Monson+-+Proposal


 * Stress and Immune Response Gene Profiles of Prespawning Coho Salmon Exposed to Pesticide Chemical Cocktails**

Monson, Chris. UW-SAFS Fish 541 Fall 2010

Surveys since the late 1990’s have indicated a surprisingly high mortality rate in returning, prespawned, Coho salmon (//Oncorynchus kisutch//). While numerous hypotheses have been put forth to explain these findings, it is as of yet unclear which, if any or all factors are involved and NOAA (2007) estimates have shown instances of anywhere from 20% to 90% prespawning mortality rates in urban watersheds while non-urban die-offs appear to be few and far between. This is backed up in studies by the Wild Fish Conservancy – Northwest (2008) that found significantly higher rates of egg retention in deceased individuals associated with urban watersheds. Similarly studies have shown increased prespawning mortality associated with high temperatures in Chinook salmon (Keefer et al. 2008) and that general stressors can affect spawning behavior in Sockeye salmon(Cook et al. 2006). While temperature changes and other stressors may be associated with urban watersheds, the role of pesticides, especially the additive and synergistic effects of multiple pesticides, in prespawning mortality is not well understood. It is clear that the mixtures of pesticides, from various applications, are common in the aquatic environment (Gilliom 2007), however, the current EPA mandates use single-chemical models for studying exposures of aquatic animals. This could lead to underestimations of the effects of stressors caused by the additive and synergistic effects of those pesticides to returning salmon.
 * INTRODUCTION**

This proposal aims to further understand the additive and synergistic potential effects to returning salmon of exposure to a mixture of pesticides. Salmon exposed to organophosphate and carbamate pesticides show reduced acetylcholinesterase (AChE) activity which is can result in behavioral changes as AChE inhibitors interfere with cholinergic neurotransmission (Fulton and Key 2001). Greater than dose-additive effects have also been observed in juvenile salmon with increasing concentrations of organophosphate and carbamate pesticides (Laetz et al. 2009). While AChE activity is a primary measure of exposure to such pesticides, there is little to nothing known about the physiological stress responses to such exposures. This study aims to shed light on the basic physiological stress and immune responses associated with a concurrent multiple-chemical exposure, prior to spawning, of returning adult Coho salmon.
 * AIM**

With the use of molecular tools, we can further understand the effects that a pesticide chemical cocktail could have returning salmon physiology, even at singly sub-lethal and sub-effective levels of any given pesticide. Samples from adult Coho salmon that were exposed to a pesticide chemical cocktail were collected previously by members of Chris Grue’s lab (UW-SAFS). Data on exposure duration, chemical concentration, survival, egg quality and AChE activity is already gathered and can be used for comparison to gene expression results. Tissue samples from brain, liver and other (to be determined) were fresh frozen and can be used for basic molecular techniques. We will perform quantitative real-time PCR (qPCR) on a suite of stress response genes: HSP’s (HSP70), HSP receptors, a heat-stress induced gene - arterial natriuretic peptide (ANP – also known as salmon cardiac peptide (sCP)); genes involved in oxidative metabolism: CYP1A1, CYP2E1, and transcripts from superoxide dismutase (SOD), catalase, and glutathione peroxidase (GP); and other general stress response genes: glucose transporter-3 (GLUT3), corticotrophin releasing hormone precursors (CRH-I and CRH-II); as well as immune response genes; inflammatory cytokines: IL3, IL6, IL10. Beta-actin, GAPDH and alpha-Tubulin will be used as a positive control for all qPCR reactions and P-21 levels will be used to control for general cell death. Protein levels of HSP70 and HSP90 will also be measured by western blot immunodetection after SDS-PAGE with rabbit anti-salmon HSP70 (AS05061) and HSP90 (AS05063) modified from Rendell et al. (2006). Loading controls against GAPDH and Tubulin will be used to control from protein volume. Antioxidant defense response elements such as superoxide dismutase (SOD) can be measured by an enzyme activity assay (Cayman Chemicals, Ann Arbor, MI – which uses tetrazolium salt to detect superoxide radicals generated by xanthine oxidase in a colormetric reaction at 405nm). Although this may be above and beyond the scope of this project in the timeframe allotted.
 * METHODS**

Over a span of one week, starting November 9, 2010, primers and antibodies will be designed and ordered as will the SOD enzyme assay kit. Tissue will be sorted for use, and protein and RNA will be extracted by the second week and qPCR assays will begin on RNA samples. Immunodetection by western blotting after SDS-PAGE using the antibodies described above will take place in weeks 2-3 to allow for optimization, while by week 3, qPCR results will be analyzed. By the end of week 4, all experimentation and analysis will be complete and comparisons can be made to AChE activity, previously collected. Week 5 will consist of report writing and consultation with the other project members.
 * TIMELINE**

The substantial goal of this project is a joint manuscript between our group and the initial project members, while the scientific goals of this project are to elucidate the additive and synergistic effects caused by concurrent exposure to multiple pesticides to returning Coho salmon. The scientific goals will be achieved by using a variety of molecular techniques that are rarely used in environmental toxicology, especially in multiple exposure models.
 * DISCUSSION**

1. Cook SJ, Hinch SG, Crossin GT, Patterson DA, English KK, Shrimpton JM, Van Der Kraak G, Farrell AP. Physiology of individual late-run Fraser River sockeye salmon (//Oncorynchus nerka//) sampled in the ocean correlates with fate during spawning migration. Canadian journal of Fisheries and Aquatic Sciences. 2006. 63. 2. Laetz CA, Baldwin DH, Collier TK, Hebert V, Start JD, Sholz NL. The synergistic toxicity pesticide mixtures: Implications for rish assessment and the conservation of endangered Pacific salmon. Environmental Health Perspectives. 2009. 117(3). 3. Fulton MH, Key PB. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organo­phosphorus insecticide exposure and effects. Environmental Toxicology and Chemistry. 2001. 20(1).  4. Gilliom RJ. Pesticides in U.S. streams and groundwater. Environmental Science and Technology. 2007. 41(10).  5. Keefer ML, Taylor GA, Garletts DF, Gauthier GA, Pierce TM, Caudill CC. Prespawn mortality in adult spring Chinook salmon outplanted above barrier dams. Ecology of Freshwater Fish. 2010. 19. 6. NOAA/Northwest Fisheries Science Center 2007 http://www.nwfsc.noaa.gov/research/divisions/ec/ecotox/fishneurobiology/acutedieoffs.cfm. 7. Rendell JL, Fowler S, Cockshutt A, Currie S. Development-dependent differences in intracellular localization of stress proteins (hsps) in rainbow trout, Oncorhynchus mykiss, following heat shock. Comparitive. Biochemical. Physiology. 2006. 1. 8. Wild Fish Conservancy Northwest. Coho Prespawning Mortality Assesment in WA and OR. EPA Assistance ID: X5-96007101-0. Final Report 2008.
 * REFERENCES**