The Antarctic Krill Euphausia superba Shows Diurnal Cycles of Transcription under Natural Conditions

Abstract

BackgroundPolar environments are characterized by extreme seasonal changes in day length, light intensity and spectrum, the extent of sea ice during the winter, and food availability. A key species of the Southern Ocean ecosystem, the Antarctic krill (Euphausia superba) has evolved rhythmic physiological and behavioral mechanisms to adapt to daily and seasonal changes. The molecular organization of the clockwork underlying these biological rhythms is, nevertheless, still only partially understood.Methodology/Principal FindingsThe genome sequence of the Antarctic krill is not yet available. A normalized cDNA library was produced and pyrosequenced in the attempt to identify large numbers of transcripts. All available E. superba sequences were then assembled to create the most complete existing oligonucleotide microarray platform with a total of 32,217 probes. Gene expression signatures of specimens collected in the Ross Sea at five different time points over a 24-hour cycle were defined, and 1,308 genes differentially expressed were identified. Of the corresponding transcripts, 609 showed a significant sinusoidal expression pattern; about 40% of these exibithed a 24-hour periodicity while the other 60% was characterized by a shorter (about 12-hour) rhythm. We assigned the differentially expressed genes to functional categories and noticed that those concerning translation, proteolysis, energy and metabolic process, redox regulation, visual transduction and stress response, which are most likely related to daily environmental changes, were significantly enriched. Two transcripts of peroxiredoxin, thought to represent the ancestral timekeeping system that evolved about 2.5 billion years ago, were also identified as were two isoforms of the EsRh1 opsin and two novel arrestin1 sequences involved in the visual transduction cascade.ConclusionsOur work represents the first characterization of the krill diurnal transcriptome under natural conditions and provides a first insight into the genetic regulation of physiological changes, which occur around the clock during an Antarctic summer day.

The opsin repertoire of the Antarctic krill Euphausia superba

“…We had previously observed daily rhythms in the expression of the krill opsins EsRh1a and EsRh1b, with a peak of expression at 06:00, in specimen exposed to natural lighting conditions, despite high levels of inter-individual variability (De Pittà et al, 2013). In the present study, the use of a robust non-parametric method specifically designed for the identification of biological rhythmicity allowed us to detect a significant sinusoidal pattern of oscillation for two out of the six newly identified opsins, namely EsPeropsin and EsRh3.…”

“…Assembled contigs ranged in size from 200 bp to 13.460 bp, with a median size of 328 bp. Each putative transcript was searched locally against the NR database downloaded from the NCBI FTP site on the 8.10.2015 by using Blast-N (against NCBI nucleotide database) and Blast-X (against NCBI protein database and TrEMBL) with an e-value cut-off of 1e-6 (De Pittà et al, 2013). The first five matches from each search were grouped and priority was given to Blast-N results.…”

“…The relative levels of each RNA were calculated by the 2 − ΔΔCT method (where CT is the cycle number at which the signal reaches the threshold of detection) (Livak and Schmittgen, 2001). As previously reported (Mazzotta et al, 2010;De Pittà et al, 2013), 18S was used as housekeeping gene (QuantumRNA™ 18S Internal Standards, Ambion, Austin, TX, USA). Each CT value used for these calculations is the mean of three replicates of the same reaction.…”

“…As we had previously observed a daily variation of EsRh1a and EsRh1b mRNA expression in the head of Antarctic krill collected under natural conditions (De Pittà et al, 2013), we investigated the temporal expression of the novel opsins detected in the E. superba transcriptome. qPCR was performed on specimens collected in the Ross Sea at different times of the day (01:00, 06:00, 10:00, 15:00, and 18:00) during the Antarctic summer (Mazzotta et al, 2010), when they were exposed to an almost continuous 24 h photoperiod characterized by daily variations in solar irradiance (Fig.…”

“…Previous investigations have identified three opsin genes in E. superba (Porter et al, 2007;De Pittà et al, 2013). Here, adopting a qualitative candidate gene approach, we analysed sequences from a de novo assembled transcriptome of krill and we identified 6 novel opsins.…”