Mechanisms of petroleum hydrocarbon toxicity in fish early life history stages (Year 3)

Principal investigator: Co-investigatorJohn P. Incardona, Ph.D., M.D. Nathaniel L. Scholz, Ph.D.Environmental Conservation Division Environmental Conservation DivisionNOAA/Northwest Fisheries Science Center NOAA/Northwest Fisheries Science Center2725 Montlake Blvd E 2725 Montlake Blvd ESeattle, WA 98112 Seattle, WA 98112Phone: 206-860-3347 Phone: 206-860-3454Fax: 206-860-3335 Fax: 206-860-3335Email: john.incardona@noaa.gov Email: nathaniel.scholz@noaa.gov

Hypothesis and objectives: Studies prompted by the Exxon Valdez oil spill, which contaminated spawninggrounds for Pacific herring (Clupea pallasi) and pink salmon (Oncorhynchus gorbuscha), identified a commonsyndrome of developmental abnormalities in both species induced by petroleum-derived polycyclic aromatichydrocarbons (PAHs) [1-9]. An extensive literature has linked the toxicity of petrogenic PAHs to activation of thearyl hydrocarbon receptor (AhR) pathway and cytochrome P4501A (CYP1A) induction [10, 11]. In this model ofPAH toxicity, harmful effects could be due to direct consequences of AhR activation, or to indirect effects of toxicPAH metabolites resulting from CYP1A activity. Since exposure of fish embryos to either petrogenic PAH mixturesor potent AhR ligands such as dioxins produces superficially similar syndromes, it has been generally held thatthese two classes of compounds act on developing fish by a common pathway. However, such a commonality hasyet to be demonstrated. Knowledge of the precise mechanisms of PAH toxicity are important, because CYP1Aactivity in fish is generally the indicator used to assess exposure to oil, and this assay has been central to thedebate over lingering effects of the Exxon Valdez spill [12, 13].

To address this problem we have used zebrafish (Danio rerio), a major model system for the study of vertebratedevelopment at the molecular and genetic level. Studies conducted Years 1 and 2 of this grant identified AhR-independent disruption of embryonic cardiac function and morphogenesis as a primary pathway of toxicity forweathered crude oil in developing zebrafish [14]. Exposure of zebrafish embryos to weathered crude oil by twodifferent methods produced a syndrome of embryolarval toxicity that was distinct from the AhR-dependent effects ofdioxins. Instead, weathered crude oil caused early cardiac function defects that were consistent with the effects ofthe most abundant tricyclic PAHs (fluorenes, dibenzothiophenes, and phenanthrenes) expected from our work onindividual model 3-ring PAHs [15]. Embryos in which either the AhRs or CYP1A were inactivated with antisensemorpholino oligonucleotides (MOs) were actually more sensitive to weathered crude oil toxicity, indicating that theAhR/CYP1A pathway actually provides a measure of protection against petrogenic PAHs, rather than playing acausal role in toxicity. Therefore, direct targets of PAHs are most likely intrinsic to cardiomyocytes. In parallel work,recently funded by the NOAA/NWFSC Internal Grants Program and to be initiated in May 2005, we are using thecommercially available (Affymetrix) zebrafish DNA microarray to identify potential cardiac targets of petrogenicPAHs by gene expression profiling of embryos exposed to phenanthrene or weathered crude oil and withgenetically-derived cardiac dysfunction (see Appendix). The primary objective for Year 3 of OWCN funding is togroundtruth the cumulative findings from zebrafish studies in Pacific herring early life history stages through thefollowing specific aims:

1. Conduct a detailed phenotypic analysis of Pacific herring embryos exposed to weathered AlaskaNorth Slope (ANS) crude oil.

2. Identify and knockdown the AhR and CYP1A genes in Pacific herring embryos.

Experimental Plan Aim 1: Although the effects of weathered ANS oil on Pacific herring embryos are well-documented [6], those studies analyzed primarily late end points of toxicity in hatching stage larvae. Because thereis considerable overlap of the appearance of the late-stage effects with classic AhR-mediated dioxin toxicity, a close

Incardona 2analysis over the full developmental period is warranted, with particular attention to early cardiac function. Pacificherring embryos will be exposed to weathered ANS crude oil using the oiled gravel effluent method [6, 7, 14] withfiltered seawater at the NOAA Mukilteo field facility near Seattle. Ripe herring are available in Puget Sound Januarythrough April, and will be obtained by gill netting with gear and small boats maintained by the NWFSCEnvironmental Conservation Division. Stripped gametes will be fertilized in the lab, and the naturally adherent eggsdeposited on glass microscope slides for exposure in oiled gravel effluent. Examination of early developmental

stages will be performed after manual dechorionationwith fine iris scissors. Analysis of cardiac function willinvolve videomicroscopy of live animals, as well asstructural characterization using cardiac chamber-specific antibodies [14, 15]. In addition, we anticipatethat new cardiac-specific markers of PAH effects will beidentified through the parallel zebrafish microarraystudy. Thus a second component of this aim is to clonethe homologous markers from Pacific herring as theyare characterized in zebrafish. Because only fragmentsof cDNA are required for the synthesis of in situhybridization probes, PCR-based cloning withredundant primers should be sufficient, and generation

of a herring cDNA library unnecessary. Methods for whole mount antibody labeling and in situ hybridization forzebrafish embryos are easily adapted to herring (figure above).

Aim 2: Our findings in zebrafish combined with prior studies on a range of other fish species (herring, salmon,mummichog, medaka, rainbowfish [6, 7, 16-18]) are most consistent with a common response of teleost embryos topetrogenic PAHs. However, to determine unequivocally whether PAHs in crude oil act through the same toxicmechanism in divergent species such as herring, we plan to replicate the AhR pathway knockdown studies inPacific herring. Antisense morpholino (MO) knockdown is now a widely used technique in diverse anamnioteembryos other than zebrafish, including sea urchin, frog, and ascidian. Importantly MO knockdown wasdemonstrated to work in fish species with much more prolonged development at cold temperatures. Effective geneinhibition at 15 to 30 days post-fertilization was demonstrated in rainbow trout [19, 20], which hatch in roughly 30days at 12-15˚C. Therefore, it is most likely that herring embryos, which develop in roughly 15 days at 10˚C, areamenable to the technique. The overall development of Pacific herring and zebrafish are quite similar (whenaccounting for temperature differences); for example, the heartbeat begins at roughly one-third of development forboth species. Therefore, we anticipate that the relationship of petrogenic PAH-induced cardiac dysfunction inherring—if observed—to AhR pathway activation should be readily dissected via MO knockdown. Herring AhRs(presumably AhR1 and AhR2 homologs) and CYP1A coding sequences will be obtained through a PCR strategyusing redundant primers [21] based on conserved regions in published sequences from zebrafish, medaka,mummichog, tomcod and rainbow trout. Design of translation-blocking MOs can be based on coding sequencederived from PCR-amplified cDNA. Microinjection of herring embryos will be performed with similar methods aszebrafish, although it may require prevention of the egg envelope hardening reaction. This reaction generallyinvolves protein cross-linking and was prevented in several species with glutathione [19, 20]. If egg hardening isinsurmountable, injection could be achieved with quartz needles rather than the standard borosilicate glass. Themain drawbacks to this are higher cost and inconvenience, as a special needle puller is required, which is availableat the nearby University of Washington campus.

Significance to oiled wildlife health: Forage fish such as herring are an extremely important component ofNortheast Pacific ecosystems, providing a major prey base for marine mammals, birds, and larger fish species suchas rockfish and lingcod. Three of the major regional forage fish species, Pacific herring (Clupea pallasi), surf smelt(Hypomesus pretiosus) and sand lance (Ammodytes hexapterus), have demersal eggs that adhere to vegetation orgravel/sand substrate in nearshore or intertidal zones. These zones are generally the most heavily impacted by oilspills, dramatically demonstrated in Prince William Sound after the Exxon Valdez spill. Although some studies have

Incardona 3documented long term, chronic effects of persistent oil on the Prince William Sound ecosystem, there is stillconsiderable debate over the veracity of these findings [12, 13, 22, 23], which has impacted the response tocontemporary spills such as the November 2002 Prestige incident [24]. Despite the scale and depth of analysesapplied to a range of biological systems as a consequence of Exxon Valdez, there is still no clear understanding ofthe mechanisms through which low-level PAH exposure adversely impacts organisms, in particular developing fishembryos. Guidelines for the protection of fish spawning habitat from anthropogenic inputs of PAHs will remainuncertain without filling these apparent data gaps concerning PAH toxicity.

Project Duration: These aims represent work proposed for the final year of a 3-year study. The broader goals ofthis project are to more accurately determine the impacts of oil on marine species. We plan to use knowledge offundamental mechanisms of toxicity for PAHs identified in zebrafish to develop technology that ultimately will betransferred to West Coast marine and anandromous species. Already the first two years of this study have providedconsiderable insight into the poorly characterized mechanisms underlying PAH toxicity in fish early life historystages. We anticipate that the proposed work should have a major impact on the direction of future research on theeffects of PAHs on aquatic ecosystems.

Incardona 4Estimated Budget:

Full-time salary for new molecular biology technician: $40,000

morpholinos 1800Travel 1200Indirect costs 6450

Total budget 49,450

Literature cited:

1. Kocan, R.M., et al., Pacific herring (Clupea pallasi) embryo sensitivity to Prudhoe Bay petroleumhydrocarbons: Laboratory evaluation and in situ exposure at oiled and unoiled sites in Prince WilliamSound. Can. J. Fish. Aquat. Sci., 1996. 53: 2366-2387.

2. Norcross, B.L., et al., Distribution, abundance, morphological condition, and cytogenetic abnormalities oflarval herring in Prince William Sound, Alaska, following the Exxon Valdez oil spill. Can. J. Fish. Aquat. Sci.,1996. 53: 2376-2387.

3. Middaugh, D.P., P.J. Chapman, and M.E. Shelton, Responses of embryonic and larval inland silversides,Menidia beryllina, to a water-soluble fraction formed during biodegradation of artificially weathered AlaskaNorth Slope crude oil. Arch. Environ. Contam. Toxicol., 1996. 31(3): 410-419.

4. Marty, G.D., et al., Ascites, premature emergence, increased gonadal cell apoptosis, and cytochromeP4501A induction in pink salmon larvae continuously exposed to oil-contaminated gravel duringdevelopment. Can. J. Zool.-Rev. Can. Zool., 1997. 75(6): 989-1007.

5. Middaugh, D.P., et al., Preliminary observations on responses of embryonic and larval Pacific herring,Clupea pallasi, to neutral fraction biodegradation products of weathered Alaska North Slope oil. Arch.Environ. Contam. Toxicol., 1998. 34(2): 188-196.

6. Carls, M.G., S.D. Rice, and J.E. Hose, Sensitivity of fish embryos to weathered crude oil: Part I. Low-levelexposure during incubation causes malformations, genetic damage, and mortality in larval Pacific herring(Clupea pallasi). Environ. Toxicol. Chem., 1999. 18(3): 481-493.

7. Heintz, R.A., J.W. Short, and S.D. Rice, Sensitivity of fish embryos to weathered crude oil: Part II.Increased mortality of pink salmon (Oncorhynchus gorbuscha) embryos incubating downstream fromweathered Exxon Valdez crude oil. Environ. Toxicol. Chem., 1999. 18(3): 494-503.

8. Vines, C.A., et al., The effects of diffusible creosote-derived compounds on development in Pacific herring(Clupea pallasi). Aquat. Toxicol., 2000. 51(2): 225-239.

9. Middaugh, D.P., et al., Effects of fractions from biodegraded Alaska North Slope crude oil on embryonicinland silversides, Menidia beryllina. Arch. Environ. Contam. Toxicol., 2002. 42(2): 236-243.

10. Payne, J.F., A. Mathieu, and T.K. Collier, Ecotoxicological studies focusing on marine and freshwater fish.,in PAHs—An Ecotoxicological Perspective, P.E.T. Dauben, Editor. 2003, John Wiley and Sons. p. 191-224.

11. Whyte, J.J., et al., Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemicalexposure. Crit. Rev. Toxicol., 2000. 30(4): 347-570.

12. Peterson, C.H., et al., Long-term ecosystem response to the Exxon Valdez oil spill. Science, 2003.302(5653): 2082-2086.

13. Huggett, R.J., et al., Biomarkers in fish from Prince William Sound and the Gulf of Alaska: 1999-2000.Environ. Sci. Technol., 2003. 37(18): 4043-51.

14. Incardona, J.P., et al., Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fishdevelopment. submitted, 2005.

15. Incardona, J.P., T.K. Collier, and N.L. Scholz, Defects in cardiac function precede morphologicalabnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol. Appl. Pharmacol.,2004. 196(2): 191-205.

Incardona 516. Couillard, C.M., A microscale test to measure petroleum oil toxicity to mummichog embryos. Environ.

Toxicol., 2002. 17(3): 195-202.17. Pollino, C.A. and D.A. Holdway, Toxicity testing of crude oil and related compounds using early life stages

of the crimson-spotted rainbowfish (Melanotaenia fluviatilis). Ecotox. Environ. Safe., 2002. 52(3): 180-189.18. Rhodes, S., et al., The effects of dimethylated and alkylated polycyclic aromatic hydrocarbons on the

embryonic development of the Japanese medaka. Ecotox. Environ. Safe., 2005. 60(3): 247-258.19. Boonanuntanasarn, S., et al., Molecular cloning, gene expression in albino mutants and gene knockdown

studies of tyrosinase mRNA in rainbow trout. Pigment Cell Res, 2004. 17(4): 413-421.20. Boonanuntanasarn, S., et al., Gene Knock-down in Rainbow Trout Embryos Using Antisense Morpholino

Phosphorodiamidate Oligonucleotides. Mar. Biotechnol., 2002. 4(3): 256-266.21. Hahn, M.E. and S.I. Karchner, Evolutionary conservation of the vertebrate Ah (dioxin) receptor:

amplification and sequencing of the PAS domain of a teleost Ah receptor cDNA. Biochem. J., 1995. 310:383-387.

22. Page, D.S., et al., Hydrocarbon composition and toxicity of sediments following the Exxon valdez oil spill inPrince William Sound, Alaska, USA. Environ. Toxicol. Chem., 2002. 21(7): 1438-1450.

23. Rice, S.D., et al., Comment on "Hydrocarbon composition and toxicity of sediments following the ExxonValdez oil spill in Prince William Sound, Alaska, USA". Environ. Toxicol. Chem., 2003. 22(11): 2539-2540.

24. Whitfield, J., Oil spills: How to clean a beach. Nature, 2003. 422(6931): 464-6.

Incardona 6Appendix:

NWFSC Internal Grant Proposal, funded March 2005 (attached)

NWFSC Internal Grants 2005

Identification of physiologically relevant biomarkers of PAH exposure for fish early life historystages using the zebrafish DNA microarray

Principal investigator: John Incardona, ECDJunior TrackBudget: $21,149Start/end dates: May-Nov 2005Key words: toxicology, oil spill, embryo, heart development, cardiovascular

Incardona 7SUMMARY:

Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants in nearshore marine andaquatic habitats. Fish early life history stages (embryos and larvae) exposed to PAHs suffer detrimentaleffects, both lethal and sublethal. While PAH contamination from environmental catastrophes such asthe Exxon Valdez oil spill have clear and dramatic effects on the early life history stages of native fish, amore insidious and less conspicuous source comes from fossil fuel consumption. Due to urbanizationand suburban sprawl, PAHs are a ubiquitous contaminant in stormwater runoff, which was identified asan increasing threat to the health of coastal habitats by both the Pew Oceans Commission and the U. S.Commission on Ocean Policy. PAHs are thus a major class of contaminant that will increase in aquatichabitats in conjunction with population increases projected for the Pacific Northwest over the nextseveral decades.

Studies following the Exxon Valdez spill identified a common syndrome that occurs in fish embryosexposed to PAHs in weathered crude oil, and documented sublethal effects that reduced the marinesurvival of pink salmon that were exposed to PAHs during embryogenesis but appeared externallynormal as smolts. The very low levels of PAHs to which these animals were exposed are often exceededin urban watersheds, so it is highly likely that PAHs associated with urban growth have effects onadjacent fish populations. The long-term goal associated with this pilot project is to identify biomarkersof PAH exposure in fish early life history stages that are predictive of health effects of exposure. Priorstudies demonstrated that the current biomarker for PAH exposure, cytochrome P4501A (CYP1A), is apoor indicator of the deleterious effects of PAHs on fish embryos. Using the zebrafish model system, wehave identified the embryonic heart as the most important target tissue for PAH exposure. Here Ipropose to identify potential cardiac-specific biomarkers that reflect the physiological changes inducedby PAH exposure, using gene profiling with oligonucleotide microarrays.

Incardona 8RESEARCH QUESTION: The early life history stages (embryos and larvae) of fish can be especiallysensitive to environmental contaminants due to the rapid proliferation, differentiation, and growth oftissues. Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuel consumption, arepervasive toxic contaminants in rivers, lakes, and nearshore marine habitats. Since the embryonic andlarval stages of many fish species are physically associated with substrates which trap PAHs, they maybe particularly susceptible to PAH exposure, as demonstrated dramatically by the 1989 Exxon Valdez oilspill [1-4]. Locally, tests conducted by the Puget Sound Ambient Monitoring Program detected PAHs inPacific herring eggs from spawning sites, and PAHs are common contaminants in urban streamsundergoing major habitat restoration. Population projections indicate that consumption-related sourcesof PAHs (e.g. automobiles) will continue to rise, and oil spills continue to be a problem in the PugetSound basin. Understanding the effects of PAHs on fish development is thus especially important for theprotection of marine resources and recovery of threatened and endangered salmonids. Yet fifteen yearsafter the Exxon Valdez spill, the pathophysiology behind the lethal and sublethal consequences of PAHexposure is still unclear.

Historically, the study of PAH toxicity has focused on the aryl hydrocarbon receptor (AhR) pathway.The AhR is a ligand-activated transcription factor that controls the expression of a battery of genesinvolved in the metabolism and elimination of xenobiotic compounds, including PAHs. PAHs bind theAhR and thus induce their own metabolism by enzymes such as cytochrome P4501A (CYP1A). CYP1Agenerally is the sole biomarker used to assess PAH exposure in field samples, and it is widely held thatPAH toxicity is mediated through toxic intermediates and oxidative stress resulting from CYP1Acatalytic activity. However, studies carried out using the zebrafish model during my tenure as an NRCassociate showed that the AhR pathway and CYP1A do not play causal roles in PAH toxicity, and thatCYP1A levels in fish embryos and larvae are unrelated to the negative impacts of PAH exposure.Instead, embryonic cardiac function is the primary target of petrogenic PAH toxicity [5, 6]. Thesefindings undermine much of the utility of CYP1A as a biomarker. As exemplified by the still highlycontentious debate over potential lingering effects more than a decade after the Exxon Valdez spill [4],there remains an urgent need for biomarkers that are indicative of the health status of fish that werespawned in PAH-contaminated habitats.

I propose to address this problem with the following specific aim: Identify new biomarkers thatreflect PAH-induced changes in cardiac function using gene expression profiling with a zebrafishmicroarray representing 14,900 transcripts. Conventional studies limited to one or a few genes havealready demonstrated changes in gene expression in response to cardiac dysfunction in zebrafishembryos [7, 8], and it is well established in human heart disease. The zebrafish model is particularlywell suited for microarray studies, because the genetic capabilities overcome one of the biggestchallenges of interpreting microarray data: identifying which genes are truly biologically relevant. Thepilot study proposed here is a simple microarray experiment utilizing 15 array chips and the follow-upPCR analysis to confirm changes in the expression of candidate genes. Future studies would determinethe pathophysiological role of genes identified in this screen, and ultimately develop homologousprobes/markers for analyzing embryos from field studies targeting species of concern for NOAAFisheries. Three features of the zebrafish model make this long term goal feasible: (1) the traditionalforward genetics approach utilized in the mid 1990s to generate a large collection of mutants that affectthe development of virtually every organ system in zebrafish; (2) the ability to “knock-down” geneexpression in zebrafish embryos by injection of highly stable antisense morpholino oligonucleotides atearly cleavage stages; and (3) the ability to overexpress cloned genes in zebrafish embryos by injectionof in vitro transcribed RNA. Thus changes in gene expression detected on microarrays can be readilytranslated into biological relevance.

Incardona 9METHODS AND RESEARCH PLANOverall experimental design: The goal is to compare gene expression profiles in cardiac tissue underthree conditions at a single time point during development (48 hpf): Wild type embryos exposed toweathered crude oil (effluent from an oiled gravel column); wild type embryos exposed to phenanthrene,a tricyclic PAH representing the most abundant class of PAHs in weathered crude oil; and embryos withcardiac dysfunction due to a mutation in a cardiac-specific gene. Two control conditions will includewild type embryos reared in an incubator (in parallel with phenanthrene exposure and the cardiac mutantembryos), and wild type embryos reared in the effluent of a control (unoiled) gravel column. Therationale for analyzing a cardiac-specific mutant is that I anticipate that the study could identify twoclasses of genes; those whose expression is changed directly as a consequence of PAH exposure, andthose that are changed secondarily as a response to cardiac dysfunction. Either type of gene has apotential role as biomarkers for PAH exposure, and the inclusion of a cardiac function mutant will allowthe unequivocal identification of transcriptional responses to changes in cardiac physiology. Of thezebrafish cardiac function mutants that have been characterized at the molecular level, and are known tobe cardiac specific genes, weak atrium (wea) is probably the best candidate. Mutations in wea disruptthe atrial myosin heavy chain gene, which is required for normal myofiber structure and contractilefunction of the atrium, and produces a cardiac function phenotype with features that overlaps with earlyphases of PAH-induced cardiac dysfunction. There are also changes in ventricular function and structurein wea mutants, which are secondary and represent a response to atrial dysfunction. Therefore, it islikely that there would be some overlap between changes in cardiac gene expression between PAH-exposed embryos and wea embryos.

The proposed work will be carried out in collaboration with the laboratory of Dr. Scott Argraves atthe Medical University of South Carolina (MUSC), and will utilize the commercially available zebrafisholigonucleotide gene array (the Affymetrix GeneChip® Zebrafish Genome Array). Dr. Argraves runsthe microarray core facility at MUSC, and is known from a collaboration stemming from the PI’sprevious position prior to joining the NWFSC. MUSC and NOAA-NOS are jointly involved inoperations of the Hollings Marine Lab and the East Coast OHH Center of Excellence, and Dr. Argraves’facility also provides support for genomics projects at NOS. Zebrafish embryo exposures, dissection ofhearts, and RNA preparation will be carried out by the PI at the NWFSC. Microarray hybridizations andsubsequent data analysis will be carried out at MUSC. Follow up real time quantitative PCR will becarried out by the PI at NWFSC using the Center’s facilities overseen by Dr. Penny Swanson.

Preparation of total RNA from dissected hearts: The array experiment will be performed in triplicate,thus utilizing 15 Affymetrix oligonucleotide microarray chips (3X3 experimental, 2X3 controls). Asingle 48 hpf embryo has roughly 0.5 µg RNA. The MUSC microarray facility is routinely working withpg quantities of RNA, so we anticipate that problems of target RNA dilution by non-target tissue can bemarkedly reduced by collection of hearts isolated from as few as 50 embryos. Thus a single arrayreplicate would entail dissection of and RNA isolation from 150 hearts from the three classes ofembryos. Experimental and control hearts dissected and placed into RNAlater reagent (Qiagen) tostabilize RNAs during the time required for dissection. Hearts will then be homogenized with Rnase-free micropestles and Qiashredders (Qiagen). If necessary, homogenates will be stored at -70°C to awaitfurther tissue collection. We anticipate that an RNA amplification step will be required, and performedusing the Arcturus RiboAmp® RNA Amplification Kit (recommended for 10-40 ng total RNA).

Quality control of total RNA preparations and cRNA target preparation: Quality and purity of totalRNA preparations will be assessed by A260:A280 ratios and by quantification of 28S:18S ribosomalRNA ratios as measured by an Agilent 2100 Bioanalyzer. Total RNA preparations with A260:A280ratios of ≥1.7 and 28S:18S ratios of ≥1.6 are typically deemed acceptable. Biotin-labeled, fragmentedcRNA targets will be prepared from total RNA samples using protocols established for the Affymetrix

Incardona 10GeneChip System (Santa Clara, CA; http://www.affymetrix.com/). Detailed protocols for biotin labelingand fragmentation of cRNA targets can be downloaded as a PDF fromhttp://www.affymetrix.com/products/reagents/specific/cleanup.affx. Fragmented cRNA samples will beevaluated with the Agilent 2100 Bioanalyzer to ensure appropriate fragment length distribution (~35-200 bases).

Hybridization of cRNA targets to Affymetrix GeneChips: Labeled and fragmented cRNA targets will behybridized to Affymetrix genomic oligonucleotide arrays. Target cRNAs will be submitted to the MUSCProteogenomics Facility for all GeneChip hybridization steps, including GeneChip hybridization, post-hybridization washing, staining and scanning. The facility, which employs Affymetrix GeneChiptechnology almost exclusively, is now in its fourth year of operation and has become proficient inmicroarray experimentation [9, 10]. Target preparations will be hybridized to Affymetrix Test3 arrays toassess target quality (e.g., 3’:5’ labeling ratios) and performance (e.g., detection of housekeeping genes,and absence of problematic background hybridization signals). Targets will then be hybridized toAffymetrix GeneChip® Zebrafish Genome Arrays that contain representations of over 14,900 Daniorerio transcripts. This array was constructed using information from RefSeq (July 2003), GenBank(release 136.0, June 2003), dbEST (July 2003), and UniGene (Build 54, June 2003).

Normalization of hybridization data and determination of differentially expressed genes: Many steps inthe analysis of DNA microarray data will be executed using the R statistical environment to implementpackages available from the Bioconductor open source software project (http://www.bioconductor.org/).Normalization and quality assessment of hybridization data are critical aspects of DNA microarrayanalysis. There are many different algorithms and methods currently available for these processes, andeach may result in subtle or perhaps significant differences in the interpretation of gene expression [11,12]. Raw hybridization data will be normalized among GeneChips using the Renvironment/BioConductor implementation of Robust MultiChip Average (RMA; [13-15]). RMAnormalized expression values will be compared between control and experimental samples anddifferentially expressed genes will be assessed according to the following criteria: fold change ≥2, p-value for unpaired t-Test ≤0.05, and false discovery rate (assessed by random iterations of permutedsample assignments) <0.05.

Hierarchical clustering and functional categorization of differentially expressed genes: Hierarchicalclustering of differentially expressed genes will be performed using the R environment/BioConductorimplementation of previously described methods [16, 17] Descriptive information, gene ontologyinformation and associated annotations for differentially expressed genes will be collected andassembled using the BioConductor package AnnBuilder. Differentially expressed gene sets will beevaluated for groups of functionally related genes using Gene Ontology classification as the primarydeterminant. To augment these tasks we will employ dbSieve, a web-based program developed by theMUSC Proteogenomics Facility in association with Array Genetics, Inc.(http://www.arraygenetics.com/).

Confirmation of candidate genes by Quantitative Real Time PCR. We plan to confirm changes in geneexpression detected in the microarray study by QPCR. Up to six candidate genes will be analyzed, withreagents designed for a seventh housekeeping gene as an internal control. QPCR will be performedusing the fluorescence-based TaqMan assay, using the NWFSC QPCR system based in the Swanson labin REUT. The TaqMan assay requires the design of a hybridization probe in addition to amplificationprimers, which requires sequence data from the target gene, as well as additional expense. We anticipatethat sufficient sequence data for most candidate genes will be available in the zebrafish genome or ESTdatabase.

Incardona 11

EXPECTED DIFFICULTIES: Microarray experiments with zebrafish embryos are uncommon todate. One of the biggest challenges in microarray studies is reducing variability in apparent geneexpression levels that result from confounding, trivial factors. For example, dissection errors couldcontribute to apparent changes in gene profiles. Similarly, it may be difficult to detect changes in geneexpression if genes of interest are diluted out by a high background of total RNAs. Most microarraystudies have thus utilized tissues (from adult animals) that can be easily dissected, collected, ormeasured (e.g. sections of an organ like liver, blood). Given the very small size of zebrafish embryos(~2.5 mm at 48 hpf), dissecting individual tissues in sufficient quantities is a challenge. It is not yet clearif total RNA isolated from whole embryos can yield useful tissue-specific information in microarraystudies. However, the zebrafish heart is one tissue that is readily accessible and easy to dissect in aconsistent manner. Although we expect that the RNA amplification step should compensate for theamount of tissue, we do not yet know precisely what yields of RNA can be obtained from isolatedhearts. Nevertheless, this difficulty is not insurmountable. It may mean that larger numbers of embryos(which are not limiting) will need to be exposed, and hearts dissected and frozen from multipleexposures before RNA isolation. Similarly the time spent dissecting may limit the number of embryosfor RNA isolation from a given exposure, requiring multiple exposures. Although tedious, the cardiacdissection is much less challenging than other microdissections that were integral to the PI’s previouspost-doctoral work in chick and mouse embryos. Ideally, the studies would be performed on singleexposures as another means to reduce variability.

GUIDELINES STATEMENT: The mission of the Northwest Fisheries Science Center is to “providethe scientific basis to meet NOAA’s stewardship role to conserve and manage living marine resourcesand their habitat with emphasis on the Pacific Northwest”. Included in this vision is to make meaningfulcontributions to the stewardship of living marine resources, and to support the NMFS Strategic Plan. Amajor component of the NMFS Strategic Plan is to “protect and maintain the health of coastal marinehabitats”. Inherent to these goals is a greater understanding of the anthropogenic impacts on fishpopulations. The work proposed here will contribute directly to the implementation of these goals. Thisis a pilot project that is unlikely to be funded by another source, and would otherwise not be completed.However, the pilot data would serve to determine whether microarray analysis should be incorporated(and appropriately funded) as a main tool used by the Ecotoxicology program to address potentialimpacts of the complex array of contaminants that will encroach upon fish habitat in the PacificNorthwest over the next several decades.

BUDGET:

Affymetrix GeneChips for zebrafish, 3 5-packs 6,000Qiagen RNeasy Mini Protect Kit 234Other RNA prep reagents/consumables 400Applied Biosystems TaqMan probes, 7 genes 1,820Enzymes/consumables for QPCR 400Applied Biosystems standard PCR machine 8,995(for basic RT-PCR prior to QPCR)Microdissection tools 800Stereomicroscope for dissection 2,500

Total budget 21,149

Incardona 12Literature cited:

1. Carls, M.G., S.D. Rice, and J.E. Hose, Sensitivity of fish embryos to weathered crude oil: Part I.Low-level exposure during incubation causes malformations, genetic damage, and mortality inlarval Pacific herring (Clupea pallasi). Environ Toxicol Chem, 1999. 18(3): p. 481-493.

2. Heintz, R.A., J.W. Short, and S.D. Rice, Sensitivity of fish embryos to weathered crude oil: PartII. Increased mortality of pink salmon (Oncorhynchus gorbuscha) embryos incubatingdownstream from weathered Exxon Valdez crude oil. Environ Toxicol Chem, 1999. 18(3): p.494-503.

3. Heintz, R.A., S.D. Rice, A.C. Wertheimer, R.F. Bradshaw, F.P. Thrower, J.E. Joyce, and J.W.Short, Delayed effects on growth and marine survival of pink salmon Oncorhynchus gorbuschaafter exposure to crude oil during embryonic development. Mar Ecol Prog Ser, 2000. 208: p.205-216.

4. Peterson, C.H., S.D. Rice, J.W. Short, D. Esler, J.L. Bodkin, B.E. Ballachey, and D.B. Irons,Long-term ecosystem response to the Exxon Valdez oil spill. Science, 2003. 302(5653): p. 2082-2086.

5. Incardona, J.P., M.G. Carls, H. Teraoka, D.W. Brown, T.K. Collier, and N.L. Scholz, Arylhydrocarbon receptor-independent toxicity of weathered crude oil during fish development.under internal review, 2005.

6. Incardona, J.P., T.K. Collier, and N.L. Scholz, Defects in cardiac function precedemorphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons.Toxicol Appl Pharmacol, 2004. 196(2): p. 191-205.

7. Berdougo, E., H. Coleman, D.H. Lee, D.Y. Stainier, and D. Yelon, Mutation of weakatrium/atrial myosin heavy chain disrupts atrial function and influences ventricularmorphogenesis in zebrafish. Development, 2003. 130(24): p. 6121-6129.

8. Joseph, E.M., Zebrafish IRX1b in the embryonic cardiac ventricle. Dev Dyn, 2004. 231(4): p.720-6.

9. Scott, K.K., R.A. Norris, S.S. Potter, D.W. Norrington, M.A. Baybo, D.M. Hicklin, and M.J.Kern, GeneChip microarrays facilitate identification of Protease Nexin-1 as a target gene of thePrx2 (S8) homeoprotein. DNA Cell Biol, 2003. 22(2): p. 95-105.

10. Cowart, L.A., Y. Okamoto, F.R. Pinto, J.L. Gandy, J.S. Almeida, and Y.A. Hannun, Roles forsphingolipid biosynthesis in mediation of specific programs of the heat stress responsedetermined through gene expression profiling. J Biol Chem, 2003. 278(32): p. 30328-30338.

11. Li, C. and W.H. Wong, Model-based analysis of oligonucleotide arrays: expression indexcomputation and outlier detection. Proc Natl Acad Sci U S A, 2001. 98(1): p. 31-36.

12. Hoffmann, R., T. Seidl, and M. Dugas, Profound effect of normalization on detection ofdifferentially expressed genes in oligonucleotide microarray data analysis. Genome Biology,2002. 3(7): p. research0033.1–research0033.11.

13. Irizarry, R.A., B.M. Bolstad, F. Collin, L.M. Cope, B. Hobbs, and T.P. Speed, Summaries ofAffymetrix GeneChip probe level data. Nucleic Acids Res, 2003. 31(4): p. e15.

14. Irizarry, R.A., L. Gautier, and L. Cope, An R package for analysis of Affymetrix oligonucleotidearrays, in The Analysis of Gene Expression Data: Methods and Software, R.I.G. Parmigiani, E.S.Garrett, and S. Ziegler, Editors. 2003, Springer: Berlin. p. 102-119.

15. Irizarry, R.A., B. Hobbs, F. Collin, Y.D. Beazer-Barclay, K.J. Antonellis, U. Scherf, and T.P.Speed, Exploration, normalization, and summaries of high density oligonucleotide array probelevel data. Biostatistics, 2003. 4(2): p. 249-264.

16. Eisen, M.B., P.T. Spellman, P.O. Brown, and D. Botstein, Cluster analysis and display ofgenome-wide expression patterns. Proc Natl Acad Sci U S A, 1998. 95(25): p. 14863-14868.

Incardona 1317. Golub, T.R., D.K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J.P. Mesirov, H. Coller, M.L.

Loh, J.R. Downing, M.A. Caligiuri, C.D. Bloomfield, and E.S. Lander, Molecular classificationof cancer: class discovery and class prediction by gene expression monitoring. Science, 1999.286(5439): p. 531-537.

Incardona 14

CURRICULUM VITAE

John Patrick Incardona

Degrees:MD Case Western Reserve University, Cleveland, OH 1996PhD Genetics, Case Western Reserve University, Cleveland, OH 1995BS Honors Biology, Indiana University, Bloomington, IN 1988

Employment/Research Experience:♦ Nov 2004-present Research Toxicologist, NMFS/NOAA Northwest Fisheries Science Center, Environmental

Conservation Division, Fish Neurobiology and Development Team♦ 2002-2004 National Research Council Senior Associate, NMFS/NOAA Northwest Fisheries Science Center,

Environmental Conservation Division (T. Collier). Analysis of environmental contaminant effects on fishdevelopment. Use of zebrafish as a model system for assessing the effects of pollutants on embryonic andlarval development, physiology, and behavior.

♦ 1999-2001 Senior Fellow, Department of Biological Structure, University of Washington (H. Roelink). Cellularaspects of Sonic Hedgehog (Shh) signaling in neural development and mechanism of action of Shh-inhibitoryteratogens; roles of cholesterol in Shh signaling.

♦ 1996-1999 Medical Teratology Fellow, Division of Congenital Defects, Departments of Pediatrics and BiologicalStructure, University of Washington (H. Roelink and R. Kapur). The role of Sonic Hedgehog signaling pathwayin the pathogenesis of holoprosencephaly induced by environmental teratogens.

♦ 1989-1994 Ph.D. Thesis in Genetics, Department of Pharmacology, Case Western Reserve University,Cleveland, OH (T. Rosenberry). Genetic Analysis of Glycolipid Anchor Function Using DrosophilaAcetylcholinesterase as a Model Protein. Molecular biology, generation of transgenic flies; protein expression,purification, and generation of antibodies; enzyme kinetics; subcellular fractionation/cellular biochemistry;microdissection and culture of primary neurons; immunocytochemistry and immunoelectron microscopy;behavioral assays.

♦ 1985-1987 Undergraduate Honors Thesis, Department of Biology, Indiana University, Bloomington, IN (E.C.Raff). Production of antibodies specific for ß3-tubulin of Drosophila and localization of ß3-tubulin in vivo.Recombinant DNA methods, production of bacterial fusion protein and synthetic peptides, polyclonal antibodyproduction, affinity purification, Western blots and immunocytochemistry.

♦ 1984-1985 Research assistant, Department of Virology and Molecular Biology, St. Jude Children's ResearchHospital, Memphis, TN (R.G. Webster). 1984, Hybridoma production and selection of monoclonal antibodiesagainst influenza N2 neuraminidase molecule; 1985, Selection of antigenic variants of H5 hemagglutininmolecule from H5N2 influenza associated with 1983-84 poultry epidemic in Pennsylvania; worked in P3-levelcontainment facility.

Fellowships:2002-present National Research Council Senior Associateship, National Atmospheric and Oceanic Administration,

Seattle1996-1999 National Research Service Award Fellowship, Medical Teratology Training Program, University of

Washington, Seattle1987-1996 National Research Service Award Pre-doctoral Fellowship, Medical Scientist Training Program, Case

Western Reserve University, Cleveland, OH

Journal Reviewer:Mechanisms of DevelopmentBirth Defects Research (formerly Teratology)

Incardona 15Research Update Service writer for Current Opinion in Cell Biology/BioMedNet Reviews OnlineAquatic ToxicologyDevelopment

Competitive Grants Awarded:Project title: Evaluating the effects of forestry herbicides on early life history stages of fish.Funding agency: U.S. Forest Service, Pesticide Impact Assessment ProgramCo-Investigator: Nat Scholz, NWFSCProject Duration: 2 years, beginning FY03Total Award: $104,518

Project title: Mechanisms of petroleum hydrocarbon toxicity in fish at early life history stagesFunding agency: Oiled Wildlife Care NetworkCo-investigator: Nat Sholz, NWFSCProject duration: 3 years, beginning FY03Total award: $48,559 (year 1); $49,394 (year 2)

Project title: Identification of physiologically relevant biomarkers of PAH exposure for fish early lifehistory stages using the zebrafish DNA microarrayFunding Agency: NOAA Northwest Fisheries Science Center Internal Grants ProgramProject duration: 1 yearTotal award: $21,149

Peer-reviewed Publications:Incardona JP, Collier TK, and Scholz NL. 2004. Defects in cardiac function precede morphological abnormalities infish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol. Appl. Pharm. 196: 191-205.Incardona JP, Gruenberg J and Roelink H. 2002. Sonic hedgehog induces segregation of Patched and

Smoothened in late endosomes. Curr. Biol. 12: 983-995Incardona JP, Lee JH, Robertson CP, Enga K, Kapur RP, and Roelink H. 2000. Receptor-mediated endocytosis of

soluble and membrane-tethered forms of Sonic hedgehog by Patched-1. Proc. Natl. Acad. Sci. U.S.A. 97:12044-12049.

Incardona JP, Gaffield W, Lange Y, Cooney A, Pentchev PG, Liu S, Watson JA, Kapur RP and Roelink H. 2000.Cyclopamine inhibition of Sonic hedgehog signal transduction is not mediated through effects on cholesteroltransport. Dev. Biol. 224: 440–452.

Neufeld EB, Wastney M, Patel S, Suresh S, Cooney AM, Dwyer NK, Roff CF, Ohno K, Morris JA, Carstea ED,Incardona JP, Strauss JF III, Vanier MT, Patterson MC, Brady RO, Pentchev PG and Blanchette-Mackie EJ.1999. The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport ofmultiple lysosomal cargo. J. Biol. Chem. 274:9627-9635.

Incardona JP, Gaffield W, Kapur RP, and Roelink H. 1998. The teratogenic Veratrum alkaloid cyclopamine inhibitsSonic Hedgehog signal transduction. Development 125:3553-3562.

Incardona JP and Rosenberry TL. 1996. Replacement of the glycoinositol phospholipid anchor of Drosophilaacetylcholinesterase with a transmembrane domain does not alter sorting in neurons and epithelia but results inbehavioral defects. Molec. Biol. Cell 7:613-630

Incardona JP and Rosenberry TL. 1996. Construction and characterization of secreted and transmembrane-anchored forms of Drosophila acetylcholinesterase. A large truncation of the C-terminal signal peptide does noteliminate glycoinositol phospholipid anchoring. Molec. Biol. Cell 7:595-611.

Kimble K, Incardona JP and Raff EC. 1989. A variant ß-tubulin isoform of Drosophila melanogaster (ß3) isexpressed primarily in tissues of mesodermal origin in embryos and pupae, and is utilized in populations oftransient microtubules. Dev. Biol. 131:415-429.

Incardona 16CURRICULUM VITAE

Nathaniel L. Scholz

Degrees:Ph.D. Zoology, University of Washington, Seattle, WA 1997M.A. Biology, Boston University Marine Program, Woods Hole, MA 1991B.A. Marine Biology with Distinction, Boston University Marine Program 1991

Fellowships and Awards:♦ Special Act Award, National Oceanic and Atmospheric Administration, 2002♦ Sustained Superior Performance Award, National Oceanic and Atmospheric Administration, 2001♦ National Research Council Postdoctoral Associateship, 1998-1999, National Oceanic and Atmospheric

Administration (E. Casillas)♦ National Institute of Health Predoctoral Traineeship, Developmental Biology, 1992-1996, University of

Washington (J.W. Truman and K. Graubard)

Employment:♦ Graduate Research Assistant, 1992-1997, University of Washington, Department of Zoology (J.W. Truman and

K. Graubard). Studied mechanisms of diffusible neurotransmission and cell-cell signaling in the nervoussystem. Discovered a novel role for nitric oxide in the central production of rhythmic motor behaviors.

♦ Postdoctoral Associate, 1998-1999, National Oceanic and Atmospheric Administration (E. Casillas).Investigated the effects current use pesticides on the neurobiology and behavior of Pacific salmon. Found thatthe insecticide diazinon interferes with predator avoidance and homing behaviors in chinook salmon.

♦ Research Zoologist, 1999-present, National Oceanic and Atmospheric Administration. Supervising a researchlaboratory at the Northwest Fisheries Science Center in Seattle. Our research focuses on environmentalcontaminants and their impacts on the neurobiology and development of protected fish species.

Teaching (TA):♦ Completed Preparing Future Faculty program, 1996, University of Washington and Pew Charitable TrustsChemosensory Biology, Woods Hole (22 students, marine science majors)Experimental Cell Biology, Friday Harbor (12 students, graduate)Introductory Physiology, Seattle (18 students, freshmen and nonmajors)Marine Invertebrate Zoology, Friday Harbor (15 students, graduate)Natural History of Marine Invertebrates, Seattle (28 students, juniors and seniors)Physiology and Development, Seattle (24 students, sophomores and juniors)Sea Education Semester, Lesser Antilles (24 students, juniors and seniors)

Journal Reviewer:American ZoologistComparative Biochemistry and PhysiologyJournal of Comparative NeurologyPesticide Biochemistry and Physiology

Presentations at Professional Meetings:Georgia Basin/Puget Sound Research Conference (2003)International Congress for Neuroethology (1992, 1995, 1998)Society for Behavioral Toxicology (2002)Society for Environmental Toxicology and Chemistry (1999)Society for Integrative and Comparative Biology (1999)

Incardona 17Society for Neuroscience (1993, 1994, 1995, 1997, 1999)Society for Risk Assessment (2001)

Invited Lectures and Seminars:Pacific Northwest Threatened and Endangered Species Conference, Yakima, 1999Society for Integrative and Comparative Biology, Session on Nitric Oxide, 2000Pacific Northwest Agriculture and Water Quality Conference, Eugene, 2000Oregon Graduate Institute, Winter Seminar Series, 2001Duke University Integrated Toxicology Program, Scholar Seminar Series, 2001University of Wisconsin-Madison, Zoology Department, Spring Seminar Series, 2002Behavioral Toxicology Society, National Meeting, Research Triangle Park, NC, 2002Washington State University, Cooperative Extension Workshop, Pullman, 2002Northwest Fisheries Science Center, Winter Seminar Series, Seattle, 2003

Popular Media:“Research suggests pesticides disrupt how salmon smell”, Seattle Post-Intelligencer and

Associated Press, Aug. 1, 2000.“Pesticides enter salmon picture”, Seattle Post-Intelligencer, Jan. 30, 2001“When it rains, it pours pollutants into the waters”, Seattle Post-Intelligencer, Nov. 20,

2002.“Our troubled sound: Spawning coho are dying early in restored creeks”, Seattle Post-

Intelligencer and Associated Press, Feb. 6, 2003.“Urban runoff killing salmon in Washington”, Environment News Service, Feb. 7, 2003.

Competitive Research Grants Awarded:

Project Title: Rapid phenotypic screening in zebrafishFunding Agency: NOAA/NMFS/NWFSC, Internal Grants ProgramCo-Investigator: N/AProject Duration: 1 year (FY01)Total Award: $55,700

Project Title: Sublethal effects of the carbamate insecticide, carbaryl, on coastalcutthroat trout in Willapa Bay, Washington

Funding Agency: UFSWS, Environmental Contaminants ProgramCo-Investigator: Jay Davis, USFWS, Western Washington OfficeProject Duration: 4 years, beginning FY02Total Award: $217,520

Project Title: Effects of algal toxin exposure in early life history stages of fishFunding Agency: Ecology and Oceanography of Harmful Algal Blooms (ECOHAB)Co-Investigators: Kathi Lefebvre and Vera Trainer, NWFSCProject Duration: 3 years, beginning FY02Total Award: $276,573

Project Title: Evaluating the effects of forestry herbicides on early life historystages of fish.

Funding Agency: U.S. Forest Service, Pesticide Impact Assessment ProgramCo-Investigator: John Incardona, NWFSCProject Duration: 2 years, beginning FY03Total Award: $104,518

Incardona 18Refereed Publications:

Sandahl, J., Baldwin, D.H., Jenkins, J.J., and Scholz, N.L. (2003). Odor-evoked fieldpotentials as indicators ofsublethal neurotoxicity in juvenile coho salmon exposed to common agricultural pesticides. Canadian Journal ofFisheries and Aquatic Sciences, Submitted.

Incardona, J.P., Collier, T.K., and Scholz, N.L. (2003). Mechanisms of polycyclic aromatic hydrocarbon toxicity inearly life history stages of fish. Environmental Science and Technology, Submitted.

Baldwin, D.H., Sandahl, J.F., Labenia, J.S., and Scholz, N.L. (2003). Sublethal effects ofcopper on coho salmon:impacts on non-overlapping receptor populations in the periperhal olfactory nervous system. EnvironmentalToxicology and Chemistry. In press.

Scholz, N.L., Labenia, J., de Vente, J., Graubard, K., and Goy, M.F. (2002). Expression of nitric oxide synthaseand nitric oxide-sensitive guanylate cyclase in the crustacean cardiac ganglion. Journal of ComparativeNeurology, 454:158-167.

Scholz, N.L., de Vente, J., Truman, J.W., and Graubard, K. (2001). Neural network partitioning by NO and cGMP.Journal of Neuroscience, 21:1610-1618.

Scholz, N.L. and Tchobanoglous, G. (2001). Water pollution. In: McGraw-Hill Encyclopedia of Science andTechnology, 9th Edition. McGraw-Hill Book Co., New York.

Scholz, N.L. (2001). NO/cGMP signaling and the flexible organization of motor behavior in crustaceans. AmericanZoologist. 41:156-167.

Scholz, N.L., Truelove, N., French, B., Berejikian, B., Quinn, T, Casillas, E., and Collier, T.K. (2000). Diazinondisrupts antipredator and homing behaviors in chinook salmon (Oncorhynchus tshawytscha). Canadian Journalof Fisheries and Aquatic Sciences, 57:1911-1918.

Baro, D.J., Ayali, A., French, L., Scholz, N.L., Labenia, J, Lanning, C.C., Graubard, K., and Harris-Warrick, R.M.(2000). Molecular underpinnings of motor pattern generation: Differential targeting of shal and shaker in thepyloric motor system. Journal of Neuroscience, 20:6619-6630.

Scholz, N.L., Graubard, K., and Truman, J.W. (1998). The NO/cGMP signaling pathway and the development ofneural networks in postembryonic lobsters. Journal of Neurobiology, 34:208-226.

Prabakhar, S., Short, D.B., Scholz, N.L., and Goy, M.F. (1997). Identification of nitric oxide-sensitive andinsensitive forms of cytoplasmic guanylate cyclase. Journal of Neurochemistry, 69:1650-1660.

Scholz, N.L., Goy, M.F., Truman, J.W., and Graubard K. (1996). Nitric oxide and peptide neurohormones activatecGMP synthesis in the crab stomatogastric ganglion. Journal of Neuroscience, 16:1614-1622.

Moore, P.A., Scholz, N.L., and Atema, J. (1991). Chemical orientation of lobsters, Homarus americanus, inturbulent odor plumes. Journal of Chemical Ecology, 17:1293-1307.

Book Chapters and Other PublicationsScholz, N.L. and Truman, J.W. (2000). Invertebrate models for studying NO-mediated signaling. In: Handbook of

Chemical Neuroanatomy Vol. 17: Functional Neuroanatomy of the Nitric Oxide System (H. Steinbusch, J. DeVente, S.R. Vincent, A. Bjorklund, and T. Hokfelt eds.). Elsevier. pp. 417-441.

Scholz, N.L. and Collier, T.K. (2000). Endangered salmon and probabilistic risk assessments for organophosphatepesticides. SETAC Globe 1:32-33.

Moran, B., Friedman, R., Davis, J., LaTier, A., and Scholz, N.L. (2001). A process for evaluating pesticides inWashington State surface waters for potential impacts to salmonids. Washington State Dept. AgriculturePublication, No. 057. 30 pp.

Steel, E.A., Liermann M., McElhany P., Scholz, N.L., and Cullen, A. (2002). Managing uncertainty in salmon habitatrecovery planning. In: Ecosystem recovery planning for listed salmon: an integrated assessment approach forsalmon habitat (Eds. Beechie T.J., Roni, P., and Steel E.A.). NOAA Technical Memorandum. In press.