Role and expression of cry1 in the adductor muscle of the oyster Crassostrea gigas during daily and tidal valve activity rhythms

Abstract

Cryptochromes are flavin- and pterin-containing photoreceptors of the cryptochrome/photolyase family. They play critical roles in organisms, among are which light-dependent and light-independent roles in biological rhythms. The present work aimed at describing a cryptochrome gene in the oyster Crassostrea gigas by (i) a characterization and phylogenetic analysis and (ii) by studying its expression in the relationship to rhythmic valve behavior in different entrainment regimes. Cryptochrome expression was focused on the adductor muscle of the oyster, the effector of the valve behavior. The results suggest involvement of Cgcry1 in oyster rhythmicity as a sensor of environmental zeitgebers, associated with circadian rhythms and potentially to tidal activity. The characterized gene belongs to type 1 cryptochrome/insect-type cry. Additionally, Cgcry1 presented a daily oscillation under L:D entrainment, which disappeared in constant darkness. Transcript expression of Cgcry1 also oscillated at tidal frequency under tidal entrainment and in constant darkness. Finally, exposure of tidally entrained oysters to saxitoxin (STX)-producing alga Alexandrium minutum induced a dose effect response in oysters by first altering Cgcry1 expression and then the behavior of oysters with increasing concentrations of toxins. This study initiates the characterization of the molecular clock in the oyster C. gigas and its interactions with environmental zeitgebers.

Identification of the Molecular Clockwork of the Oyster Crassostrea gigas

“…gigas (S2 Fig). Some of which were previously characterized such as CgCry1 [9] or identified such as CgRev-erb and CgROR [17]. For instance, Voleger et al [17] analyzed nuclear receptor genes from the genome of C .…”

“…gigas using Pfam conserved domains as well as homology with clock sequences identified in other organisms [6,16]. CgCry1 was previously described and complete sequence was retrieved from Mat et al [9]. Similarly, ROR and Rev-Erb homologs in oyster were retrieved from Vogeler et al [17].…”

“…Primer sets of clock candidates were designed from full cDNA sequence, excepted for CgCry1 , CgRev-Erb and CgROR [9,17]; stability of the expression of glyceraldehyde-3-phosphate dehydrogenase ( GADPH ) and elongation factor ( EF1 ) as reference genes were verified and EF1 was used as reference gene (S1 Table). PCR efficiency (E) was assessed for each primer pair by determining the slope of standard curves obtained from serial dilution analysis of cDNA from different experimental samples.…”

“…From these studies, it appeared that some clock genes and interacting feedback loops are conserved across phyla [4 for review] but number of variations also existed and the divergence of molecular components and their function across phyla could be explain, in part, by gene duplication and loss [5]. However, few studies were focused on the molecular bases of clock systems in marine invertebrates [6,7] and more specifically in bivalves [8,9] whereas these organisms inhabited complex environments exposed to solar and lunar light entrainments (as terrestrial organisms) but also to tides [10,11]. …”

“…Moreover, a cryptochrome belonging to the molecular clock was characterized in C . gigas and experiments demonstrated its transcriptional oscillation under tidal and circadian entrainments [9]. However, no other component of the molecular clockwork in oyster was characterized despite the large volume of molecular data as well as the available genome for this bivalve [15].…”