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Antarctic krill (Euphausia superba) is a high latitude pelagic organism which plays a central role in the Southern Ocean ecosystem. E. superba shows daily and seasonal rhythms in physiology and behaviour, which are synchronized with the environmental cycles of its habitat. Recently, the main components of the krill circadian machinery have been identified and characterized. However, the exact mechanisms through which the endogenous timing system operates the control and regulation of the overt rhythms remains only partially understood. Here we investigate the involvement of the circadian clock in the temporal orchestration of gene expression by using a newly developed version of a krill microarray platform. The analysis of transcriptome data from krill exposed to both light-dark cycles (LD 18:6) and constant darkness (DD), has led to the identification of 1,564 putative clock-controlled genes. A remarkably large proportion of such genes, including several clock components (clock, period, cry2, vrille, and slimb), show oscillatory expression patterns in DD, with a periodicity shorter than 24 hours. Energy-storage pathways appear to be regulated by the endogenous clock in accordance with their ecological relevance in daily energy managing and overwintering. Our results provide the first representation of the krill circadian transcriptome under laboratory, free-running conditions.

Light regime is an important zeitgeber for Antarctic krill (Euphausia superba Dana, 1850), which seems to entrain an endogenous timing system that synchronizes its life cycle to the extreme light conditions in the Southern Ocean. To understand the flexibility of Antarctic krill’s seasonal cycle, we investigated its physiological and behavioural responses to different light regimes and if an endogenous timing system was involved in the regulation of these seasonal processes. We analysed growth, feeding, lipid content, and maturity in a 2-year laboratory experiment simulating the latitudinal light regimes at 52°S and 66°S and constant darkness under constant food level. Our results showed that light regime affected seasonal cycles of growth, feeding, lipid metabolism, and maturity in Antarctic krill. Seasonal patterns of growth, feeding, and maturity persisted under constant darkness, indicating the presence of an endogenous timing system. The maturity cycle showed differences in critical photoperiods according to the simulated latitudinal light regime. This suggests a flexible endogenous timing mechanism in Antarctic krill, which may determine its response to future environmental changes.

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.

The Southern Ocean ecosystem is characterized by extreme seasonal changes in environmental factors such as day length, sea ice extent and food availability. The key species Antarctic krill (Euphausia superba) has evolved metabolic and behavioural seasonal rhythms to cope with these seasonal changes. We investigate the switch between a physiological less active and active period for adult krill, a rhythm which seems to be controlled by internal biological clocks. These biological clocks can be synchronized by environmental triggers such as day length and food availability. They have evolved for particular environmental regimes to synchronize predictable seasonal environmental changes with important life cycle functions of the species. In a changing environment the time when krill is metabolically active and the time of peak food availability may not overlap if krill's seasonal activity is solely determined by photoperiod (day length). This is especially true for the Atlantic sector of the Southern Ocean where the spatio-temporal ice cover dynamics are changing substantially with rising average temperatures. We developed an individual-based model for krill to explore the impact of photoperiod and food availability on the growth and demographics of krill. We simulated dynamics of local krill populations (with no movement of krill assumed) along a south-north gradient for different triggers of metabolic activity and different levels of food availability below the ice. We also observed the fate of larval krill which cannot switch to low metabolism and therefore are likely to overwinter under ice. Krill could only occupy the southern end of the gradient, where algae bloom only lasts for a short time, when alternative food supply under the ice was high and metabolic activity was triggered by photoperiod. The northern distribution was limited by lack of overwintering habitat for krill larvae due to short duration of sea ice cover even for high food content under the ice. The variability of the krill's length-frequency distributions varied for different triggers of metabolic activity, but did not depend on the sea ice extent. Our findings suggest a southward shift of krill populations due to reduction in the spatial sea ice extent, which is consistent with field observations. Overall, our results highlight the importance of the explicit consideration of spatio-temporal sea ice dynamics especially for larval krill together with temporal synchronization through internal clocks, triggered by environmental factors (photoperiod and food) in adult krill for the population modelling of krill.

The Antarctic krill, Euphausia superba, has a key position in the Southern Ocean food web by serving as direct link between primary producers and apex predators. The southwest Atlantic sector of the Southern Ocean, where the majority of the krill population is located, is experiencing one of the most profound environmental changes worldwide. Up to now, we have only cursory information about krill’s genomic plasticity to cope with the ongoing environmental changes induced by anthropogenic CO2 emission. The genome of krill is not yet available due to its large size (about 48 Gbp). Here, we present two cDNA normalized libraries from whole krill and krill heads sampled in different seasons that were combined with two datasets of krill transcriptome projects, already published, in order to produce the first knowledgebase krill ‘master’ transcriptome. The new library produced 25% more E. superba transcripts and now includes nearly all the enzymes involved in the primary oxidative metabolism (Glycolysis, Krebs cycle and oxidative phosphorylation) as well as all genes involved in glycogenesis, glycogen breakdown, gluconeogenesis, fatty acid synthesis and fatty acids β-oxidation. With these features, the ‘master’ transcriptome provides the most complete picture of metabolic pathways in Antarctic krill and will provide a major resource for future physiological and molecular studies. This will be particularly valuable for characterizing the molecular networks that respond to stressors caused by the anthropogenic CO2 emissions and krill’s capacity to cope with the ongoing environmental changes in the Atlantic sector of the Southern Ocean.

Adult Euphausia superba survive winter without or with little feeding. It is not exactly known whether the scarcity of food or an internal clock, set by the natural Antarctic light regime, are responsible for non-feeding. Our research questions were therefore the following: (1) How will physiological and biochemical conditions of krill change during long-term starvation at constant light regime? (2) If and how do enzyme activities change during such starvation? (3) What is the influence of food availability versus that of light regime? To answer these questions, adult krill were starved under laboratory conditions for 12 weeks with constant light regime (12:12; dark/light) and the impact on physiological functions was studied. Initial experimental condition of krill resembled the condition of late spring krill in the field with fully active metabolism and low lipid reserves. Metabolic activity and activities of enzymes catabolising lipids decreased after the onset of starvation and remained low throughout, whereas lipid reserves declined and lipid composition changed. Mass and size of krill decreased while the inter-moult period increased. Depletion of storage- and structural metabolites occurred in the order of depot lipids and glycogen reserves after onset of starvation until proteins were almost exclusively used after 6–7 weeks of starvation. Results confirmed various proposed overwintering mechanisms such as metabolic slowdown, slow growth or shrinkage and use of lipid reserves. However, these changes were set in motion by food shortage only, i.e. without the trigger of a changing light regime.

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 [2-5]. 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].

A dominant Antarctic ecological paradigm suggests that winter sea ice is generally the main feeding ground for krill larvae. Observations from our winter cruise to the southwest Atlantic sector of the Southern Ocean contradict this view and present the first evidence that the pack-ice zone is a food-poor habitat for larval development. In contrast, the more open marginal ice zone provides a more favourable food environment for high larval krill growth rates. We found that complex under-ice habitats are, however, vital for larval krill when water column productivity is limited by light, by providing structures that offer protec- tion from predators and to collect organic material released from the ice. The larvae feed on this sparse ice-associated food during the day. After sunset, they migrate into the water below the ice (upper 20 m) and drift away from the ice areas where they have previously fed. Model analyses indicate that this behaviour increases both food uptake in a patchy food environment and the likelihood of overwinter transport to areas where feeding conditions are more favourable in spring.

18 pages, 9 figures, 10 tables, We investigated physiological parameters (elemental and biochemical composition, metabolic rates, feeding activity and growth) of adult Antarctic krill in the Lazarev Sea in late spring (December), mid autumn (April) and mid winter (July and August) to evaluate proposed hypotheses of overwintering mechanisms. Our major observations are: (1) respiration rates were reduced by 30 to 50% in autumn and winter, compared to values in late spring; (2) feeding activity was reduced by 80 to 86% in autumn and winter, compared to late spring, at similar food concentrations; (3) feeding was omnivorous during winter; (4) with each moult, krill grew by 0.5 to 3.8% in length; (5) body lipids and, to a small extent, body proteins were consumed during winter. Adult Euphausia superba thus adopt metabolic slowdown and omnivorous feeding activity at low rates to survive the winter season in the Lazarev Sea. By mid autumn, metabolic activity is reduced, most likely being influenced by the Antarctic light regime, which is accompanied by a reduction in feeding activity and growth. Although at a low level, the feeding activity during winter seems to provide an important energy input, This work was supported by funding from the German Ministry of Education and Research (BMBF) through project 03F0400A, Subproject 4 of the Lazarev Sea Krill Study (LAKRIS) Project, the International Bureau of the BMBF through project SUA 05/008, and the Natural Environment Research Council (United Kingdom, AFI 5/09). The LAKRIS Project is the German contribution to the Southern Ocean-Global Ocean Ecosystem Dynamics (SO-GLOBEC) program

A change in photoperiod has been implicated in triggering a transition from an active to a quiescent state in Antarctic krill. We examined this process at the molecular level, to identify processes that are affected when passing a photoperiodic threshold. Antarctic krill captured in the austral autumn were divided into two groups and immediately incubated either under a photoperiod of 12 h light:12 h darkness (LD), simulating the natural light cycle, or in continuous darkness (DD), simulating winter. All other conditions were kept identical between incubations. After 7 days of adaptation, krill were sampled every 4 h over a 24 h period and frozen. Total RNA was extracted from the heads and pooled to construct a suppression subtractive hybridisation library. Differentially expressed sequences were identified and annotated into functional categories through database sequence matching. We found a difference in gene expression between LD and DD krill, with LD krill expressing more genes involved in functions such as metabolism, motor activity, protein binding and various other cellular activities. Eleven of these genes were examined further with quantitative polymerase chain reaction analyses, which revealed that expression levels were significantly higher in LD krill. The genes affected by simulated photoperiodic change are consistent with known features of quiescence, such as a slowing of moult rate, a lowering of activity levels and a reduction in metabolic rate. The expression of proteases involved in apolysis, where the old cuticle separates from the epidermis, showed particular sensitivity to photoperiod and point to the mechanism by which moult rate is adjusted seasonally. Our results show that key processes are already responding at the molecular level after just 7 days of exposure to a changed photoperiodic cycle. We propose that krill switch rapidly between active and quiescent states and that the photoperiodic cycle plays a key role in this process.

The effect of different light regimes on the development of sexual maturity and body composition (carbon, nitrogen, lipid and protein) of Antarctic krill, Euphausia superba, was studied over 12 weeks under laboratory conditions. Krill were exposed to light-cycle regimes of variable intensity to simulate Southern Ocean summer, autumn and winter conditions, respectively using: (1) continuous light (LL; 200 lux max), (2) 12-h light and 12-h darkness (LD 12:12; 50 lux max), and (3) continuous darkness (DD). The sexual maturity of female and male krill exposed to LL and LD 12:12 showed an accelerated succession of external maturity stages during the experimental period, while krill exposed to continuous darkness showed no changes in external maturity during the course of the study. Changes in the maturity development of krill between the different light regimes are reflected in changes in body composition. Krill exposed to LL and LD 12:12 showed an increase in lipid utilization, indicating that the development of external maturation may be fuelled preferentially by lipid reserves. In contrast, values of total lipid content of krill held under continuous darkness indicated an unchanged lipid catabolism during the course of the study. Thus, the maturity development of krill was affected either directly or indirectly by the different simulated light conditions. Based on these results, and observations on the effects of simulated light regimes on feeding and metabolic rates of krill available from a previous study, we suggest that the Antarctic light regime is an essential cue governing the seasonal cycle of krill physiology and maturity, and highlight the importance of this environmental factor in the life history of krill.

The Antarctic krill Euphausia superba experiences almost all marine photic environments throughout its life cycle. Antarctic krill eggs hatch in the aphotic zone up to 1000m depth and larvae develop on their way to the ocean surface (development ascent) and are exposed to different quality (wavelength) and quantity (irradiance) of light. Adults show a daily vertical migration pattern, moving downward during the day and upward during the night within the top 200m of the water column. Seawater acts as a potent chromatic filter and animals have evolved different opsin photopigments to perceive photons of specific wavelengths. We have investigated the transcriptome of E. superba and, using a candidate gene approach, we identified six novel opsins. Five are r-type visual opsins: four middle-wavelength-sensitive (EsRh2, EsRh3, EsRh4 and EsRh5) and one long-wavelength-sensitive (EsRh6). Moreover, we have identified a non-visual opsin, the EsPeropsin. All these newly identified opsin genes were significantly expressed in compound eyes and brain, while only EsPeropsin and EsRh2 were clearly detected also in the abdomen. A temporal modulation in the transcription of these novel opsins was found, but statistically significant oscillations were only observed in EsRrh3 and EsPeropsin. Our results contribute to the dissection of the complex photoreception system of E. superba, which enables this species to respond to the daily and seasonal changes in irradiance and spectral composition in the Southern Ocean.

Since the 1920s, E. superba is one of the best studied species in the Southern Ocean in terms of their general biology. The main driver for this research focus has been the fisheries’ requirements for stock forecasting and conservation measures. Nowadays this is joined by concerns over climate change effects and the requirement to take a more holistic view to understand food web structures. So far, however, we do not have a clear understanding of the physiological response of krill and hence their adaptability to cope with ongoing environmental changes, caused by the anthropogenic carbon emissions. This is due to the extreme lack of intense studies on krill physiology, especially of their larval stages in relation to their seasonal environment. A major aim of this book chapter is on the one hand to summarize how physiological functions such as lipid accumulation and utilisation, metabolic activity and growth change with ontogeny and season and to demonstrate which environmental factors are the main drivers for seasonal variability of these functions in adult and larval krill. On the other hand, we draw the attention to the importance of photoperiod (day length) as an entrainment cue for endogenous rhythms and clocks in the life cycle of krill. Furthermore, we give an overview of the current knowledge on the impact of elevated seawater temperature and ocean acidification on krill.

International audience; Antarctic krill Euphausia superba (hereafter `krill') occur in regions undergoing rapid environmental change, particularly loss of winter sea ice. During recent years, harvesting of krill has in creased, possibly enhancing stress on krill and Antarctic ecosystems. Here we review the overall impact of climate change on krill and Antarctic ecosystems, discuss implications for an ecosystem-based fisheries management approach and identify critical knowledge gaps. Sea ice decline, ocean warming and other environmental stressors act in concert to modify the abundance, distribution and life cycle of krill. Although some of these changes can have positive effects on krill, their cumulative impact is most likely negative. Recruitment, driven largely by the winter survival of larval krill, is probably the population parameter most susceptible to climate change. Predicting changes to krill populations is urgent, because they will seriously impact Antarctic ecosystems. Such predictions, however, are complicated by an intense inter-annual variability in recruitment success and krill abundance. To improve the responsiveness of the ecosystem-based management approach adopted by the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR), critical knowledge gaps need to be filled. In addition to a better understanding of the factors influencing recruitment, management will require a better understanding of the resilience and the genetic plasticity of krill life stages, and a quantitative understanding of under-ice and benthic habitat use. Current precautionary management measures of CCAMLR should be maintained until a better understanding of these processes has been achieved. [GRAPHICS] .

Antarctic krill, Euphausia superba, shapes the structure of the Southern Ocean ecosystem. Its central position in the food web, the ongoing environmental changes due to climatic warming, and increasing commercial interest on this species emphasize the urgency of understanding the adaptability of krill to its environment. Krill has evolved rhythmic physiological and behavioral functions which are synchronized with the daily and seasonal cycles of the complex Southern Ocean ecosystem. The mechanisms, however, leading to these rhythms are essentially unknown. Here, we show that krill possesses an endogenous circadian clock that governs metabolic and physiological output rhythms. We found that expression of the canonical clock gene cry2 was highly rhythmic both in a light-dark cycle and in constant darkness. We detected a remarkable short circadian period, which we interpret as a special feature of the krill's circadian clock that helps to entrain the circadian system to the extreme range of photoperiods krill is exposed to throughout the year. Furthermore, we found that important key metabolic enzymes of krill showed bimodal circadian oscillations (∼9–12 h period) in transcript abundance and enzymatic activity. Oxygen consumption of krill showed ∼9–12 h oscillations that correlated with the temporal activity profile of key enzymes of aerobic energy metabolism. Our results demonstrate the first report of an endogenous circadian timing system in Antarctic krill and its likely link to metabolic key processes. Krill's circadian clock may not only be critical for synchronization to the solar day but also for the control of seasonal events. This study provides a powerful basis for the investigation into the mechanisms of temporal synchronization in this marine key species and will also lead to the first comprehensive analyses of the circadian clock of a polar marine organism through the entire photoperiodic cycle.

The utilization of dietary proteins in crustaceans is facilitated by a set of peptide hydrolases which are often dominated by “trypsin-like” serine proteinases. As expected, the North Sea shrimps Crangon crangon and Crangon allmani showed in their midgut glands high proteolytic activities. However, the majority of animals lacked trypsin and chymotrypsin. Conversely, a minority of about 10% of the animals had elevated trypsin activities. The appearance of trypsin was neither related to the mode of feeding nor to the nutritive state of the animals. When present, trypsin was expressed in both species as a single isoform of apparently 20 kDa. The lack of serine proteinases was also confirmed by inhibitor assays. AEBSF, a serine proteinase inhibitor, slightly reduced total proteinase activity by less than 10%. In contrast E 64, a cysteine proteinase inhibitor, caused a reduction of more than 70% of total proteinase activity, indicating that a substantial share of proteolytic activity is caused by cysteine proteinases. Cathepsin L-like proteinases were identified as major cysteine proteinases. A comparison with the eucarid crustaceans Pandalus montagui , Pagurus bernhardus , Cancer pagurus and Euphausia superba showed a similar high level of total proteinase activity in all species. Trypsin, however, varied significantly between species showing lowest activities in Caridea and the highest activity in E. superba . E 64 suppressed total proteinase activity by more than 70% in Crangon species but not in C. pagurus and E. superba . In contrast, the serine proteinase inhibitor AEBSF had only little effect in Caridea but was most effective in P. bernhardus , C. pagurus and E. superba . The results may indicate different traits of food utilization strategies in some eucarid crustaceans. Caridea may express predominantly cysteine proteinase, while in Anomura, Brachyura and Euphausiacea, serine proteinases may prevail.