The molecular basis of diel and seasonal rhythmicity in the copepod Calanus finmarchicus

The copepod Calanus finmarchicus has an ecological key position in the northern Atlantic pelagic food web and its life is characterized by diel and seasonal rhythmicity. Neither diel nor seasonal rhythmicity of C. finmarchicus are understood with regard to their mechanistic regulation. Endogenous clock systems are central in controlling rhythms in various terrestrial species, but have hardly been investigated in marine organisms. This thesis shows that C. finmarchicus possesses an endogenous circadian clock, that regulated 24h rhythms of gene expression, metabolic activity and vertical migration behavior. The thesis further suggests that clock-based day length measurement and an endogenous annual clock is involved in the regulation of seasonal rhythmicity. The findings on C. finmarchicus’ timing systems are further related to the extreme light conditions in polar environments, discussing potential effects of climate chance on the copepods rhythmicity and biology

Calanus finmarchicus diel and seasonal rhythmicity in relation to endogenous timing under extreme polar photoperiods

Changing environmental conditions cause poleward distribution shifts in many marine organisms including the northern Atlantic key zooplankton species Calanus finmarchicus. The copepod has diel cycles of vertical migration and feeding, a seasonal life cycle with diapause in winter and a functioning circadian clock. Endogenous clock mechanisms control various aspects of rhythmic life and are heavily influenced by environmental light conditions. Here we explore how the extreme seasonal change in photoperiod (day length) in a high Arctic fjord affects circadian clock functioning as well as diel and seasonal cycles in C. finmarchicus. Expression of clock genes was measured in the active life phase at the end of midnight sun, in early diapause when photoperiod was ~12 h, and in late diapause during the polar night. While the clock maintained diel rhythmicity under extremely long photoperiods, it became arrhythmic during diapause. This was probably not due to a lack of light but was related to the physiological state of diapause. Seasonal expression analyses of 35 genes show distinct patterns for each investigated life phase. C. finmarchicus is able to maintain diel clock rhythmicity at photoperiods close to 24 h, and it is discussed how this may be related to the nature of the marine environment. The work also evaluates the potential negative consequences of rigid clock-based seasonal timing in a polar environment exposed to climate change and with high interannual variability

Calanus finmarchicus seasonal cycle and diapause in relation to gene expression, physiology, and endogenous clocks: Calanus finmarchicus seasonal rhythmicity

The copepod Calanus finmarchicus plays a crucial role in the north Atlantic food web. Its seasonal life cycle involves reproduction and development in surface waters before overwintering in diapause at depth. Although diapause has been studied for more than a century, the factors responsible for the initiation and termination of it are still unclear. Endogenous clocks have been identified as potent tools for photoperiod measurement and seasonal rhythmicity in many terrestrial species, but knowledge of these remains scarce in the marine realm. Focusing on the dominant CV copepodid stage, we sampled a population of C. finmarchicus from a Scottish sea loch to characterize population dynamics, several physiological parameters, and diel and seasonal expression rhythms of 35 genes representing different metabolic pathways, including the circadian clock machinery. This generated a detailed overview of the seasonal cycle of C. finmarchicus including the most extensive field dataset on circadian clock gene expression in a marine species to date. Gene expression patterns revealed distinct gene clusters upregulated at different phases of the copepod's seasonal cycle. While diel clock cycling was restricted to the active spring/summer phase, many clock genes exhibited the highest expression during diapause. Our results provide new insights into diapause on physiological and genetic levels. We suggest that photoperiod, in interaction with internal and external factors (lipid content, temperature, food availability) and the endogenous clock mechanism, plays an important role in the timing of diapause in C. finmarchicus

Circadian Clock Involvement in Zooplankton Diel Vertical Migration

Biological clocks are a ubiquitous ancient and adaptive mechanism enabling organisms to anticipate environmental cycles and to regulate behavioral and physiological processes accordingly [1]. Although terrestrial circadian clocks are well understood, knowledge of clocks in marine organisms is still very limited [2, 3, 4, 5]. This is particularly true for abundant species displaying large-scale rhythms like diel vertical migration (DVM) that contribute significantly to shaping their respective ecosystems [6]. Here we describe exogenous cycles and endogenous rhythms associated with DVM of the ecologically important and highly abundant planktic copepod Calanus finmarchicus. In the laboratory, C. finmarchicus shows circadian rhythms of DVM, metabolism, and most core circadian clock genes (clock, period1, period2, timeless, cryptochrome2, and clockwork orange). Most of these genes also cycle in animals assessed in the wild, though expression is less rhythmic at depth (50–140 m) relative to shallow-caught animals (0–50 m). Further, peak expressions of clock genes generally occurred at either sunset or sunrise, coinciding with peak migration times. Including one of the first field investigations of clock genes in a marine species [5, 7], this study couples clock gene measurements with laboratory and field data on DVM. While the mechanistic connection remains elusive, our results imply a high degree of causality between clock gene expression and one of the planet’s largest daily migrations of biomass. We thus suggest that circadian clocks increase zooplankton fitness by optimizing the temporal trade-off between feeding and predator avoidance, especially when environmental drivers are weak or absent [8]

Clock genes in a north Atlantic key zooplankter - Expression during overwintering in a high Arctic fjord

The copepod Calanus finmarchicus plays a crucial role in the north Atlantic food web, channelling energy from phytoplankton primary production to higher trophic levels including commercially important fish stocks like herring and cod. The copepod species is spreading northward into the Arctic due to ocean warming. The activity phase of C. finmarchicus in spring/summer is characterized by diel vertical migration, meaning that the animals migrate to surface waters around sunset to feed, and back to deeper layers around sunrise to hide from visual predators. This rhythmic vertical migration behaviour is characteristic for zooplankton communities all around the world. At the end of the activity phase in autumn, C. finmarchicus enters an overwintering mode and inactively dwell in deep waters until next spring when it starts a new generation cycle. Although both rhythms (diel and seasonal) have been studied for more than a century, the exact factors controlling them are still unclear. Molecular techniques have precisely described genetic clockworks in numerous species and there is clear evidence that clock genes are not only involved in the regulation of diel 24h rhythms, but also in the entrainment of the seasonal cycle. We present first records of clock gene expression in Calanus finmarchicus from a high Arctic fjord in Svalbard at 79°N and compare gene activity between specimen in the early and late phase of overwintering. Copepods were sampled from overwintering depth (>220 m) in September 2014 when surface photoperiod was about 10 hours and during polar night in January 2015 when no light was present. Samples were analysed by quantitative real-time PCR (qRT-PCR) using custom designed Taqman® low-density array cards. The results show clear 24h oscillations in most genes for September, whereas gene expression is almost completely arrhythmic during the polar night in January. It furthermore appears that in September most of the investigated clock genes show distinct expressions patterns, which often match pattern previously observed in other (model) species. For example, expression of period (1 & 2) is highest around sunset (per1) or early night (per2) whereas activity of clock sharply increases around sunrise and peaks in the afternoon. Expression of cryptochrome 1 is highest around midnight while expression of cryptochrome 2 shows patterns similar to those of the period genes. The results strongly point towards the existence of a light-entrained genetic clock in Calanus finmarchicus that becomes arrhythmic during the constant darkness of the polar night. Our work presents an example on how the vast mechanistic knowledge about endogenous timekeeping gained from model organisms can be transferred to field studies on non-model species of high ecological relevance

First records of clock gene activity in Calanus finmarchicus – Expression patters during overwintering in a high Arctic fjord

The copepod Calanus finmarchicus is a dominant zooplankter in the north Atlantic and is spreading northward into the Arctic due to ocean warming. The copepods life is characterized by diel vertical migration as well as a seasonal cycle with overwintering in deep waters. Although both phenome have been studied for more than a century, the exact factors controlling these rhythms are still unclear. Molecular techniques have precisely described genetic clockworks in several, mostly terrestrial species and there is clear evidence that clock genes are not only involved in the regulation of diel 24h rhythms, but can also play an important role in the synchronisation (entrainment) of the seasonal cycle. We present first records of clock gene expression in Calanus finmarchicus from Kongsfjorden, Svalbard and compare gene activity between specimen in the early and late phase of overwintering. Copepods were sampled from overwintering depth (>220 m) in September 2014 when day length was about 10 hours and during polar night in January 2015. The results show clear 24h oscillations in most genes for September, whereas gene expression is generally lower and almost completely arrhythmic during the polar night. The results strongly point towards the existence of a light-entrained genetic clock in Calanus finmarchicus. As the regulators of seasonal timing in this species are still unclear, understanding the mechanism of the clock could help assessing the adaptability of this boreal species to the strongly fluctuating light conditions at high latitudes. This could be crucial in predicting future seasonal mismatches and ecosystem consequences

Metabolic suppression during protracted exposure to hypoxia in the jumbo squid, \u3cem\u3eDosidicus gigas\u3c/em\u3e, living in an oxygen minimum zone

The jumbo squid, Dosidicus gigas, can survive extended forays into the oxygen minimum zone (OMZ) of the Eastern Pacific Ocean. Previous studies have demonstrated reduced oxygen consumption and a limited anaerobic contribution to ATP production, suggesting the capacity for substantial metabolic suppression during hypoxic exposure. Here, we provide a more complete description of energy metabolism and explore the expression of proteins indicative of transcriptional and translational arrest that may contribute to metabolic suppression. We demonstrate a suppression of total ATP demand under hypoxic conditions (1% oxygen, PO2=0.8 kPa) in both juveniles (52%) and adults (35%) of the jumbo squid. Oxygen consumption rates are reduced to 20% under hypoxia relative to air-saturated controls. Concentrations of arginine phosphate (Arg-P) and ATP declined initially, reaching a new steady state (~30% of controls) after the first hour of hypoxic exposure. Octopine began accumulating after the first hour of hypoxic exposure, once Arg-P breakdown resulted in sufficient free arginine for substrate. Octopine reached levels near 30 mmol g−1 after 3.4 h of hypoxic exposure. Succinate did increase through hypoxia but contributed minimally to total ATP production. Glycogenolysis in mantle muscle presumably serves to maintain muscle functionality and balance energetics during hypoxia. We provide evidence that post-translational modifications on histone proteins and translation factors serve as a primary means of energy conservation and that select components of the stress response are altered in hypoxic squids. Reduced ATP consumption under hypoxia serves to maintain ATP levels, prolong fuel store use and minimize the accumulation of acidic intermediates of anaerobic ATP-generating pathways during prolonged diel forays into the OMZ. Metabolic suppression likely limits active, daytime foraging at depth in the core of the OMZ, but confers an energetic advantage over competitors that must remain in warm, oxygenated surface waters. Moreover, the capacity for metabolic suppression provides habitat flexibility as OMZs expand as a result of climate change

Circadian clock involvement in zooplankton diel vertical migration, link to supplementary material

Genetic clocks are a ubiquitous ancient and adaptive mechanism enabling organisms to anticipate environmental rhythms and to regulate behavioral, physiological and behavioral processes accordingly. Whilst terrestrial circadian clocks are well studied and understood, knowledge about the clock systems in marine organisms is still limited. This is particularly true for abundant species displaying large-scale rhythms like diel vertical migration (DVM) that contribute significantly to shaping their respective ecosystems. Here, we describe endogenous and highly rhythmic patterns in the biology of the ecologically important and highly abundant planktic copepod Calanus finmarchicus. This species shows circadian rhythms of DVM, metabolism, and most core circadian clock genes (clock, period1, period2, timeless, cryptochrome2, clockwork orange) in the laboratory. In the field, copepods from shallow water (0-50m) have more robust rhythmic clock gene oscillations than those caught in deeper water (140-50m). Further, peak expressions of clock genes generally occurred at either sunset or sunrise coinciding with peak migration times. Providing one of the first field investigations of clock gene rhythmicity in a marine species this study further couples clock genes measurements with laboratory and field data on DVM. While the mechanistic connection remains elusive, our results imply a high degree of causality between clock gene expression and one of the planet's largest daily migration of biomass. This could increase zooplankton fitness by optimizing the temporal trade-off between feeding and predator avoidance