Urchin+Proposal+RW


 * STRESS RESPONSE OF THE RED SEA URCHIN TO ELEVATED CO2: A MOLECULAR PERSPECTIVE **


 * Introduction **

Global climate change is likely to have myriad effects on a wide variety of marine organisms over the next several decades and into the foreseeable future. More intense weather patterns, increased sea surface temperature and elevated levels of pCO2 have the potential to disrupt life histories, limit food resources and alter the physiological makeup of organisms at all points in the marine food web. Increased pCO2 in the oceans resulting in ‘ocean acidification’ is of particular concern for calcifying organisms which rely on dissolved calcium carbonate in the water column to produce integral parts of their anatomy. Sea urchins which are common members of many marine habitats are one such organism. Increased pCO2 in the water column has been shown to impair the urchin’s ability to produce its calcium carbonate-derived test and spines in addition to disrupting its larval development. Sea urchins are distributed worldwide and occupy key niches within their habitats as grazers of both algae and invertebrates. In the Pacific Northwest, urchins play an important role in subtidal communities by maintaining space (a limiting resource) on subtidal rock walls, processing detrital macroalgae into particulate organic matter and dissolved organic matter for use by other organisms, and providing a food source for many fish species and large predators. As levels of CO2 in our oceans begin to rise, it’s vital that we understand the effects this will have on urchins and how they function in their ecosystem. Many studies have looked at the effects of elevated pCO2 on urchins from a purely developmental point of view (Brennand et al. 2010), but few have dealt with the underlying molecular stress response including gene transcription rates, protein activity and DNA methylation that elevated pCO2 may evoke (// but see work by //Hoffman, O’Donnell & Todgham). Molecular-based techniques provide a powerful tool that can uncover novel response pathways and help predict an organism’s ability to cope with stress in a changing environment. This study will attempt to describe underlying molecular responses to elevated CO2 in sea urchins of the Pacific Northwest. Specifically it will attempt to answer the following questions: 1. Do urchins show increased transcription rates of genes associated with stress when exposed to elevated pCO2? 2. What are the activity levels of proteins associated with these genes? 3. Do urchins demonstrate a methylation response to elevated pCO2?
 * Materials and Methods **

Sea urchins (n=30) will be collected and held in common sea tables for 48 hours in order to minimize imposed stress prior to experimentation. Urchins will then be assigned randomly to one of three treatments, two replicates each, 5 urchins per replicate:

1. Ambient CO2; 380ppm CO2 (control) 2. Slightly raised CO2; 540ppm CO2 (after O’Donnell et al. 2009) 3. Greatly raised CO2; 970ppm CO2 (after O’Donnell et al. 2009) // From Todgham et al. 2009  //

Urchins will be held in treatments for 9 days before tissue extraction for molecular analysis. Tanks will be aerated with premixed gas from a commercial supplier in order to simplify the mechanics of CO2 manipulation. All tanks will be aerated 24 hours prior to urchin additions to ensure sufficient mixing. The pH of each tank and two carbonate chemistry parameters will be checked daily as per EPOCA guidelines (EPOCA 2010) and urchins will be supplied with the same kelp diet across treatments. Potential genes to explore for differential expression in elevated CO2 treatments include those involved in calcification processes, acid–base compensation and ion regulation, the cellular stress response, apoptosis, cell cycle, development, metabolism, translational control of proteins and cell signaling (as suggested by Hoffman et al. 2008). Proteins such as cytochrome P450, GST and Cysteine Proteases are likely candidates for differential expression under stress. Quantifying methylation of urchin genome segments through dot blot and other analyses will also provide insight into potential epigenetic response to CO2.


 * Timeline **

__November__ __December__
 * 9 ** (tue) begin tank set-up /order gas (Praxair)/ source urchins
 * 12 **(fri) acclimate urchins for the weekend
 * 15 ** (mon) finish setting up CO2-treatments / begin aerating CO2 treatments
 * 16 **(tue) move urchins into treatments
 * 16-24 **monitor pH / feed urchins
 * 24 **(wed) remove urchins and take tissue for analysis
 * 29 **(mon) begin extractions for cDNA, methylation, protein analysis
 * 30 **(tue) continue extractions and begin processing as appropriate
 * 2 **(thu) all qPCR, protein assays, dot blots complete
 * 3 **(fri) statistical analyses begin
 * 6-15 **stats complete / write up


 * References **

Brennand HS, Soars N, Dworjanyn SA, Davis AR, Byrne M (2010) Impact of ocean warming and ocean acidification on larval development and calcification in the sea urchin // Tripneustes gratilla //. PLoS ONE 5(6):e11372 EPOCA (2010) Guide to best practices for ocean acidification research and data reporting. European Commission, Brussels Hoffman GE, O’Donnell MJ, Todgham AE (2008) Using functional genomics to explore the effects of ocean acidification on calcifying marine organisms. Mar Ecol Prog Ser 373:219-225 O’Donnell MJ, Hammond LM, Hofmann GE (2009) Predicted impact of ocean acidification on a marine invertebrate: elevated CO2 alters response to thermal stress in sea urchin larvae. Mar Biol 156:439-446 Todgham AE, Hofmann GE (2009) Transcriptomic response of sea urchin larvae // Strongylocentrotus purpuratus // to CO2-driven seawater acidification. J Exp Biol 212:2579-2594