Project description:The copepod, Calanus finmarchicus is a keystone species for the North Atlantic. Because of recent changes in the geographic distribution of this species, there are questions as to how this organism responds physiologically to environmental cues. Molecular techniques allow for examination and new understanding of these physiological changes. Here, we describe the development of a microarray for high-throughput studies of the physiological ecology of C. finmarchicus. An EST database was generated for this species using a normalized cDNA library derived from adult and sub-adult individuals. Sequence data were clustered into contigs and annotated using Blastx. Target transcripts were selected, and unique, 50 base-pair, oligomer probes were generated for 995 genes. Blast2GO processing provided detailed information on gene function. The selected targets included broad representation of biological processes, cellular components, and molecular functions. The microarray was tested in two sets of comparisons: adult females maintained at different food concentrations and field-caught sub-adults showing differences in lipid storage. Up-regulated and down-regulated transcripts were identified for both comparisons. Only a small subset of the genes up-regulated in low food individuals were also up-regulated in lipid-poor animals; no overlap was seen between the genes down-regulated in the two comparisons.
Project description:Physiological changes in response to environmental cues are not easily documented in pelagic copepods using traditional methods. Molecular biological tools provide new approaches to the investigation of difficult to sample organisms such as oceanic zooplankton. Here, we describe the development of a species-specific microarray for high-throughput studies of the physiological ecology of the North Atlantic copepod Calanus finmarchicus. An EST database was generated for this species using a normalized cDNA library derived from adult and sub-adult individuals from the Gulf of Maine. Sequence data were clustered into contigs and annotated using Blastx. Target transcripts were selected, and unique, 50 base-pair long, oligomer probes were designed and synthesized for 995 genes. Bioinformatic processing using Blast2GO software provided detailed information on gene function. The selected targets include a broad representation of biological processes, cellular components, and molecular functions. The microarray was tested on both experimental and ecological samples, i.e. food abundance and two morphotypes exhibiting distinct lipid stores, respectively. Differentially regulated transcripts were identified for both comparisons. Two comparisons were performed: 1) Lipid-rich (fat) and Lipid-poor (thin) morphotypes 2) Copepods kept under high food and low food experimental conditions
Project description:The marine copepod Calanus finmarchicus is the most abundant zooplankton species in the northern regions of the Atlantic Ocean and the Barents Sea. Very little is known about the molecular mechanisms underlying critical processes associated with this species’ complex life history (e.g., ontogenetic development, reproduction, molting, diapause) and physiology (e.g., digestion, neural processes, and membrane physiology). This study analyzed patterns of gene expression of C. finmarchicus samples collected from the Gulf of Maine (Northwest Atlantic Ocean) using a 1,000 expressed sequence tag (EST) microarray designed to assay genes of known physiological function and hypothesized ecological importance for C. finmarchicus. Replicate analyses compared adult females and final-stage juveniles (Copepodite-5) collected from surface (0-30m) and deep (130-170m) layers. Environmental data include detailed characterization of biological, chemical, and physical oceanographic parameters in the sampled water packets. All data were screened for artifacts, normalized and selected using a fold-change filter prior to analysis. Replicate comparisons were analyzed by Significance Analysis of Microarrays (SAM; Stanford University Labs) with a control for False Discovery Rate (FDR) and with Principle Component Analysis with evaluation of significance by one- or two-sample t-test in Acuity Microarray Informatics Software (Molecular Devices, Inc.). Gene Ontology Enrichment Analysis was carried out using GOEAST (http://omicslab.genetics.ac.cn/GOEAST/index.php) to assess functional relationships of selected genes and/or proteins. The results indicated: up-regulation of genes involved in cell division, protein synthesis and mating in deep females and juveniles; up-regulation of genes related to cellular homeostasis, circadian behavior and nervous system development in surface females; and up-regulation of genes related to muscle development and protein catabolism in deep juveniles versus deep females. KEGG pathway analysis using the Blast2GO suite (http://www.blast2go.org/) indicated: up-regulation of genes encoding enzymes related to the citrate cycle and anaerobic metabolism in deep females and juveniles; and up-regulation of genes encoding enzymes related to energy metabolism and osmoregulation in surface females.