Alberto Biscontin

The Antarctic krill Euphausia superba shows diurnal cycles of transcription under natural conditions.

Background Polar 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 Findings The 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. Conclusions Our 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.

Pyrosequencing and de novo assembly of Antarctic krill (Euphausia superba) transcriptome to study the adaptability of krill to climate-induced environmental changes.

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 south‐west 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 krills 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 data sets of krill transcriptome projects, already published, 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 krills capacity to cope with the ongoing environmental changes in the Atlantic sector of the Southern Ocean.

New miRNA labeling method for bead-based quantification

BackgroundmicroRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies.ResultsHere we have applied with an innovative approach, the Luminex® xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies.ConclusionsWe demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex® xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.

The opsin repertoire of the Antarctic krill Euphausia superba

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.

Leigh Syndrome in Drosophila melanogaster: MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF Surf1 POST-TRANSCRIPTIONAL SILENCING*

Background: Mutations in SURF1, a human gene important for the assembly of cytochrome-c-oxidase (COX) causes Leigh Syndrome, a mitochondrial encephalopathy. Results: Knockdown of Surf1 in Drosophila matches the biochemical features of the human disease. Conclusion: In Drosophila SURF1 is essential to maintain mitochondrial function and its silencing leads to COX deficiency. Significance: Drosophila offers new tools to investigate the pathogenic mechanism of Leigh Syndrome. Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LSSurf1 patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R+ cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS.

Functional characterization of drim2, the Drosophila melanogaster homolog of the yeast mitochondrial deoxynucleotide transporter

Background: Carrier-mediated influx of cytosolic deoxynucleotides is a major source of precursors for mitochondrial DNA synthesis. Results: dRIM2 is required to maintain normal deoxynucleotide pools in Drosophila mitochondria, and its knock-out is lethal at the larval stage. Conclusion: dRIM2 is a deoxynucleotide carrier and is essential to maintain mitochondrial function. Significance: Our data provide the first animal model of RIM2 deficiency. The CG18317 gene (drim2) is the Drosophila melanogaster homolog of the Saccharomyces cerevisiae Rim2 gene, which encodes a pyrimidine (deoxy)nucleotide carrier. Here, we tested if the drim2 gene also encodes for a deoxynucleotide transporter in the fruit fly. The protein was localized to mitochondria. Drosophila S2R+ cells, silenced for drim2 expression, contained markedly reduced pools of both purine and pyrimidine dNTPs in mitochondria, whereas cytosolic pools were unaffected. In vivo drim2 homozygous knock-out was lethal at the larval stage, preceded by the following: (i) impaired locomotor behavior; (ii) decreased rates of oxygen consumption, and (iii) depletion of mtDNA. We conclude that the Drosophila mitochondrial carrier dRIM2 transports all DNA precursors and is essential to maintain mitochondrial function.

KrillDB: A de novo transcriptome database for the Antarctic krill (Euphausia superba)

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 projects. In particular, we have built a de novo transcriptome assembly using more than 360 million 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.

Diurnal preference, mood and the response to morning light in relation to polymorphisms in the human clock gene PER3

PER3 gene polymorphisms have been associated with differences in human sleep-wake phenotypes, and sensitivity to light. The aims of this study were to assess: i) the frequency of allelic variants at two PER3 polymorphic sites (rs57875989 length polymorphism: PER34, PER35; rs228697 SNP: PER3C, PER3G) in relation to sleep-wake timing; ii) the effect of morning light on behavioural/circadian variables in PER34/PER34 and PER35/PER35 homozygotes. 786 Caucasian subjects living in Northern Italy donated buccal DNA and completed diurnal preference, sleep quality/timing and sleepiness/mood questionnaires. 19 PER34/PER34 and 11 PER35/PER35 homozygotes underwent morning light administration, whilst monitoring sleep-wake patterns and the urinary 6-sulphatoxymelatonin (aMT6s) rhythm. No significant relationship was observed between the length polymorphism and diurnal preference. By contrast, a significant association was observed between the PER3G variant and morningness (OR = 2.10), and between the PER3G-PER34 haplotype and morningness (OR = 2.19), for which a mechanistic hypothesis is suggested. No significant differences were observed in sleep timing/aMT6s rhythms between PER35/PER35 and PER34/PER34 subjects at baseline. After light administration, PER34/PER34 subjects advanced their aMT6s acrophase (p < 0.05), and showed a trend of advanced sleep-wake timing. In conclusion, significant associations were observed between PER3 polymorphic variants/their combinations and both diurnal preference and the response to light.

Functional characterization of the circadian clock in the Antarctic krill, Euphausia superba

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 identified sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to define the role of the main clock components. Our findings 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.

Modulation of miR-210 alters phasing of circadian locomotor activity and impairs projections of PDF clock neurons in Drosophila melanogaster

Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.