealing with marine organisms are very scarce. At the daily level, the best studied endogenous clock is the circadian clock, which is based on molecular feedback loops generating a rhythm with a period of approximately 24 h. Specific light-sensitive proteins promote the entrainment of the circadian clock with the day/night cycle, ensuring effective synchronization of rhythmic output functions according to daily recurring environmental changes. In krill, a circadian clock has been recently identified and characterized, and its influence on daily rhythms of metabolism and transcription has been demonstrated in the laboratory and in natural conditions. At the seasonal level, the regulation of rhythmic functions is less well understood, also in terrestrial species. An endogenous circannual clock seems to be involved, but the molecular mechanisms underlying its functioning are still unclear. Due to its ability to measure changes in day length, the circadian clock might contribute to the seasonal entrainment of the circannual clock. In krill, a circannual rhythm (i.e. a rhythm promoted by a circannual clock) might be involved in the regulation of the seasonal shifts in sexual maturity and metabolic activity observed in the field in summer and winter. During this dissertation, I investigated the involvement of endogenous clocks and rhythms in the regulation of rhythmic functions in krill at the daily and seasonal levels. Moreover, I also examined the role played by photoperiod in the entrainment of those clocks and rhythms. The work focused on three main research topics, which resulted in three publications: 1) the impact of the extreme seasonal photoperiodic cycle of the Southern Ocean on the activity of the circadian clock of krill at different times of the year (Publication I); 2) the involvement of an endogenous circannual rhythm and the role played by photoperiod in the regulation of the seasonal metabolic activity cycle of krill (Publication II); and 3) the involvement of the circadian clock and the role played by photoperiod in the regulation of diel vertical migration (DVM) in krill (Publication III). In publication I, I investigated the activity of the circadian clock of krill in different simulated seasonal Antarctic light conditions. The extreme variability displayed by the seasonal photoperiodic cycle in the Southern Ocean might cause a problem for the photoperiodic entrainment of the clock in different seasons. Especially during summer and winter, when overt light/dark cues are missing, the clock might get disrupted and the clock output might become arrhythmic. Indeed, laboratory work demonstrated that under simulated mid-summer and mid-winter conditions, when overt photoperiodic cues were missing, the circadian clock of krill was arrhythmic, and the metabolic output was de-synchronized. Conversely, under simulated early-autumn and late-winter conditions, when overt photoperiodic cues were present, the circadian clock of krill was active, and the metabolic output was synchronized with the light/dark cycle. This suggested that major changes are occurring during the year in the entraining process of the circadian clock of krill, depending on the different seasonal light conditions to which krill are exposed. In publication II, I investigated the involvement of an endogenous circannual rhythm in the regulation of the seasonal metabolic activity cycle of krill. Moreover, I also examined the role played by photoperiod in the entrainment of this rhythm. In response to the strong seasonal variability displayed by light and food availability in the Southern Ocean, krill display seasonal differences in metabolic rates, feeding activity and growth. During summer, when light and food availability is high, krill metabolic and feeding activity is enhanced, and krill growth rates are positive. During winter, when light and food conditions are low, krill metabolic and feeding activity is reduced, and krill show reduced growth or even shrinkage (i.e. reduction of size). It has been hypothesized that an endogenous rhythm entrained by the seasonal Antarctic light regime might be responsible for the regulation of the seasonal metabolic cycle of krill. Krill exposed to different long-term simulated natural seasonal light conditions, showed seasonal patterns of growth, enzyme activity and gene expression of key metabolic genes, which were also observed in krill exposed to constant darkness. The results strongly suggested the involvement of a circannual clock in the regulation of the seasonal metabolic cycle of krill. However, major differences were observed in the seasonal patterns of oxygen consumption, suggesting that exposition of krill to specific seasonal light cues might be necessary for the effective entrainment of the circannual clock. In publication III, I investigated the involvement of an endogenous circadian rhythm in the regulation of krill diel vertical migration (DVM). Moreover, I also examined the role played by photoperiod in the entrainment of krill DVM. DVM is a mass migratory movement displayed by many zooplankton species worldwide. During the night, the animals come to the surface to graze on phytoplankton, while during the day they sink to deeper layers to escape from visual predators. The environmental factors involved in the regulation of DVM are photoperiod, food availability and presence/absence of predators. However, DVM occurs also in constantly dark environments (e.g. the deep sea and the Arctic ocean during the polar night), suggesting the involvement of an endogenous rhythm of regulation. Using krill exposed to different light/dark (LD) and constant darkness (DD) conditions, I found that krill DVM was driven by an endogenous rhythm, with krill moving upward during the light phase and downward during the dark phase. A similar rhythm was found in krill oxygen consumption, confirming the presence of an endogenous rhythm of activity associated with DVM. Rhythmic expression of clock genes related to the circadian clock was found in the eyestalks of krill entrained to similar LD conditions, suggesting that an involvement of the circadian clock in the regulation of krill DVM would be possible. Major differences were observed among individual krill in the rhythmic regulation of DVM and oxygen consumption, suggesting that the circadian system of krill might display high degrees of individual plasticity. In conclusion, this dissertation improves our knowledge about the mechanisms regulating daily and seasonal rhythmic functions in the Antarctic krill, E. superba. The implication of endogenous rhythmicity was demonstrated for krill DVM at the daily level, and for krill seasonal metabolic cycle at the seasonal level. Photoperiod proved to be a most fundamental factor for the entrainment of krill DVM and krill seasonal metabolic cycle, as well as for the modulation of the activity of the circadian clock of krill at different times of the year. This work provides an example of how techniques which have been developed to study the molecular biology and chronobiology of terrestrial model species can be applied to the study of ecologically relevant species in the marine environments. In the future, understanding the regulation of rhythmic functions in ecological key marine species like Antarctic krill will help us to understand how these species will adapt to environmental changes driven by climate change.

Description: Antarctic krill (Euphausia superba) are high latitude pelagic organisms which play a key ecological role in the ecosystem of the Southern Ocean. To synchronize their daily and seasonal life-traits with their highly rhythmic environment, krill rely on the implementation of rhythmic strategies which might be regulated by a circadian clock. A recent analysis of krill circadian transcriptome revealed that their clock might be characterized by an endogenous free-running period of about 12–15 h. Using krill exposed to simulated light/dark cycles (LD) and constant darkness (DD), we investigated the circadian regulation of krill diel vertical migration (DVM) and oxygen consumption, together with daily patterns of clock gene expression in brain and eyestalk tissue. In LD, we found clear 24 h rhythms of DVM and oxygen consumption, suggesting a synchronization with photoperiod. In DD, the DVM rhythm shifted to a 12 h period, while the peak of oxygen consumption displayed a temporal advance during the subjective light phase. This suggested that in free-running conditions the periodicity of these clock-regulated output functions might reflect the shortening of the endogenous period observed at the transcriptional level. Moreover, differences in the expression patterns of clock gene in brain and eyestalk, in LD and DD, suggested the presence in krill of a multiple oscillator system. Evidence of short periodicities in krill behavior and physiology further supports the hypothesis that a short endogenous period might represent a circadian adaption to cope with extreme seasonal photoperiodic variability at high latitude.

Description: An endogenous circadian clock influences metabolic output rhythms in the Antarctic krill (Euphausia superbaDana, 1850), a key species in the Southern Ocean ecosystem. Seasonal changes in photoperiod in Antarctica, ranging from midnight sun (24 h light) during mid-summer to very short days (3–4 h light) during mid-winter, represent a challenge for the synchronization of the krill circadian clock. We analyzed clock gene activity and clock output functions in krill exposed to different light conditions during a long-term photoperiodic simulation in the laboratory. In simulated early-autumn (light/dark or LD 16:8) and late-winter (LD 8:16) conditions, the circadian clock of krill was functional and the metabolic output was synchronized to the light/dark cycle, the clock genes Esper and Esclk peaked in antiphase around simulated dusk/dawn and most metabolic-related genes showed upregulation around simulated dusk. In contrast, in simulated mid-summer (light/light or LL) and mid-winter (LD 3:21) conditions, the synchronization of the circadian clock and the metabolic output appeared to be weaker, with clock gene expression becoming arrhythmic and upregulation of metabolic genes occurring at different times during the day. Early-autumn and late-winter photoperiodic cues in the laboratory thus seem to be sufficient to entrain the krill clock and promote metabolic synchronization, whereas mid-winter and mid-summer photoperiodic cues seem to be insufficient for krill entrainment. Krill in the field may overcome the seasonal lack of overt photoperiodic cycle occurring during mid-summer and mid-winter by using alternative light-related Zeitgebers (i.e., varying light intensity rather than the presence or absence of light) to promote basic homeostatic rhythms over 24 h.

Description: Biological clocks are universal to all living organisms on Earth. Their ubiquity is testament to their importance to life: from cells to organs and from the simplest cyanobacteria to plants and primates, they are central to orchestrating life on this planet. Biological clocks are usually set by the day–night cycle, so what happens in polar regions during the Polar Night or Polar Day when there are periods of 24! hours of darkness or light? How would a biological clock function without a timekeeper!cycle? This chapter details evidence that biological clocks are central to structuring daily and seasonal activities in organisms at high latitudes. Importantly, despite a strongly reduced or absent day–night cycle, biological clocks in the Polar Night still appear to be regulated by background illumination. Here we explore evidence for highly cyclic activity, from behaviour patterns to clock gene expression, in copepods, krill and bivalves. The ultimate goal will be to understand the role of endogenous clocks in driving important daily and seasonal life cycle functions and to determine scope for plasticity in a rapidly changing environment.

Description: Antarctic krill (Euphausia superba) are high latitude pelagic organisms which play a key ecological role in the ecosystem of the Southern Ocean. To synchronize their daily and seasonal life-traits with their highly rhythmic environment, krill rely on the implementation of rhythmic strategies which might be regulated by a circadian clock. A recent analysis of krill circadian transcriptome revealed that their clock might be characterized by an endogenous free-running period of about 12–15 h. Using krill exposed to simulated light/dark cycles (LD) and constant darkness (DD), we investigated the circadian regulation of krill diel vertical migration (DVM) and oxygen consumption, together with daily patterns of clock gene expression in brain and eyestalk tissue. In LD, we found clear 24 h rhythms of DVM and oxygen consumption, suggesting a synchronization with photoperiod. In DD, the DVM rhythm shifted to a 12 h period, while the peak of oxygen consumption displayed a temporal advance during the subjective light phase. This suggested that in free-running conditions the periodicity of these clock-regulated output functions might reflect the shortening of the endogenous period observed at the transcriptional level. Moreover, differences in the expression patterns of clock gene in brain and eyestalk, in LD and DD, suggested the presence in krill of a multiple oscillator system. Evidence of short periodicities in krill behavior and physiology further supports the hypothesis that a short endogenous period might represent a circadian adaption to cope with extreme seasonal photoperiodic variability at high latitude.