Description: The Antarctic krill, Euphausia superba, has evolved seasonal rhythms of physiology and behaviour to survive under the extreme photoperiodic conditions in the Southern Ocean. However, the molecular mechanisms generating these rhythms remain far from understood. The aim of this study was to investigate seasonal differences in gene expression in three different latitudinal regions (South Georgia, South Orkneys/Bransfield Strait, Lazarev Sea) and to identify genes with potential regulatory roles in the seasonal life cycle of Antarctic krill. The RNA-seq data were analysed (a) for seasonal differences between summer and winter krill sampled from each region, and (b) for regional differences within each season. A large majority of genes showed an up-regulation in summer krill in all regions with respect to winter krill. However, seasonal differences in gene expression were less pronounced in Antarctic krill from South Georgia, most likely due to the milder seasonal conditions of the lower latitudes of this region, with a less extreme light regime and food availability between summer and winter. Our results suggest that in the South Orkneys/Bransfield Strait and Lazarev Sea region, Antarctic krill entered a state of metabolic depression and regressed development (winter quiescence) in winter. Moreover, seasonal gene expression signatures seems to be driven by a photoperiodic timing system that may adapt the flexible behaviour and physiology of Antarctic krill to the highly seasonal environment according to the latitudinal region. However, at the lower latitude South Georgia region, food availability might represent the main environmental cue influencing seasonal physiology.

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) 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.

Description: Antarctic krill (Euphausia superba) is a key species in Southern Ocean ecosystem where it plays a central role in the Antarctic food web. Available information supports the existence of an endogenous timing system in krill enabling it to synchronize metabolism and behavior with an environment characterized by extreme seasonal changes in terms of day length, food availability, and surface ice extent. A screening of our transcriptome database “KrillDB” allowed us to identify the putative orthologues of 20 circadian clock components. Mapping of conserved domains and phylogenetic analyses strongly supported annotations of the identi ed sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to de ne the role of the main clock components. Our ndings provide an overall picture of the molecular mechanisms underlying the functioning of the endogenous circadian clock in the Antarctic krill and shed light on their evolution throughout crustaceans speciation. Interestingly, the core clock machinery shows both mammalian and insect features that presumably contribute to an evolutionary strategy to cope with polar environment’s challenges. Moreover, despite the extreme variability characterizing the Antarctic seasonal day length, the conserved light mediated degradation of the photoreceptor EsCRY1 suggests a persisting pivotal role of light as a Zeitgeber.

Description: 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 1000 m 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 200 m 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-wavelengthsensitive (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 systemof E. superba, which enables this species to respond to the daily and seasonal changes in irradiance and spectral composition in the Southern Ocean.

Description: 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.

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: Antarctic krill (Euphausia superba) is a key species in the Southern Ocean with an estimated biomass between 100 and 500 million tonnes. Changes in krill population viability would have catastrophic effect on the Antarctic ecosystem. One looming threat due to elevated levels of anthropogenic atmospheric carbon dioxide (CO2) is ocean acidification (lowering of sea water pH by CO2 dissolving into the oceans). The genetics of Antarctic krill has long been of scientific interest for both for the analysis of population structure and analysis of functional genetics. However, the genetic resources available for the species are relatively modest. We have developed the most advanced genetic database on Euphausia superba, KrillDB, which includes comprehensive data sets of former and present transcriptome proj- ects. In particular, we have built a de novo transcriptome assembly using more than 360 mil- lion Illumina sequence reads generated from larval krill including individuals subjected to different CO2 levels. The database gives access to: 1) the full list of assembled genes and transcripts; 2) their level of similarity to transcripts and proteins from other species; 3) the predicted protein domains contained within each transcript; 4) their predicted GO terms; 5) the level of expression of each transcript in the different larval stages and CO2 treatments. All references to external entities (sequences, domains, GO terms) are equipped with a link to the appropriate source database. Moreover, the software implements a full-text search engine that makes it possible to submit free-form queries. KrillDB represents the first large- scale attempt at classifying and annotating the full krill transcriptome. For this reason, we believe it will constitute a cornerstone of future approaches devoted to physiological and molecular study of this key species in the Southern Ocean food web.