Gene Expression During Development of Clam Leukemia: Interactive effects of temperature and ocean acidification on viral loading and onset of disease
The soft-shell clam (Mya arenaria) fishery has a landings value of more than $20 million annually with more than 3.9 million pounds harvested largely from the state of Maine. Additional commercial landings occur in Massachusetts and Rhode Island. In New Hampshire, the soft-shell clam fishery is an important recreational activity with hundreds of local and vacationing participants annually. In 1997, researchers estimated a harvestable soft-shell clam population in Hampton/Seabrook harbor, N.H. of 25,000 bushels, but that number has since fallen to only 5,400 bushels. Clearly, clams are far from their peak abundance in N.H. coastal environments and many scientists from Maine through Massachusetts have reported similar declines. This loss of productivity has caused considerable concern among both scientists and commercial fishermen. As water temperatures continue to rise and as winters get warmer, biologists have observed crabs, that eat spat (clams in larval stage) combined with seasonal and regional coastal acidification, significantly impacting clam abundance. These dual environmental stressors (temperature and pH stress) occur in a background of infection with a retrovirus to impact clams at two years of age when they are first reproductive. Here we will investigate potentially interacting contributors to the etiology of clam leukemia including: a retroviral infection and increasing seawater temperature both with and without ocean acidification. Linkages between specific environmental parameters and cancer are often difficult to demonstrate for lack of an appropriate model system. Clam leukemia represents an opportunity to contribute to and may even lead to the demonstration of such a model. The genetic analysis of clam hemocyte leukemia using transcriptomics (methods to determine which genes are being expressed in an organisms cells at a given time period) will provide an indication of changing expression of stress related genes and gene products (mRNAs) during initiation and progression of this disease in the soft-shell clam. Ideally, results from this analysis will highlight unique gene expression patterns that can be traced to the environmental stressors of seawater temperature and ocean acidification.
Transcriptome studies of the oyster have already identified an extensive set of genes related to environmental stressors (Zhang et al., 2012). As pointed out in their recent article in the journal Nature, “Expression of genes coding for heat shock protein 70 (Hsp70 = proteins that bind to, fold and escort other proteins) and inhibitors of apoptosis (programmed cellular death in response to severe temperature and other stressors) is probably central to the oyster’s adaptation to sessile life in the highly stressful intertidal zone.” Also the recent completion of the oyster (Zhang et al., 2012) and soft-shell (Walker lab, unpublished) entire draft genomes reveals that bivalves are particularly good indicators of environmental stress (Walker et al., 2009; 2011; Zhang et al., 2012). An increased understanding of the etiology of clam leukemia resulting from the project proposed in this proposal will contribute to the N.H. Sea Grant mission in Healthy Coastal Ecosystems and to the commercial fishing industry, resource managers and other end users for the following reasons:
1) Identified environmental stressors, such as viral infection, rising seawater temperature and/or low pH and possible linkage to clam leukemia could be considered when local communities and conservation programs develop restoration protocols or assess the effectiveness of restoration in critical habitats.
2) Outreach programs focused on educating resource managers and the fishing industry, that link clam leukemia to possible environmental stressors could be developed, leading to more sustainable management of the resource.
Additionally, this project leverages the information from transcriptomic studies on other ecologically and commercially important bivalve species such as hard clams (Mercenaria mercenaria), oysters (Crossastrea virginica and edulis), the blue mussel (Mytilus edulis), the Atlantic surf clam (Spisula solidissima), the Atlantic sea scallop (Placopecten magellanicus) and the arctic surf clam (Mactromeris polynyma) as well as Mya arenaria currently being conducted with the support of N.H. Sea Grant Development Funds (M/D-1203 to M. Lesser).
1. To generate transcriptome data from clam hemocytes sampled during the transition from normal to leukemic hemocytes while exposed to high temperature, low pH, or both during an increase in viral load (i.e., viremia).
2. To develop outreach programs to transfer critical habitat information gained from this study regarding the effects of temperature and pH stressors during generation of clam leukemia, as well as more broadly the linkage between environmental stressors and shellfish productivity, to resource managers, conservation biologists and coastal communities.
Leukemia free clams will be induced to develop leukemia via inoculation with lysed hemolymph/hemocytes under treatment with increased temperature or pH or simultaneous treatment with increased temperature and pH and increasing viral loading. Clams held under these conditions will be sampled over a four day period and those that develop 100% leukemia will be prepared as transcriptomes to determine differential expression of genes related to stress from temperature, pH and viral infection. Control clams not held under stressors will also be sampled at the same time periods and prepared as transcriptomes for comparison with results on gene expression from those held under stressors.
Comparative studies at the molecular level using next nucleotide generation sequencing hold the best opportunity to identify critical pathways affected by increasing sea water temperature and ocean acidification that have not yet been considered. Potentially important genetic markers (eg., mortalin, Steamer, p53 related genes, etc.) that could be used to determine the effects of ocean temperature and/or acidification on mortality or product quality (e.g., decrease in tissue biomass) could lead to specific action (e.g., harvesting and/or remediation in land based facilities with seawater equilibrated to normal temperature and pH) by those involved in bivalve aquaculture.