Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

This project focused on developing molecular tools based on relative gene expression to investigate physiological ecology of Calanus finmarchicus (Calanoida: Copepoda) in the Gulf of Maine. For this project, a custom microarray was developed for this species and tested on field and experimental data. Using RNA-seq technology, a de novo transcriptome was obtained from RNA extracted from different developmental stages of C. finmarchicus from embryo to adult female.

Datasets generated through this project were deposited at NCBI (www.ncbi.nlm.nih.gov) in 5 separate NCBI BioProjects:

1. BioProject PRJNA151477 (www.ncbi.nlm.nih.gov/bioproject/PRJNA151477): Microarray platform information and relative expression data for lipid-rich vs. lipid-poor pre-adults (stage CV) and adult females maintained at high- and low-food levels.
2. BioProject PRJNA149523 (www.ncbi.nlm.nih.gov/bioproject/PRJNA1495230): Microarray platform information and relative expression data for pre-adults (stage CV) and adult females collected from depth (130-170 m) and from surface waters (0-30 m).
3. BioProject PRJNA236528 (www.ncbi.nlm.nih.gov/bioproject/PRJNA236528): RNA-Seq data. Data includes biosample description, raw sequence data for each sample, and transcriptome shotgun assembly of sequences. Calanus finmarchicus transcriptome from 6 developmental stages (embryo, early nauplius, late nauplius, early copepodite, late copepodite and adult females).
4. BioProject PRJNA312028 (http://www.ncbi.nlm.nih.gov/bioproject/PRJNA312028): RNA-Seq data. Data includes biosample description, raw sequence data for each sample, and transcriptome shotgun assembly of sequences. Calanus finmarchicus adult female transcriptome of Alexandrium fundyense response.
5. Bioproject: PRJNA356331(http://www.ncbi.nlm.nih.gov/bioproject/PRJNA356331): RNA-Seq data. Data includes biosample description, raw sequence data for each sample, and transcriptome shotgun assembly of sequences Calanus finmarchicus late nauplii (NV-NVI) transcriptome of Alexandrium fundyense response.

Dataset Related References:

Lenz, P.H., Roncalli, V., Hassett, R.P., Wu, L.-S., Cieslak, M.C., Hartline, D.K. and Christie, A.E. (2014) De novo assembly of a transcriptome for Calanus finmarchicus (Crustacea, Copepoda) – the dominant zooplankter of the North Atlantic Ocean. Plos One 9: e885389 DOI: 10.1371/journal.pone.0088589)

Roncalli, V., Cieslak, M.C., Lenz, P.H. Transcriptomic responses of the calanoid copepod Calanus finmarchicus to the saxitoxin producing dinoflagellate Alexandrium fundyense. Scientific Reports 6, Article number: 25708 (2016) doi:10.1038/srep25708.

Roncalli V., Cieslak M.C., Lenz P.H. (2016) Data from: Transcriptomic responses of the calanoid copepod Calanus finmarchicus to the saxitoxin producing dinoflagellate Alexandrium fundyense. Dryad Digital Repository. http://dx.doi.org/10.5061/dryad.11978

Roncalli, V., Jungbluth, M.J., Lenz, P.H. (2016). Glutathione S-transferase regulation in Calanus finmarchicus feeding on the toxic dinoflagellate Alexandrium fundyense. PloS One, 11(7): e0159563.

Roncalli, V., Turner, J.T., Kulis, D., Anderson, D.M., Lenz, P.H. (2016). The effect of the toxic dinoflagellate Alexandrium fundyense on the fitness of the calanoid copepod Calanus finmarchicus. Harmful Algae, 51: 56-66

Roncalli, V., Lenz, P.H., Cieslak, M.C., Hartline, D.K. (2017). Complementary mechanisms for neurotoxin resistance in a copepod. Scientific Reports, 2017; 7: 14201, doi: 10.1038/s41598-017-14545-z

Methodology References:

Microarray

Lenz, P.H., Unal, E., Hassett, R.P., Smith, C.M., Bucklin, A., Christie, A.E. and Towle, D.W. (2012) Functional genomics resources for the North Atlantic copepod, Calanus finmarchicus: EST database and physiological microarray. Comparative Biochemistry Physiology Part D, Genomics & Proteomics, 7:110-123

Unal, E., Bucklin, A., Lenz, P.H. and Towle, D.W. (2013) Gene expression of the marine copepod Calanus finmarchicus: Responses to small-scale environmental variation in the Gulf of Maine (NW Atlantic Ocean). Journal of Experimental and Marine Biology and Ecology, 446:76-85

Gene discovery studies using C. finmarchicus transcriptome:

Christie, A.E., Fontanilla, T.M., Nesbit, K.T. and Lenz, P.H. (2013) Prediction of the protein components of a putative Calanus finmarchicus (Crustacea, Copepoda) circadian signaling system using a de novo assembled transcriptome. Comparative Biochemistry and Physiology Part D, Genomics & Proteomics, 8:165-193

Christie, A.E., Roncalli, V., Wu, L.-S., Garrote, C.L., Doak, T. and Lenz, P.H. (2013) Peptidergic signaling in Calanus finmarchicus (Crustacea: Copepoda): in silico identification of putative peptide hormones and their receptors using a de novo assembled transcriptome. General and Comparative Endocrinology, 187:117-135

Christie, A.E., Roncalli, V., Batta Lona, P., McCoole, M.D., King, B.L., Bucklin, A., Hartline, D.K. and Lenz, P.H. (2013) In silico characterization of the insect diapause-associated protein couch potato (CPO) in Calanus finmarchicus (Crustacea: Copepoda). Comparative Biochemistry and Physiology. Part D, Genomics & Proteomics, 8:45-57.

Christie, A.E., Fontanilla, T.M., Roncalli, V., Cieslak, M.C. and Lenz, P.H. (2014) Diffusible gas transmitter signaling in the copepod crustacean Calanus finmarchicus: Identification of the biosynthetic enzymes of nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) using a de novo assembled transcriptome. General and Comparative Endocrinology 202: 76-86

Christie, A.E., Fontanilla, T.M., Roncalli, V., Cieslak, M.C. and Lenz, P.H. (2014) Identification and developmental expression of the enzymes responsible for dopamine, histamine, octopamine and serotonin biosynthesis in the copepod crustacean Calanus finmarchicus. General and Comparative Endocrinology 195: 28-39.

Roncalli, V., Cieslak, M.C., Passamaneck, Y., Christie, A.E., & Lenz, P.H. (2015). Glutathione S-Transferase (GST) Gene diversity in the crustacean Calanus finmarchicus - contributors to cellular detoxification. PloS One, 10(5): e012332.

See publications for detailed information on sample acquisition, experimental treatment and methodology.

Source of C. finmarchicus:

1. Mid- and late copepodites and adults were collected during the summer (June and July, 2009, 2010, 2011, 2012) in the Gulf of Maine (Lat: 44°2′N; Long: 68°3′W) by towing a 75 cm diameter (560 μm mesh) net vertically from 75 m depth.  These individuals were used in microarray (2009, 2010) and RNA-Seq (2011, 2012) studies.
2. Embryos, nauplii and early copepodites were obtained from offspring of field-collected adult females raised in laboratory cultures. These individuals were used in RNA-Seq studies.
3. Late stage copepodites (CV) for microarray analysis (Unal et al. 2013) were collected in April 17, 2008 from Wilkinson Basin in the Gulf of Maine as part of a multi-year time-series study by the Center for Coastal Ocean Observation and Analysis (COOA) at the University of New Hampshire, USA.  Vertically-stratified plankton tows (0-30 m and 130-170 m) were taken using a 1/4-m MOCNESS equipped with 150 μm mesh nets.

Brief summary of methods

RNA was extracted from fresh and preserved samples.  Samples were preserved either in RNALater or in liquid nitrogen.  RNA was extracted using the Qiagen RNeasy Mini or Mini Plus kits for both microarray and RNASeq. Quality and quantity of RNA was checked in an Agilent 2100 Bioanalyzer prior to further processing of samples as described in the references.  Microarray hybridization incubations were performed in a Micro Array User Interface (MAUI) Hybridization Chamber (BioMicro Systems). For the RNASeq, high quality total RNA samples were sent to the Georgia Genomics Facility at U. of Georgia. Multiplex cDNA gene libraries were prepared from the samples, and these were sent to Alpha Hudson Institute for Biotechnology for paired 100-base-pair sequencing on an Illumina HISeq 2000 instrument.

The RNASeq data were used to assemble and annotate a de novo transcriptome. Raw reads were analyzed for quality: low quality and over-represented sequences were removed, and sequences were trimmed to remove the random primer sequences (first 9 bases) prior to assembly using Trinity (Trinity 2012-03-17-IU_ZIH_TUNED software, on the National Center for Genome Analysis Support’s [NCGAS; Indiana University, Bloomington, IN, USA] Mason Linux cluster; each node of this computer system is composed of four Intel Xeon L7555 8-core processors running at 1.87 GHz with 512 GB of memory. Assembled contigs were annotated using Blast2GO software and targeted gene discovery workflows.

This project focused on developing molecular tools based on relative gene expression to investigate physiological ecology of Calanus finmarchicus (Calanoida: Copepoda). For this project, a custom microarray was developed and tested for this species. In addition, using RNA-seq technology, a de novo transcriptome was obtained from RNA extracted from different developmental stages of C. finmarchicus from embryo to adult female.  

Description from NSF award abstract:
This project will develop transcriptomics approaches to investigate gene regulation as a function of environmental cycles and in response to experimental manipulation. Currently, there are few tools to establish physiological state of marine zooplankton, in particular for oceanic species. Molecular approaches based on quantifying the transcriptome could serve as powerful tools to obtain a physiological profile for individuals and groups of individuals collected in the field. In combination with laboratory experiments, transcriptome analysis will provide a new approach to understanding organism-environment interactions in the pelagic zone.

The PI will focus on a model planktonic crustacean, Calanus finmarchicus, to develop the molecular tools. C. finmarchicus, a calanoid copepod, is highly abundant in the North Atlantic, with populations extending from the Gulf of Maine and Labrador Sea to the North Sea. Pyrosequencing and microarray technologies will be used to develop a diagnostic tool to determine physiological state in C. finmarchicus. The goal of having a measurement of physiological state is to determine if individuals in the population are growing, are synthesizing or catabolizing storage lipids, and are metabolically active and/or experiencing environmental stress. Specific objectives of this project include:

1. High throughput sequencing of C. finmarchicus transcriptome from pre-adult (copepodid stage V [CV]) individuals representing distinct phases of the annual cycle (late spring-early summer, early fall, diapausing individuals).

2. Analysis of the sequence data for discovery of seasonally regulated genes for the development of an ecologically relevant microarray. Probes for this microarray will include seasonally regulated genes, genes involved in the environmental stress response and control genes.

3. Preliminary testing of microarray on existing samples collected from the Gulf of Maine and stored in liquid nitrogen, as well as on experimentally manipulated animals.