Identification and expression of differentially expressed genes in the hard clam, Mercenaria mercenaria, in response to quahog parasite unknown (QPX)

<p>Abstract</p> <p>Background</p> <p>The hard clam, <it>Mercenaria mercenaria</it>, has been affected by severe mortality episodes associated with the protistan parasite QPX (Quahog Parasite Unknown) for several years. Despite the commercial importance of hard clams in the United States, molecular bases of defense mechanisms in <it>M. mercenaria</it>, especially during QPX infection, remain unknown.</p> <p>Results</p> <p>Our study used suppression subtractive hybridization (SSH), as well as the construction of cDNA libraries from hemocytes to identify genes related to the defense of the hard clam against its parasite. Hard clams were experimentally infected with QPX and SSH was performed on mRNA samples extracted from mantle and gill tissues at different times post-challenge. A total of 298 clones from SSH libraries and 1352 clones from cDNA libraries were sequenced. Among these sequences, homologies with genes involved in different physiological processes related to signal transduction, stress response, immunity and protein synthesis were identified. Quantitative PCR revealed significant changes in the expression of several of these genes in response to QPX challenge and demonstrated significant correlations in terms of levels of gene expression between intermediates of signalling pathways and humoral defense factors, such as big defensin and lysozyme.</p> <p>Conclusion</p> <p>Results of this study allowed the detection of modifications caused by QPX at the transcriptional level providing insight into clam immune response to the infection. These investigations permitted the identification of candidate genes and pathways for further analyses of biological bases of clam resistance to QPX allowing for a better understanding of bivalve immunity in general.</p

Oysters expressed circadian cycles of valve activity behavior.

<p>A. Group level analysis. Left panel (A1): Double-plotted actograms of mean hourly opening duration (%) of the group (n = 15 oysters) submitted to 15 days of L:D (10:14) and 15 days of D:D. Right panel (A2): Period determined by spectral analysis (Lomb and Scargle periodogram; dotted line for <i>p</i>-value = 0.05) and percent rhythm (PR) of the Cosinor model. B. Individual analysis. Distribution of rhythmic (R) and arrhythmic (AR) oysters among LD and DD conditions. Details of periods in rhythmic oysters (circadian: 20-28h and ultradian <20h) are provided.</p

Identification of the Molecular Clockwork of the Oyster <i>Crassostrea gigas</i>

<div><p>Molecular clock system constitutes the origin of biological rhythms that allow organisms to anticipate cyclic environmental changes and adapt their behavior and physiology. Components of the molecular clock are largely conserved across a broad range of species but appreciable diversity in clock structure and function is also present especially in invertebrates. The present work aimed at identify and characterize molecular clockwork components in relationship with the monitoring of valve activity behavior in the oyster <i>Crassostrea gigas</i>. Results provided the characterization of most of canonical clock gene including <i>clock</i>, <i>bmal</i>/<i>cycle</i>, <i>period</i>, <i>timeless</i>, vertebrate-type <i>cry</i>, <i>rev</i>-<i>erb</i>, <i>ror</i> as well as other members of the cryptochrome/photolyase family (<i>plant-like cry</i>, <i>6–4 photolyase</i>). Analyses of transcriptional variations of clock candidates in oysters exposed to light / dark regime and to constant darkness led to the generation of a putative and original clockwork model in <i>C</i>. <i>gigas</i>, intermediate of described systems in vertebrates and insects. This study is the first characterization of a mollusk clockwork. It constitutes essential bases to understand interactions of the different components of the molecular clock in <i>C</i>. <i>gigas</i> as well as the global mechanisms associated to the generation and the synchronization of biological rhythms in oysters.</p></div

Variation of expression levels of clock genes.

<p>Relative transcription levels (RQ, mean ± SEM, n = 9 oysters) of <i>Cg6-4photolyase</i>, <i>CgCry1</i>, <i>CgpCry</i>, <i>CgCry2</i>, <i>CgClock</i>, <i>CgBmal</i>, <i>CgPeriod</i>, <i>CgTim</i>, <i>CgRev-Erb</i> and <i>CgROR</i> RNA in gill tissues of oysters exposed for 26 h to L:D 10:14 (□) and for 26 h to constant darkness (■). Gray areas referred to scotophase during L:D cycles. Significant differences at <i>p</i> < 0.05 between light regimes are indicated and asterisks denote significant time-specific differences between light regime treatments.</p

Phylogenetic analysis based on timeless sequences.

<p>The tree was generated by Maximum Likelihood method using Mega 6 program and based on the multiple alignments performed with Clustal Omega. Percentage of bootstraps based on 1,000 replicates were indicated with only values > 50%. Swi1 from <i>Schizosaccharomyces</i>. <i>pombe</i> was used as outgroup to root the tree. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169790#pone.0169790.s004" target="_blank">S2 Table</a> for sequence details and accession numbers.</p

Spearman analysis of clock gene expression under L:D regime (n = 72 oysters).

<p>Spearman analysis of clock gene expression under L:D regime (n = 72 oysters).</p

Phylogenetic analysis based on period sequences.

<p>The tree was generated by Maximum Likelihood method using Mega 6 program and based on the multiple alignments performed with Clustal Omega. Percentage of bootstraps based on 1,000 replicates were indicated with only values > 50%. Neuronal PAS domain-containing protein (NPas) from <i>C</i>. <i>gigas</i> was used as outgroup to root the tree. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169790#pone.0169790.s004" target="_blank">S2 Table</a> for sequence details and accession numbers.</p

Phylogenetic analysis based on clock and cycle/Bmal sequences.

<p>The tree was generated by Maximum Likelihood method using Mega 6 program and based on the multiple alignments performed with Clustal Omega. Percentage of bootstraps based on 1,000 replicates were indicated with only values > 50%. Hypoxia-inducible factor (HIF) from <i>C</i>. <i>gigas</i> was used as outgroup to root the tree. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169790#pone.0169790.s004" target="_blank">S2 Table</a> for sequence details and accession numbers.</p