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44X44 Poster: Umass Dartmouth Profiling spatial & temporal gene expression responses of a copepod (Calanus pacificus) from the Northern California current using a novel RNA-Seq procedure Mark DeSimone1, Dave P. Jacobson2, Dr. Michael Banks2, Dr. Kym Jacobson3, Dr. Vera L. Trainer4, Brian D. Bill4 1 Department of Bioengineering, University of Massachusetts Dartmouth, North Dartmouth, MA 02747; 2 Hatfield Marine Science Center, Oregon State University, Newport, OR 97365; 3 Hatfield Marine Science Center, NOAA NWFSC, Newport, OR 97365, 4Marine Biotoxins Program, NOAA NWFSC, Seattle, WA 98112 Abstract Results Differentially Expressed Genes in 2018 Copepods are the most abundant metazoans on Earth and play an important role in • 19,255 potential genes were assembled from three spatially discrete sites • Up regulation of the energy metabolism genes related to oxidative marine food webs and the carbon cycle. As the dominant group of zooplankton, in 2017 and 2018 using two technical replicates for each site phosphorylation, such as subunits of NADH dehydrogenase, copepods are crucial secondary producers serving either directly or indirectly as • ~8,500 received a gene match by BLASTing to the National Center for Cytochrome C Oxidase, and ATP Synthase complexes. Other food sources for most commercially important fish species.[4] Through their vertical Biotechnology Information’s non-redundant protein database for Metazoa studies have found that this is a response to environmental migrations, copepods indirectly transfer carbon from the atmosphere into the deep • Over 5,000 of the 8,500 annotated genes came from a single species, variation and limited food.[9], [14] [7] Eurytemora affinis, which is a calanoid copepod sea where it can be stored. In addition, copepods are ecological indicators of climate • Up regulation of the muscle contraction proteins tropomyosin and • 34 genes were significantly differentially expressed (DE) when considering change, with warming ocean temperatures affecting copepod community structure, dystrophin. One study found similar results in low food conditions.[9] abundance, distribution, and seasonal timing.[13] Although they are abundant in the both the year and location Source: The Nervous System in Action by Dr. Michael D. Mann • Down regulation of ferritin, a transcriptional stress biomarker and an Northern California current throughout the winter and spring, the copepod species Volcano Plot: 34 Differentially Expressed Genes Calanus pacificus has been the subject of very few transcriptome studies. iron storage protein that protects proteins against oxidative damage. Previous studies found that domoic acid is used to increase the Using a novel RNA-Seq technique, TagSeq, the transcriptomes were sequenced for uptake of iron in Pseudo-nitzchia diatoms, which are a potential C. pacificus samples from three locations off Oregon and Northern California in May food source for C. pacificus and were present in high concentrations 2017 and 2018. Bioinformatic techniques were utilized to curate the sequences, in May 2017 but greatly decreased in 2018 as seen in Figure 5.[5], [12] assemble them into potential genes, annotate, and analyze for differential gene • Down regulation of cuticle protein which is related to a copepod’s expression. Thirty-four genes had significantly different expression profiles among exoskeleton. Adult copepods do not molt; however, females are years and locations. Various genes related to oxidative phosphorylation and muscle known to deposit granules of exoskeleton proteins into their eggs contraction were upregulated while a stress biomarker, ferritin, and a cuticle protein to form a protective layer. Lower production of cuticle protein could gene were downregulated in 2018 compared to 2017. During this time period there mean lower egg production, reduced frequency of spawning, or Source: Zirbel & Miller, 2007 was a significant change in phytoplankton abundance, determined by chlorophyll weaker eggs. fluorescence, and water nutrients at all locations. In particular, the increased presence of Pseudo-nitzchia diatoms and domoic acid may have led to the increased ferritin Conclusions expression and reduced egg production in C. pacificus copepods. By further • Differential gene expression analysis on Calanus pacificus transcriptomes, investigating gene expression responses of C. pacificus spatially and temporally, the Figure 2: The volcano plot shown above displays the 34 differentially expressed genes revealed thirty-four significantly different profiles including genes related to physiological state of the copepods can be determined to assess the health of the that had an adjusted p-value < 0.05 and a log2 fold-change > 1. DE genes with available energy metabolism, muscle contraction, stress, and exoskeleton formation. surrounding marine ecosystem. annotations are labeled. • Environmental conditions such as nutrient levels, phytoplankton abundance, and Heat Map: DE Genes Show Spatial & Temporal Variation domoic acid concentrations differed greatly between May 2017 and 2018. • A potential explanation is that in 2018 Calanus pacificus likely increased energy production and muscle contractions, while reducing egg production in response to food limitations. The copepods also experienced less oxidative stress from domoic acid and iron in 2018. Source: Center for Ocean Life • Future goals include comparing more Calanus pacificus transcriptomes collected Objective with greater spatial and temporal variation to determine how gene expression profiles vary as environmental conditions change. In addition, sequencing the Analyze the gene expression profiles of Calanus pacificus and associate the gene genome of Calanus pacificus and other Calanus species will help provide more expression findings with alternate environmental states to better inform fishery index gene annotations enabling greater correlation with their potential functions. predictions in the Pacific Northwest • Long-term aim is that the information gathered from the base of marine food webs Methods will improve predictions on likely outcomes of unforeseen oceanographic states. The first step in this project was collecting live copepod samples. This was done May 17-24th, 2017 and May 4-10th, 2018 at three References 1. Andrews, S. 2010. FastQC: a quality control tool for high throughput sequence data. Available online at: Figure 3: The heatmap shown above illustrates the spatial and temporal variation of the DE http://www.bioinformatics.babraham.ac.uk/projects/fastqc locations off the coast of Oregon and California: 2. Baumgartner M. F., Tarrant, A. M. 2017. The Physiology and Ecology of Diapause in Marine Copepods Annu. Rev. Mar. genes. Red is up regulated and green is down regulated with the darker colors indicating S c i , 9 , 387– 4 11 . • Cape Meares, OR - 28km offshore, Depth 98m 3. Bolger, A. M., Lohse, M., & Usadel, B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, stronger up or down regulation. Gene names for each row are listed on the right with NA 30( 1 5 ) , 2 11 4 – 2 1 2 0 . signifying that no gene annotation was available. To make this graphic the average expression 4. Bron, J. E., Frisch, D., Goetze, E., Johnson, S. C., Lee, C. E., & Wyngaard, G. A. 2011. Observing copepods through a • Heceta Head, OR - 33km offshore, Depth 105m genomic lens. Frontiers in Zoology , 8 ( 1 ) , 2 2 . for each gene is taken across all samples and the values shown are the z-scores. 5. Bruland, K.W., Rue, E.L., Smith, G.J. 2001. Iron and macronutrients in California coastal upwelling regimes: implications for diatom blooms . Limnol. Oceanogr . 46, 1661 – 1 6 7 4 . • Trinidad Head, CA - 15km offshore, Depth 98m 6. Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., … Regev, A. 2011. Full - l e n g t h transcriptome assembly from RNA - Seq data without a reference genome. Nature Biotechnology , 29( 7 ) , 6 4 4 – 6 5 2 . Environmental Nutrient Levels 7. Jónasdóttir, S. H., Visser, A. W., Richardson, K., & Heath, M. R. 2015. Seasonal copepod lipid pump promotes carbon sequestration in the deep North Atlantic. Proceedings of the National Academy of Sciences , 11 2 (39), 12122. 8. Langmead, B., Salzberg, S. 2012. Fast gapped - read alignment with Bowtie 2. Nature Methods, 9, 357- 3 5 9 . 9. Lenz, P. H., Unal, E., Hassett, R. P., Smith, C. M., Bucklin, A., Christie, A. E., & Towle, D. W. 2012. Functional genomics resources for the North Atlantic copepod, Calanus finmarchicus: EST database and physiological microarray. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics , 7 ( 2 ) , 11 0 - 1 2 3 . 10. Li, B., & Dewey, C. N. 2011. RSEM: Accurate transcript quantification from RNA - Seq data with or without a reference Source: Flanders Marine Institute g e n o m e . BMC Bioinformatics, 12 ( 1 ) . 11. Lohman, B. K., Weber, J. N., & Bolnick, D. I. 2016. Evaluation of TagSeq, a reliable low ‐ cost alternative for RNAseq. Mol Ecol Resour, 16 : 1 3 1 5 - 1 3 2 1 . 12. Miesner, A. K., Lundholm, N., Krock, B., & Nielsen, T. G. 2016. The effect of Pseudo - nitzschia seriata on grazing and Samples were collected using a bongo net and stored in RNAlater solution. TagSeq fecundity of Calanus finmarchicus and Calanus glacialis. Journal of Plankton Research , 38( 3 ) , 5 6 4 – 5 7 4 . 13. Northwest Fisheries Science Center (NOAA Fisheries). 2018. Ocean Ecosystem Indicators. Retrieved from was used to sequence the pooled transcriptomes of eight adult female C. pacificus https://www.nwfsc.noaa.gov/research/divisions/fe/estuarine/oeip/index.cfm
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