Yungkul Kim

Eric N Powell

Terry L Wade

Bobby J Presley

Relationship of Parasites and Pathologies to Contaminant Body Burden in Sentinel Bivalves: NOAA Status and Trends `Mussel Watch' Program

The 1995-1998 database from NOAA's National Status and Trends `Mussel Watch' Program was used to compare the distributional patterns of parasites and pathologies with contaminant body burdens. Principal components analysis (PCA) resolved ve groups of contaminants in both mussels and oysters: one dominated by PAHs, one dominated by pesticides, and three dominated by metals. Metals produced a much more complex picture of spatial trends in body burden than did either the pesticides or PAHs. Contrasted to the relative simplicity of the

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Final xDraft { September 11, 2007 2 contaminant groupings, PCA exposed a suite of parasite/pathology groups with few similarities between the sentinel bivalve taxa. Thus, the relationship between parasites/pathologies and contaminants di ers signi cantly between taxa despite the similarity in contaminant pattern. Moreover, the combined e ects of many contaminants and parasites may be important, leading to complex biologicalcontaminant interactions with synergies both of biological and chemical origin.

Overall, correlations between parasites/pathologies and contaminants were more frequent with metals, frequent with pesticides, and less frequent with PAHs in mussels. In oysters, correlations with pesticides and metals were about equally frequent, but correlations with PAHs were still rare. In mytilids, correlations with metals predominated. Negative and positive correlations with metals occurred with about the same frequency in both taxa. The majority of correlations with pesticides were negative in oysters; not so for mytilids. Of the many signi cant correlations involving parasites, few involved single-celled eukaryotes or prokaryotes. The vast majority involved multi-cellular eukaryotes and nearly all of them either cestodes, trematode sporocysts, or trematode metacercariae. The few correlations for singlecelled parasites all involved proliferating protozoa or protozoa reaching high body burdens through transmission. The tendency for the larger or more numerous parasites to be involved suggests that unequal sequestration of contaminates between host and parasite tissue is a potential mediator. An alternative is that contaminants di erentially a ect parasites and their hosts by varying host susceptibility or parasite survival.

Introduction

Bivalve mollusks, such as mussels or oysters, are often used as sentinel organisms in contaminant monitoring programs [START_REF] Phillips | The use of biological indicator organisms to monitor trace metal pollution in marine and estuarine environments -a review[END_REF][START_REF] Farrington | US Mussel Watch 1976-1978: An overview of the trace metal, DDE, PCB, hydrocarbon and arti cial radionuclide data[END_REF][START_REF] Green | Use of freshwater mussels (Bivalvia: Unionidae) to monitor the nearshore environment of lakes[END_REF][START_REF] Oliver | Appraisal of prospective bivalve immunomarkers[END_REF][START_REF] Peterson | The \Exxon Valdez" oil spill in Alaska: Acute, indirect and chronic e ects on the ecosystem[END_REF]. Bivalves are preferentially chosen as sentinel organisms for their ability to concentrate both metals and organic contaminants, their immobility, their limited ability to metabolize accumulated contaminants, their relatively wide distribution among habitats including those relatively heavily polluted, their abundance, their persistence, and their ease of collection, all of which make them good long-term integrators of the surrounding environment [START_REF] Phillips | The use of biological indicator organisms to monitor trace metal pollution in marine and estuarine environments -a review[END_REF][START_REF] O'connor | National distribution of chemical concentrations in mussels and oysters in the USA[END_REF].

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The National Status and Trends `Mussel Watch' Program has been monitoring contaminants of environmental concern since 1986 using bivalves as sentinel organisms [START_REF] O'connor | National distribution of chemical concentrations in mussels and oysters in the USA[END_REF]. As Mussel Watch was designed to document the geographic distribution and temporal trends of contamination on a national scale, most sites were placed away from known point sources of contamination, poorly ushed industrial waterways, and pilings or metal buoys [START_REF] O'connor | Recent trends in coastal environmental quality: Results from the Mussel Watch Project[END_REF][START_REF] O'connor | Trends in chemical concentrations in mussels and oysters collected along the U.S. coast from 1986 to 1993[END_REF]. Sampling sites of an earlier Mussel Watch program [START_REF] Goldberg | The Mussel Watch[END_REF][START_REF] Goldberg | Mussel Watch: 1977-1978 results on trace metals and radionuclides[END_REF] were, when possible, incorporated into the current Mussel Watch program [START_REF] O'connor | The National Oceanic and Atmospheric Administration (NOAA) National Status and Trends Mussel Watch Program: National monitoring of chemical contamination in the coastal United States[END_REF].

Bivalves are presently collected every other year at a network of over 250 estuarine, lakeshore, and coastal locations along the U.S. Great Lakes, East, West, and Gulf coasts, with approximately half being visited during each annual sampling. Bivalve samples are analyzed for a variety of contaminants, including polycyclic aromatic hydrocarbons (PAHs), chlorinated pesticides, polychlorinated biphenyl (PCB) congeners, and major and trace elements. The Mussel Watch program also monitors the health of bivalve populations through the measurement of a variety of biological indices, including the prevalence and infection intensity of parasites, diseases, and tissue pathologies. The database thus obtained provides an opportunity to examine the relationships between parasites and pathologies on the one hand and contaminants on the other over a large geographic scale. The question of the relationship of contaminant body burden to parasites and pathologies is a complex one. Extrapolating from laboratory and eld experimentation comparing parasites/pathologies with contaminant body burden to inferences at geographic scales has been di cult, due to the multiplicity of contaminants and parasites present in most environments and the limited geographic scale of most present-day databases inclusive of both variables.

Certain contaminants may increase parasite body burden favoring the propagation of parasites by excluding their natural predators, by reducing the resistance of their hosts, or by providing improved living conditions for their intermediate hosts (M oller, 1987). Contaminants may also interfere with parasite transmission or proliferation within hosts and thus reduce parasite burden [START_REF] La Erty | How environmental stress a ects the impacts of parasites[END_REF]. The interaction of parasite and pathology with contaminant body burden is known ACCEPTED MANUSCRIPT Final xDraft { September 11, 2007 4 primarily from controlled experiments. [START_REF] Barszcz | Chronic e ects of three crude oils on oysters suspended in estuarine ponds[END_REF] showed that host oysters exposed to crude oil had an increase in prevalence and intensity of infection by parasites, for example. Experimental studies by [START_REF] Pascoe | The e ect of parasitism on the toxicity of cadmium to the three-spined stickleback, Gasterosteus aculeatus L[END_REF] and [START_REF] Boyce | E ects of a parasite, Eubothrium salvelini (Cestoda: Pseudophyllidea), on the resistance of juvenile sockeye salmon, Oncorhynchus nerka, to zinc[END_REF] showed that parasitized host shes were more sensitive to Cd and Zn, respectively. Ciliate epibionts increased host mortality in copepods exposed to PAHs [START_REF] Puckett | Ciliate epibiont e ects on feeding, energy reserves, and sensitivity to hydrocarbon contaminants in an estuarine harpacticoid copepod[END_REF]. In contrast, [START_REF] Khan | E ects of crude oil on the gastrointestinal parasites of two species of marine sh[END_REF] found reduced infections of endoparasites in host sh exposed to crude oil.

Whether the in uence of the contaminant is directly on the parasite or mediated through the host is often unclear. The immune systems of host animals can be a ected by contaminants [START_REF] Sindermann | Pollution associated diseases and abnormalities of sh and shell sh: A review[END_REF][START_REF] Rohde | Ecology of marine parasites: An introduction to marine parasitology[END_REF], lowering the resistance of a host to a variety of parasites and increasing susceptibility to parasitic invasion and disease [START_REF] Barszcz | Chronic e ects of three crude oils on oysters suspended in estuarine ponds[END_REF][START_REF] Galli | Water quality as a determinant of the composition of sh parasite communities[END_REF]. [START_REF] Thulin | Can sh parasites be used to monitor pollution?[END_REF] pointed out that ectoparasites may be particularly sensitive to contaminants because they are in intimate contact with the external environment. Such parasites may be useful indicator organisms for early detection of adverse environmental e ects [START_REF] Thulin | Can sh parasites be used to monitor pollution?[END_REF][START_REF] Mackenzie | Parasites as indicators of water quality and the potential use of helminth transmission in marine pollution studies[END_REF][START_REF] Galli | Water quality as a determinant of the composition of sh parasite communities[END_REF].

The cause and e ect explanation is most often the inference from observed correlations between parasite body burden and contaminant body burden. More recently, however, research has begun documenting an alternative explanation; the di erential in body burden between parasites and their hosts may accrue from di erential a nities for contaminant accumulation. [START_REF] Zimmerman | Experimental studies on lead accumulation in the eel-speci c endoparasites Anguillicola crassus (Nematoda) and Paratenuisentis ambiguus (Acanthocephala) as compared with their host, Anguilla anguilla[END_REF], [START_REF] Sures | Parasites as accumulation indicators of heavy metal pollution[END_REF], and [START_REF] Heinonen | Temperature-and parasite-induced changes in toxicity and lethal body burdens of pentachlorophenol in the freshwater clam Pisidium amnicum[END_REF] provide salient examples. Although this potential is not yet included in models of contaminant body burden (e.g., [START_REF] Fraysse | E ects of Cd and Zn waterborne exposure on the uptake and depuration of 57 Co, 110m Ag and 134 Cs by the Asiatic clam (Corbicula uminea) and the zebra mussel (Dreissena polymorpha){whole organism study[END_REF][START_REF] Lee | Prediction of time-dependent PAH toxicity in Hyalella azteca using a damage assessment model[END_REF][START_REF] Yamamoto | Application of a twocompartment one-toxin model to predict the toxin accumulation in Paci c oysters in Hiroshima Bay, Japan[END_REF], this realization leaves the obvious quandary for sentinel bivalves of whether parasite body burden by their tissue contribution in uences the measured value for the host organism as a whole. Parasites may contribute a signi cant fraction of host biomass (e.g., [START_REF] Powell | In uence of parasitism in controlling the health, reproduction and PAH body burden of petroleum seep mussels[END_REF]; thus, di erential a nities could be important. This possibility o ers one explanation for the frequent nding that contaminant body burden is non-normally distributed among individuals at a site, such that a few animals have unusually high contaminant body burdens [START_REF] Boyden | Seasonal variation and inherent variability of trace elements in oysters and their implications for indicator studies[END_REF][START_REF] Daskalakis | Variability of metal concentrations in oyster tissue and implications to biomonitoring[END_REF][START_REF] Baldwin | Spatial and temporal variation of selenium concentration in ve species of intertidal molluscs from Jervis Bay, Australia[END_REF]. Parasites tend to be skewed similarly in their distribution among hosts [START_REF] Munger | Host genetics as a cause of overdispersion of parasites among hosts: How general a phenomenon[END_REF][START_REF] Jones | Fitting the negative binomial distribution to parasitological data[END_REF].

ACCEPTED MANUSCRIPT Final xDraft { September 11, 2007 5 The interaction between tissue pathology and contaminant body burden has also received some attention. [START_REF] Lowe | Chronic exposure of oysters to DDT, toxaphene and parathion[END_REF] demonstrated that oysters exposed chronically to the pesticide DDT showed edema, lesions, and necrosis of the digestive tubule epithelium. [START_REF] Farley | Ultrastructural observations on epizootic neoplasia and lytic virus infection in bivalve mollusks[END_REF] suggested the possible induction of epizootic neoplasia in bivalve molluscs by carcinogenic compounds, such as pesticides (aldrin and dieldrin). [START_REF] Couch | The American oyster (Crassostrea virginica) as an indicator of in the aquatic environment[END_REF] found that oysters exposed to the carcinogen benzo(a)pyrene showed in ammation of the connective tissue and proliferation of a cell resembling a sarcomatoid neoplasm. [START_REF] Wintermyer | Dioxin/furan and polychlorinated biphenyl concentrations in Eastern oyster (Crassostrea virginica, Gmelin) tissue and the e ects on egg fertilization and development[END_REF] and [START_REF] Sv Ardh | Incidence of hemocytes and parasites in coastal populations of blue mussels (Mytilus edulis){testing correlations with area, season, and distance to industrial plants[END_REF] provide additional examples. The distribution of pathologies in the eld, however, can rarely be ascribed unequivocally to individual contaminants [START_REF] Sindermann | An examination of some relationships between pollution and disease[END_REF][START_REF] Johnson | National benthic surveillance project: Northeast coast. Fish histopathology and relationships between lesions and chemical contaminants (1987-89)[END_REF][START_REF] Landsberg | Neoplasia and biotoxins in bivalves: Is there a connection[END_REF]. The establishment of parasitism and disease in aquatic populations is usually not due to a simple cause-and-e ect relationship, but is dependent upon a complex interaction between organism and environment.

Thus, the use of parasites or pathologies as indicators of pollution stress has rarely been unequivocal (M oller, 1987). One important constraint is the rarity of surveys that include both contaminant and health assessments. [START_REF] Yevich | Histopathology as a monitor for marine pollution. Results of histopathological examinations of the animals collected for the US 1976 Mussel Watch Program[END_REF] correlated stations with high parasitism with stations with high PCB concentrations in host bivalves collected from natural coastal and estuarine waters in the rst Mussel Watch Program. [START_REF] Wilson | Spatial and temporal distributions of contaminant body burden and disease in Gulf of Mexico oyster populations: The role of local and large-scale climatic controls[END_REF] and [START_REF] Kim | The geographic distribution of population health and contaminant body burden in Gulf of Mexico oysters[END_REF] examined the relationship of contaminant body burden to health indices at Gulf of Mexico Mussel Watch sites. [START_REF] Johnson | National benthic surveillance project: Northeast coast. Fish histopathology and relationships between lesions and chemical contaminants (1987-89)[END_REF] and [START_REF] Schmidt | Parasites of ounder (Platichthys esus L.) from the German Bight, North Sea, and their potential use in biological e ects monitoring. C. Pollution e ects on the parasite community and a comparison to biomarker responses[END_REF] obtained promising results relating sh pathologies to contaminant burdens along the northeast coast of the U.S. and in the North Sea, respectively. The Mussel Watch Program is a unique multi-year study and the rst of its kind that covers a large geographic area, multiple parasite species, and multiple contaminants. Here, we used the Mussel Watch database for 1995-1998 to examine the relationships between parasitism, pathology, and contaminant body burdens on regional scales with emphasis on identifying parasite assemblages and contaminant suites that resolve as signi cant relationships within the complex medley of potential contaminant/parasite/pathology combinations. Such relationships can provide the basis for mapping the results of individual cause and e ect experiments to integrated population level processes observed in the eld.

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Materials and Methods

Species Sampled and Sample Collection Species Sampled and Sample Collection Species Sampled and Sample Collection

The bivalve samples were collected annually from a network of sites established along the United States coastline. Bivalves were dredged or hand-picked in intertidal to shallow subtidal areas. Sampling targeted the largest animals available at each site. Each sampled site subsequently was assigned to one of 126 bays see [START_REF] Lauenstein | Mussel Watch Project site descriptions through 1997[END_REF] for further details of site location]. A `bay', as termed herein, represents all the sites in a single estuary, estuarine reach of a large estuary, or group of neighboring sites on an open coastline. Details on the allocation of sites to bays can be found in the Appendix of Kim and Powell (2006).

The introduced zebra mussel, Dreissena polymorpha, and quagga mussel, D. bugensis, were sampled at sites in the Great Lakes (Bays 1-10) and Hudson River (Bay 11). Zebra mussels were rst discovered in the Great Lakes in 1988 [START_REF] Hebert | Ecological and genetic studies on Dreissena polymorpha (Pallas): A new mollusc in the Great Lakes[END_REF]. Quagga mussels were found in 1991 [START_REF] May | Genetic identi cation and implications of another invasive species of dreissenid mussel in the Great Lakes[END_REF]. Both likely were introduced from Europe in ship ballast water [START_REF] Hebert | Ecological and genetic studies on Dreissena polymorpha (Pallas): A new mollusc in the Great Lakes[END_REF]. Both species spread rapidly throughout the Great Lakes system and then into the major river basins of the central and eastern U.S. [START_REF] Gri Ths | Distribution and dispersal of the zebra mussel (Dreissena polymorpha) in the Great Lakes Region[END_REF][START_REF] Strayer | Projected distribution of the zebra mussel, Dreissena polymorpha, in North America[END_REF][START_REF] Mills | Exotic species in the Hudson River Basin: A history of invasions and introductions[END_REF]. Recently, the quagga mussel has displaced the zebra mussel in many areas [START_REF] Stoeckmann | Physiological energetics of Lake Erie dreissenid mussels: A basis for the displacement of Dreissena polymorpha by Dreissena bugensis[END_REF]. [START_REF] Rosenberg | A nomenclatural review of Dreissena (Bivalvia: Dreissenidae), with identi cation of the quagga mussel as Dreissena bugensis[END_REF] discussed the taxonomy. Mussel Watch sites include all Great Lakes except Lake Superior [START_REF] Lauenstein | Mussel Watch Project site descriptions through 1997[END_REF].

Mytilid mussel taxa were collected from the Northeast (Bays 12-36) and West (Bays 94-125) coasts, including Alaska. According to [START_REF] Hilbish | Origin of the antitropical distribution pattern in marine mussels (Mytilus spp.): Routes and timing of transequatorial migration[END_REF], mussels collected on the East coast were Mytilus edulis sensu stricto, as M. edulis is the predominant species from central Maine south [START_REF] Rawson | Species composition of blue mussel populations in the northeastern Gulf of Maine[END_REF] to Cape Hatteras [START_REF] Wells | The seasonal occurrences of Mytilus edulis on the Carolina coast as a result of transport around Cape Hatteras[END_REF]. On the West coast, three mussel taxa were collected, M. californianus and two species referable to the M. edulis complex, M. galloprovincialis and M. trossulus. The taxonomic status of species in the M. edulis complex is uncertain [START_REF] His | The assessment of marine pollution { bioasssays with bivalve embryos and larvae[END_REF]. Mytilus californianus was collected at 31 sites mostly located at jetties, points or capes among the 58 total West coast sites excluding Alaska (viz., 27 sites out of 36 in California, two sites among six in

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Oregon and two sites among 16 in Washington). Mytilus trossulus was collected at the vast majority of the more northern stations from Oregon to Alaska. Mytilus galloprovincialis was introduced into the eastern Paci c in the 1880s and now occurs from central California to Baja California, with some populations probably farther north [START_REF] Mcdonald | The mussels Mytilus galloprovincialis and M. trossulus on the Paci c coast of North America[END_REF][START_REF] Koehn | The genetics and taxonomy of species in the genus Mytilus[END_REF][START_REF] Seed | Systematics evolution and distribution of mussels belonging to the genus Mytilus: An overview[END_REF], including a population in Puget Sound established at least by 1988 [START_REF] Wonham | Mini-review: Distribution of the Mediterranean mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) and hybrids in the northeast Paci c[END_REF]. Thus, M. galloprovincialis was collected at some California sites and may have been present in some Puget Sound collections. In addition, some central and northern California sites yielded mussels that probably were hybrids of M. galloprovincialis and M. trossulus [START_REF] Hilbish | Origin of the antitropical distribution pattern in marine mussels (Mytilus spp.): Routes and timing of transequatorial migration[END_REF]. Four oyster taxa were sampled, Crassostrea virginica, C. rhizophorae, C. gigas, and Dendostrea sandvichensis. Crassostrea virginica was sampled from coastal and estuarine areas of the Mid-Atlantic and southeast coasts (Bays 37-61) and the Gulf of Mexico (Bays 63-93). Crassostrea rhizophorae was collected in Puerto Rico (Bay 62), and C. gigas and D. sandvichensis were collected in Hawaii (Bay 126). Considerable disagreement exists as to the taxonomic status of C. virginica and C. rhizophorae [START_REF] Newball | Systematic relationship of the oysters Crassostrea rhizophorae and C. virginica: A comparative ultrastructural study of the valves[END_REF][START_REF] Ladr On De Guevara | Comparative karyology of four American oyster species[END_REF][START_REF] Lap Egue | Trans-Atlantic distribution of a mangrove oyster series revealed by 16S mtDNA and karyological analyses[END_REF], whereas C. gigas and D. sandvichensis are clearly distinct from the other two. In total, however, C. virginica was collected at 55 of 57 bays where oysters were collected.

To simplify discussion when referring to groups of these taxa, the following inclusive terms will be used: dreissenid in reference to the combination of D. bugensis and D. polymorpha; oyster jointly for C. virginica, C. rhizophorae, C. gigas, and D. sandvichensis; mytilid for M. edulis on the East coast and the combination of M. edulis, M. californianus, M. galloprovincialis, and M. trossulus on the West coast.

Except in the Great Lakes, the sampling sites were visited annually during winter months with each site occupied within 30 days of an annual target date [START_REF] O'connor | The National Oceanic and Atmospheric Administration (NOAA) National Status and Trends Mussel Watch Program: National monitoring of chemical contamination in the coastal United States[END_REF]. The sampling was done in winter to minimize the in uence of reproduction on contaminant body burden as spawning may in uence contaminant body burden [START_REF] Jovanovich | Seasonal variation in uptake and depuration of anthracene by the brackish water clam Rangia cuneata[END_REF][START_REF] Ellis | Sources of local variation in polynuclear aromatic hydrocarbon and pesticide body burden in oysters (Crassostrea virginica) from Galveston Bay, Texas[END_REF]Baldwin and Mayer, 1997). In the Great Lakes, dreissenid mussels were collected in late August through Final xDraft { September 11, 2007 8 September. Access to sampling sites in this area is di cult in winter as the Great Lakes are frequently frozen over during this period. As a consequence of sampling program design, analysis of the Mussel Watch dataset may be biased for those parasites that exhibit a strong seasonal cycle of infection intensity (e.g., [START_REF] Wallet | Enquête sur la r epartition et l' evolution du parasitisme a Bucephalus polymorphus Baer, 1827 chez le mollusque Dreissena polymorpha dans le sud-est de la France[END_REF][START_REF] Burreson | Epizootiology of Perkinsus marinus disease of oysters in Chesapeake Bay, with emphasis on data since 1985[END_REF][START_REF] Ford | Modeling the MSX parasite in eastern oyster (Crassostrea virginica) populations. I. Model development, implementation, and veri cation[END_REF].

Parasite/Pathology Quanti cation Parasite/Pathology Quanti cation Parasite/Pathology Quanti cation Tissue preparation followed the original NOAA Status and Trends protocols (Ellis et al., 1998a), as delineated by Kim and Powell (2006). Tissue samples were embedded in para n after dehydration and clearing, sectioned at 5 m, and the sections stained in a pentachrome series (Ellis et al., 1998a).

Major tissue types examined included gill, mantle, gonad and gonoducts, digestive gland tubules, stomach/digestive gland, and connective tissue. For oysters, Perkinsus marinus was assayed by the more precise thioglycollate method [START_REF] Powell | Sampling and analytical methods of the National Status and ACCEPTED MANUSCRIPT Final xDraft[END_REF]. All other parasites and pathologies were scored microscopically for intensity based on either a quantitative or semiquantitative scale. Quantitative scores were used for parasites, pathologies, and selected morphological conditions, including prokaryotic inclusions (rickettsia, chlamydia, etc.), ciliates, gregarines, nematodes, cestodes and metacercariae of trematodes, that could be tallied individually, following procedures described by Ellis et al. (1998b) and Kim et al. (2006). Ciliates were quanti ed by tissue type (viz., gill and digestive tract), as were the gregarines (viz., body, gill, and mantle). Each nematode cross-section observed was counted, although a single individual may be responsible for a number of tissue cross-sections. Certain tissue pathologies and tissue components were also quanti ed by direct counts, including cases of hemocytic in ltration that were scored separately as focal and di use [START_REF] Kim | Surfclam histopathology survey along the Delmarva mortality line[END_REF], granulocytomas [START_REF] Lowe | The cytology and occurrence of granulocytomas in mussels[END_REF], and ceroid bodies or pigment cells [START_REF] Mackin | Histopathology of infection of Crassostrea virginica (Gmelin) by Dermocystidium marinum Mackin, Owen, and Collier[END_REF][START_REF] Stein | A study of the nature of pigment cells of oysters and the relation of their numbers to the fungus disease caused by Dermocystidium marinum[END_REF]. Some parasites and morphological conditions, such as ramifying trematode sporocysts and the disease-producing protozoans, P. marinus and Haplosporidium nelsoni, were assigned to semiquantitative scales depending on the intensity or extensiveness of the a ected area. De nitions of scale values can be found in Ellis et ACCEPTED MANUSCRIPT Final xDraft { September 11, 20079 al. (1998b) and Kim et al. (2006). A semiquantitative 0-to-4-point scale was used for ramifying trematode sporocysts of the families Fellodistomidae and Bucephalidae due to the extensiveness of infections and the di culty in obtaining quantitative counts of the large branching sporocysts. Intensity of P. marinus infection was assigned using the semiquantitative 0-to-5-point scale of Mackin as modi ed by [START_REF] Craig | Distribution of Perkinsus marinus in Gulf Coast oyster populations[END_REF]. Haplosporidium nelsoni infection intensity was scored on a 0-to-4-point scale of Kim et al. (2006) adapted from [START_REF] Ford | E ects of sublethal infection by the parasite Haplosporidium nelsoni (MSX) on gametogenesis, spawning, and sex ratios of oysters in Delaware Bay, U.S.A[END_REF].

For each specimen examined, the presence of neoplasms and unusual digestive tubules was recorded. Abnormal gonadal development, characterized by unusual development at the base of the follicles and by presence of foreign cells and cellular debris in the follicles, was given a semiquantitative 0-to-4-point score related to the spatial coverage of the condition (Kim et al., 2006). For digestive gland atrophy, a condition known to be caused by a variety of stressors most likely related to poor nutrition [START_REF] Palmer | A histological and histochemical study of digestion in the bivalve Arctica islandica L[END_REF][START_REF] Winstead | Digestive tubule atrophy in eastern oysters, Crassostrea virginica (Gmelin, 1791), exposed to salinity and starvation stress[END_REF][START_REF] Kim | Surfclam histopathology survey along the Delmarva mortality line[END_REF], the average degree of thinning of the digestive tubule walls was assigned a numerical rating on a 0-to-4-point scale (Kim et al., 2006).

Contaminant Analysis Contaminant Analysis Contaminant Analysis

The contaminant analyses were carried out on pooled samples of twenty bivalves from each site using methods developed for the National Status and Trends Program [START_REF] Lauenstein | Sampling and Analytical Methods of the National Status and Trends Program National Benthic Surveillance and Mussel Watch Projects: 1984-1992[END_REF]. Only data which passed rigorous QA/QC tests using blanks, spikes, and reference materials were used. In addition, each analytical laboratory successfully completed yearly QA/QC programs involving analysis of blind samples supplied by the National Institute of Standards and Technology and the National Research Council of Canada [START_REF] Cantillo | NOAA National Status and Trends Program quality assurance program[END_REF]. Detailed descriptions of analytical methods for contaminant analyses can be found in [START_REF] Lauenstein | Sampling and Analytical Methods of the National Status and Trends Program Mussel Watch Projects: 1993-1996 Update[END_REF].

Oysters were sampled from the Housatonic River (Bay 37) and Port Je erson (Bay 38), New York, in only one of the four years (1998). Consequently, data from these collections were deleted from analysis.

Statistical Analysis Statistical Analysis Statistical Analysis

Data for 24 contaminant categories were used for data analysis: 11 trace metals Bay mean values, determined using all measurements from all sites within each designated bay, were obtained geometrically for contaminants. Mean values of individual contaminants for each bay are given in Figures 12345678. Data for 17 parasite/pathology groups were used for data analysis: rare parasites, prokaryotic inclusions, Haplosporidium nelsoni, Perkinsus marinus, gill ciliates, gut ciliates, body, mantle, and gill gregarines, nematodes, trematode sporocysts, trematode metacercariae, cestodes, major pathologies, tissue pathologies, ceroid bodies, and digestive gland atrophy. The sum of the counts of the rare parasites was obtained by excluding the common taxa (e.g. gregarines, ciliates, nematodes, cestodes and prokaryotic inclusions). The category of total prokaryotes included prokaryotic inclusions of chlamydia-like organisms and rickettsia-like organisms. Pathologies were grouped into two categories, major and tissue. Major pathologies included neoplasms, unusual digestive tubules, and gonadal abnormalities. Tissue pathologies included focal and di use hemocytic in ltration and granulocytomas as described in [START_REF] Villalba | Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the R ias of Galicia (NW Spain)[END_REF].

Parasite assemblages vary signi cantly between mussels, oysters, and dreissenids. In addition, mussels and oysters are characterized by inherently di erent contaminant a nities, at least for a number of metals [START_REF] Rainbow | Trace metal accumulation in marine invertebrates: Marine biology or marine chemistry[END_REF][START_REF] O'connor | Mussel Watch results from 1986 to 1996[END_REF][START_REF] Valette-Silver | Elevated arsenic concentrations in bivalves from the southeast coasts of the USA[END_REF][START_REF] O'connor | National distribution of chemical concentrations in mussels and oysters in the USA[END_REF]. For these reasons, no betweentaxon comparisons are presented.

The largest bivalves were chosen due to the larger tissue volume required for multiple chemical analyses and the increased likelihood of equilibrium between contaminant exposure and contaminant body burden (e.g., [START_REF] Lee | Petroleum hydrocarbons: uptake and discharge by the marine mussel Mytilus edulis[END_REF][START_REF] Roesijadi | Behavior of trace metals in Mytilus edulis during a reciprocal transplant eld experiment[END_REF][START_REF] Hinch | The e ects of source and destination on growth and metal uptake in freshwater clams reciprocally transplanted among ACCEPTED MANUSCRIPT Final xDraft { September 11, 2007 35 south central Ontario lakes[END_REF][START_REF] Palmer | Field studies using the oyster Crassostrea virginica to determine mercury accumulation and depuration rates[END_REF][START_REF] Abbe | Pathways of silver uptake and accumulation by the American oyster (Crassostrea virginica) in Chesapeake Bay[END_REF]. Final xDraft { September 11, 2007 11 These bivalves, being older, can also be presumed to have an increased incidence of some parasites and pathologies (e.g., [START_REF] Anderson | Processes in uencing the distribution of parasite numbers within host populations with special emphasis on parasiteinduced host mortalities[END_REF][START_REF] Anderson | Experimental studies of age-prevalence curves for Schistosoma mansoni infections in populations of Biomphalaria glabrata[END_REF][START_REF] Aguirre-Macedo | Diversity of metazoan parasites of the introduced oyster species Crassostrea gigas in the Exe Estuary[END_REF][START_REF] Curtis | Longevity of oversized individuals: Growth, parasitism, and history in an estuarine snail population[END_REF][START_REF] Wilson-Ormond | E ects of gas-producing platforms on continental shelf megafauna in the northwest Gulf of Mexico: Reproductive status and health[END_REF]. Animal size and age vary between habitats and with latitude, as does the relationship of size with age (e.g., [START_REF] Hofmann | Modeling oyster populations II. Adult size and reproductive e ort[END_REF][START_REF] Weinberg | Age-structure, recruitment, and adult mortality in populations of the Atlantic surfclam, Spisula solidissima, from 1978-1997[END_REF]. Some parasites have extended life spans; others do not [START_REF] Powell | In uence of parasitism in controlling the health, reproduction and PAH body burden of petroleum seep mussels[END_REF][START_REF] Curtis | Longevity of oversized individuals: Growth, parasitism, and history in an estuarine snail population[END_REF][START_REF] Poulin | The diversity of parasites[END_REF], but the life spans of most bivalve parasites are very poorly known. The degree to which variation in the relationship of host size and age, as well as parasite transmission rate and life span, among Mussel Watch sites a ects parasite and contaminant body burden cannot be evaluated; however, the focus on the largest animals can be expected to enhance the likelihood of identifying a relationship between parasite and contaminant.

Analysis of parasite/pathology occurrence began with the computation of prevalence and infection intensity. Prevalence, the fraction of individuals with the parasite or pathology, was calculated as: prevalence = number animals a ected number animals analyzed : Infection intensity, the average number of occurrences of a parasite or pathology in the a ected individuals only, was calculated as:

infection intensity = P n i=1 number of occurrences of parasite or pathology number a ected individuals :

For conditions rated using semiquantitative scales, the scale rating replaced the number of occurrences in this calculation. If a parasite or condition did not occur in a bay, infection intensity was unde ned for that bay. A value of zero was not assigned.

Correlations were common among parasites for both variables (Kim and Powell, 2006) and occurred commonly as well among the contaminants (see also [START_REF] Ringwood | Cellular responses of oysters, Crassostrea virginica, to metal-contaminated sediments[END_REF][START_REF] Green | Bivalve molluscs as response systems for modelling spatial and temporal environmental patterns[END_REF][START_REF] Green | Relating sets of variables in environmental studies: The sediment quality triad as a paradigm[END_REF]. Accordingly, to evaluate the potential relationships between parasites/pathologies and contaminant body burden in sentinel bivalves, principal components analysis (PCA) was used to reduce the many correlated variables in each dataset down to a few orthogonal variables for further analysis. Accomplishing this requires data points for each variable in each of the 126 bays. Infection intensity, unfortunately, is not de ned for some variables in Final xDraft { September 11, 2007 12 some bays because prevalence was zero. A full set of values exists for prevalence, but this variable does not provide any information about infection intensity. To resolve this dilemma, we calculated weighted prevalence or mean abundance, a measure often used in the bivalve literature (e.g., [START_REF] Ford | Host-parasite interactions in eastern oysters selected for resistance to Haplosporidium nelsoni (MSX) disease: Survival mechanisms against a natural pathogen[END_REF][START_REF] Kim | Surfclam histopathology survey along the Delmarva mortality line[END_REF], that combines prevalence and infection intensity:

weighted prevalence = prevalence infection intensity: Bay average prevalences and infection intensities are reported in Kim and Powell (in press).

Separate PCAs were conducted for each species using the weighted prevalence of 17 parasite/pathology categories and 24 contaminant categories. Each variable was standardized to x = 0 and s 2 = 1 prior to using them in the PCA. Factor scores for factors having eigenvalues above 1.0 obtained from the weighted prevalence and contaminant body burden datasets were compared by ANOVA. The suite of individual variables with factor loads exceeding 0.5 for each signi cant contaminant factor weighted prevalence factor in ANOVA were then compared in a posteriori tests to resolve potential reasons for the signi cant relationships between factor scores.

The a posteriori comparisons were conducted in two ways. First, we tested the signi cance of correlations between variables with factor loading scores 0.5 for factors identi ed as signi cantly related by ANOVA using Spearman's Rank correlation tests. We con rmed that such correlations did not occur by chance using binomial tests [START_REF] Conover | Practical nonparametric statistics[END_REF]. Second, double-zero pairs could not be removed from the PCA because all variables were simultaneously analyzed. These doublezero pairs are ambiguous. In some cases, double zeros occur simply because the bay was beyond the range of both parasites. In other cases, they occur if no a ected animals were found for either condition in an area where each could occur. For this analysis, double-zero pairs introduced a possible bias because some relationships elucidated by PCA and a posteriori tests might exist due to the coincidence of low or zero values. Relationships of substance should be characterized by the cooccurrence of two high values more frequently, for positive correlations, or less frequently, for negative correlations, than expected by chance. We de ned a `high' value as a value exceeding the 75 th percentile of all values for a variable. So, the Final xDraft { September 11, 2007 13 results of signi cant Spearman's Rank tests were examined further by tallying the number of observations for each biological and contaminant variable greater than the 75 th percentile value of all observations of that variable. Then, the occurrence rate for cases where the two variables were simultaneously high was compared to that expected by chance using a binomial test.

Results

Parasites and pathologies were rarely observed in dreissenid mussels, likely due to their recent introduction into the U.S. (Kim and Powell, in press, 2006). Oysters from Puerto Rico and Hawaii were di erent species and these sites were not readily assigned to geographic regions. As a consequence, dreissenids were not further analyzed and oyster analyses were limited to the East and Gulf coasts where C. virginica was sampled.

Principal Components Analysis Principal Components Analysis Principal Components Analysis

PCAs were run separately for mytilids and oysters. Factor axes for parasites/pathologies were determined by substantively di erent groups of parasites/pathologies in mytilids and oysters (Table 1). Similarities are remarkably few, and this is in keeping with the divergent geographic trends among the parasitic fauna, observed in these two taxa (Kim and Powell, in press). In mytilids, Factor 1 (P1) contained the three gregarines (Table 1). Kim and Powell (in press) suggested that mantle and body gregarines might be the same species, but the evidence supports a separation between these two and most gregarines occurring in the gill see also Sprague andOrr (1952, 1955)]. The gregarines did not fall on the same factor axis in oysters. Factor 2 (P2) in mytilids was determined by the pathologies and trematode sporocysts. The rare parasites and prokaryotic inclusions determined Factor 3 (P3). Trematode metacercariae, digestive gland atrophy, and ceroid bodies determined Factor 4 (P4). Factor 5 (P5) was determined by the ciliates. Again, in oysters, as for the gregarines, the ciliates did not fall on the same axis as they did for the mytilids. For oysters, P1 was determined by gill gregarines and P. marinus. Prokaryotic inclusions and major pathologies determined P2. The remaining gregarines determined P3. P4 combined the tissue pathologies, ceroid bodies, and cestodes. Trematode sporocysts and gut ciliates fell on P5. Kim andFinal xDraft { September 11, 2007 14 Powell (2006, in press) discuss the geographic relationships of these parasites and pathologies in more detail.

The contaminant PCAs were much more consistent between the two taxa (Table 1). Each contained a PAH-dominated factor (C1), a pesticide-dominated factor (C3), and three metal-dominated factors. Ag and Zn fell on the same PCA axis in both PCAs. Mirex and Pb fell on the same PCA axis in both PCAs. Cu and Hg did likewise, as did Zn, Ni, and Cd. Total chlordanes, for both species, contributed signi cant in uence to both the PAH-dominated and the pesticide-dominated axis. Finally, the PAH-dominated axis was de ned by 6 contaminants common to both taxa and the pesticide-dominated axis was de ned by 4 contaminants common to both taxa (Table 1). For the mytilids, Cu, Hg and Pb combined with two pesticides on Factor axis 2 (C2). Cd, Cr, Fe, Ni, and Zn loaded on Factor axis 4 (C4). Ag, As and Zn de ned Factor axis 5 (C5). In oysters, Ag, Cd, Cu, Ni and Zn loaded on C2; Cr, Cu and Hg on C4; and Fe, Pb, and Se, with mirex, on C5. Common to C1 in both PCAs were phenanthrene, uoranthene, pyrene, chrysene, total chlordanes, and total PAHs. Total PCBs contributed to this axis in oysters. Common to C3 were total DDTs, aldrin, dieldrin, and total chlordanes. Lindane and total PCBs contributed to this axis in oysters. [START_REF] Presley | Trace elements in Gulf of Mexico oysters[END_REF] also observed correlations between Cu and Zn in oysters from Gulf of Mexico Mussel Watch sites, in agreement with their co-occurrence on Factor axis 2. The tendency for the metals to be distributed independently of the organics also conforms to earlier analyses of Mussel Watch data [START_REF] Wilson | Spatial and temporal distributions of contaminant body burden and disease in Gulf of Mexico oyster populations: The role of local and large-scale climatic controls[END_REF][START_REF] Kim | The geographic distribution of population health and contaminant body burden in Gulf of Mexico oysters[END_REF]. Interestingly, one common contaminant, naphthalene, did not fall on any of the ve primary PCA axes in either taxon suggesting an inherent di erence in the distribution of this contaminant in comparison to the others.

Signi cant relationships between contaminants and parasites/pathologies were examined by ANOVAs comparing PCA factors. A total of 25 tests, 5 parasite/pathology factors 5 contaminant factors, were performed for each taxon. Six of these tests were signi cant for mytilids (Table 2), considerably more than anticipated by chance at the least signi cant signi cance level obtained (Table 2) (tested probability p, signi cance level P ; p = 0:015; P < 0:0001). Five were signi cant for oysters. Once again, this number is considerably more than anticipated

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Final xDraft { September 11, 2007 15 by chance (p = 0:029; P < 0:0001). We also di erentiated the oysters on the East and Gulf coasts and the mytilids on the East and West coasts. For the oysters, at this geographic scale, 5 tests were signi cant in the Gulf and 2 on the East coast.

The number of signi cant comparisons for Gulf oysters exceeded that expected by chance (p = 0:026; P < 0:0001). The number on the East coast did not. Thus, signi cant results overall were principally a function of the Gulf of Mexico portion of the oyster dataset. For the mytilids, 3 comparisons were signi cant on the East coast. This exceeded the number anticipated by chance (p = 0:033; P < 0:01). The frequency on the West coast, however, did not exceed the frequency expected by chance.

The PAH-dominated factor, C1, generated signi cant comparisons with parasite/pathology factors in both oysters and mussels. For oysters, the signi cant comparison was with P4 (tissue pathologies, cestodes, ceroid bodies). Interestingly, for mussels, the signi cant factor, P3, contained a very di erent parasite complement, rare parasites and prokaryotic inclusions. Thus, PAHs were associated with a number of pathologies and tissue components, ceroid bodies, thought to have detoxi cation abilities [START_REF] Zaroogian | The role of the red gland in Mercenaria mercenaria in detoxi cation[END_REF][START_REF] Zaroogian | E ect of selected inhibitors on cadmium, nickel, and benzo(a)pyrene uptake into brown cells of Mercenaria mercenaria[END_REF] in oysters, but not in mussels. The pesticide-dominated factor (C3) had signi cant comparisons also with P4 in oysters, but with no mytilid parasite/pathology factors.

Among the metals, a mytilid factor containing gill gregarines (P1) was significantly related to a factor containing the metals Ag and Zn (C5). Cu and Hg fell on the same axis on both mytilid and oyster PCAs. In mytilids, C2 that included Cu, Hg, Pb, and mirex demonstrated signi cant comparisons with P1 (gregarines) and P2 (pathologies and trematode sporocysts). The factor containing Cu and Hg in oysters (C4), however, showed no signi cant comparisons, but C5 that contained Pb and mirex (plus Se, Fe) showed signi cance with P1, also a gregarine containing factor, and P2, a pathology containing factor (with prokaryotic inclusions). Ni, Zn, and Cd fell on the same PCA axis in both mytilid and oyster PCAs. For oysters, this axis (C2) was signi cantly related to P4 (tissue pathologies, ceroid bodies, and cestodes). For mytilids, C4 was signi cantly related to P2 (pathologies) and P4 (trematode metacercariae, ceroid bodies and digestive gland atrophy). Final xDraft { September 11, 2007 16

A Posteriori Spearman's Correlations A Posteriori Spearman's Correlations A Posteriori Spearman's Correlations

A posteriori tests were con ned to cases where signi cant ANOVA comparisons occurred more frequently than expected by chance. For the mytilids, six signi cant relationships existed between contaminant and parasite/pathology PCA factors out of a possible 25 (Table 2), an incidence that far exceeds the incidence anticipated by chance. From these, 43 signi cant contaminant-parasite/pathology a posteriori correlations were obtained out of a possible 81. Among the 43 pairs with signi cant relationships, 30 achieved signi cant levels at 0:01 (Table 3). Thirty of 81 far exceeds the incidence expected by chance (p = 0:01; P < 0:0001). Forty-three of 81 reached signi cance at 0:05, a frequency also exceeding chance (P < 0:0001). Of these 43, 26 were negative, a frequency not exceeding chance. Thus, negative and positive correlations were about equally likely.

The majority of signi cant correlations in the mytilids involved metals, 33 of 43 (Table 3). Correlation tests with metals involved 70% of the 81 possible tests. Both signi cance tests with metals and with organics occurred more frequently than expected by chance (metals: 33 of 57, p = 0:05; P < 0:0001; organics: 10 of 24, p = 0:05; P < 0:0001). Taking into consideration the di erential in the number of tests, 57 versus 24, signi cant di erences with metals occurred signi cantly more frequently than with organics (P < 0:0001). Finally, positive correlations occurred in 7 of 10 signi cant tests for the organics, a frequency no di erent than chance. For the metals, the ratio was 10 of 33, a frequency much lower than expected by chance (P < 0:02); hence signi cant correlations with metals were signi cantly more likely to be negative. A substantial number of the signi cant correlations for mytilids involved the gregarines; however, gregarines were absent from East-coast mytilids and this absence might have reinforced an East coast-West coast di erence in metal body burden yielding the observed signi cant a posteriori tests. Removing the suspect gregarines does not materially change conclusions deduced from this battery of tests, with one exception. The frequency of positive versus negative correlations no longer di ers from that expected by chance.

Excluding the suspect gregarines, the majority of the correlations with metals, whether positive or negative, involved major pathologies, tissue pathologies (mostly granulocytomas), and trematode sporocysts and metacercariae. Single-celled para-Final xDraft { September 11, 2007 17 sites o ered no signi cant correlations with metals in mytilids. Among organic contaminants, mirex was positively associated with tissue pathologies. Fluoranthene, pyrene, and total PAHs showed weak positive relationships with rare parasites. Total PCBs were positively correlated with major/tissue pathologies and trematode sporocysts. Excluding the gregarines, all correlations with organics were positive, higher weighted prevalence being associated with higher contaminant body burden. Among the metals, Cd, Cr, Fe, Ni, and Zn exclusively demonstrated signi cant negative correlations, whereas Cu, Hg, and Pb were exclusively involved in signi cant positive correlations. Such an outcome would not be expected by chance.

Limiting the analysis to East-coast M. edulis produced signi cant relationships in ANOVA more frequently than expected by chance. From these, nine signi cant contaminant-parasite/pathology pairs were identi ed out of 33 possible, a frequency that is greater than expected by chance (p = 0:05; P < 0:0001). About one-third were positive correlations. With one exception, all involved digestive gland atrophy, ceroid bodies, and trematode metacercariae (Table 4). All of the latter two were negative. All for digestive gland atrophy were positive. Such an outcome would not be expected by chance. The single signi cant ANOVA for West-coast mytilids may have occurred by chance; nevertheless, we include the a posteriori tests in Table 4 because of their unusual nature; all involve PAHs and major pathologies, and all are positive.

The ANOVAs for oysters yielded ve signi cant relationships between contaminant and parasite/pathology factors (Table 5), an incidence not anticipated by chance. A posteriori Spearman's Rank correlations identi ed 19 signi cant individual contaminant-parasite/pathology pairs out of 70 possible. Ten achieved signi cance levels exceeding = 0:01 (Table 5). Ten of 70 possible far exceeds the incidence expected by chance (p = 0:01; P < 0:0001), as does 19 of 70 at p = 0:05. Of these, 8 were positive, a frequency possibly obtained by chance. Negative and positive correlations were equally likely.

Among the 19 correlations in oysters, nine signi cant comparisons occurred with the metals, and ten with organic contaminants (Table 5). Correlation tests with metals involved 38.6% of the 70 possible tests. Both signi cance tests with metals and with organics occurred more frequently than expected by chance (metals:

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Final xDraft { September 11, 2007 18 9 of 27, p = 0:05; P < 0:0001; organics: 10 of 43, p = 0:05; P < 0:0001). Taking into consideration the di erential in the number of tests, 27 versus 43, signi cant di erences with metals occurred less frequently than with organics (P < 0:05).

Finally positive correlations occurred in 3 of 10 signi cant tests for the organics, a frequency no di erent than chance. For the metals, the ratio was 5 of 9, a frequency also no di erent than expected by chance. All negative correlations in oysters were with metals and pesticides or PCBs (Table 5). The vast majority of these signi cant results involved cestodes and tissue pathologies. All positive correlations were with metals and PAHs. Interestingly, these covered a range of parasite and pathology types. Tissue pathologies were more frequently signi cant with metals than with pesticides, whether the correlation was positive or negative.

Overall ANOVAs were driven principally by the Gulf oyster dataset. Not surprisingly, restricting a posteriori tests to the Gulf dataset yielded essentially the same results, with one important exception. Body and mantle gregarines were negatively correlated with a variety of pesticides (Table 6).

Re-examination of Spearman's Rank Correlations Re-examination of Spearman's Rank Correlations Re-examination of Spearman's Rank Correlations Correlations obtained from the Spearman's Rank correlation tests may occur because of frequent co-occurrences of low parasite/pathology-low contaminant values. The most important correlations should be those produced by a coincidence of high parasite/pathology and high contaminant values exceeding (positive) or falling below (negative) the number expected by chance. We re-examined each signi cant correlation focusing on the values that exceeded the 75 th percentile value of all observations.

Restricting signi cant correlations to those meeting this more stringent criterion decreased the number of signi cant results for the mytilids from 43 to 22 (Table 3). The overall distribution of signi cant results was not much changed, however. Some correlations with gregarines were lost, but some remained. The frequency of signi cant results with major pathologies and tissue pathologies was much reduced, but these two biological variables continued to manifest important relationships with contaminants. No pesticide or PAH relationship survived this

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Final xDraft { September 11, 2007 19 analysis, however, with one exception, mirex. Only metals and PCBs remained. Separate analyses for East-and West-coast mytilids also remained qualitatively the same, although the more stringent criterion retained only 57% of the original Spearman's Rank correlations.

The more stringent 75 th percentile requirement retained only ve of the 19 signi cant Spearman's Rank correlations for oysters. These involved metals and pesticides exclusively; no PAH correlation was retained. These dominantly involved cestodes; no relationship with tissue pathologies or ceroid bodies was retained. Restricting the analysis to the Gulf of Mexico oysters reduced the 20 signi cant Spearman's Rank correlations to three, again involving exclusively pesticides and metals; no PAH correlation was retained.

Discussion

Approach Approach Approach [START_REF] Mcdowell | The e ects of lipophilic organic contaminants on reproductive physiology and disease processes in marine bivalve molluscs[END_REF] argued that establishing a causal relation between parasites/pathologies observed in bivalve populations and a speci c contaminant is inherently di cult, as di erent types of contaminants simultaneously occur as mixtures, as organisms are exposed to these complex environmental mixtures, not individual contaminants, and as synergistic or antagonistic e ects of several classes of contaminants may occur. We add that this same complication exists with parasites and pathologies: most animals are parasitized by multiple taxa (Kim and Powell, 2006); some taxa promote certain pathologies [START_REF] Kim | Surfclam histopathology survey along the Delmarva mortality line[END_REF]Kim and Powell, in press); and the infection process is inherently and substantially inuenced by processes external to prospective hosts (e.g., [START_REF] Mollison | Spatial dynamics of parasitism[END_REF][START_REF] Ford | Diseases and defense mechanisms[END_REF]. Bearing these points in mind, we resolve possible relationships between biological and contaminant variables in this study by rst utilizing PCA to identify relationships between multiple contaminants and multiple parasites/pathologies (Table 2); we then investigate these relationships in more detail. Because the co-occurrence of low values of parasite/pathology and contaminant might occur for many reasons, a stepwise progression was used to remove from consideration parasite/pathology-contaminant pairs that might originate strictly from frequent co-occurrences of low parasite/pathology-low contaminant pairs. The nal Final xDraft { September 11, 2007 20 stringent culling of statistical results focused solely on the frequency of co-occurrence of high values of parasite/pathology and contaminant (Tables 3456).

Positive associations surviving the nal cull are cases in which a high contaminant and a high parasite/pathology value co-occurred more frequently than expected by chance. A diversity of possibilities exists to explain these ndings: contaminant stresses may reduce host resistance thus increasing susceptibility to parasitism; some parasites may preferentially accumulate certain contaminants in their tissues to a greater extent than their hosts; environmental in uences may simultaneously increase contaminant availability and the chance of infection; some parasites may increase host contaminant body burden by modulating accumulation rate; and/or some parasites, through their in uence on reproduction or physiology, may decrease depuration or detoxi cation rate. Negative relationships surviving the nal cull are cases in which a high contaminant value and a high parasite/pathology value co-occurred less frequently than expected by chance. A diversity of possibilities exists to explain these ndings as well: some contaminants may negatively impact the parasite directly; environmental in uences may increase contaminant availability and simultaneously reduce the chance of infection; and/or lower tissue a nity in the parasite may lower apparent host body burden at high infection intensity.

One caveat applies to the comparisons that include data from multiple coasts. Parasites/pathologies vary between coasts; the most obvious case being the absence of gregarines in mytilids on the East coast (Kim and Powell, in press). Some relationships might simply be due to the coincidence of changes in parasite availability and transmission process and the availability of contaminants in the watershed. As one cannot, in most cases, discount a more causative interaction, we include these larger-scale analyses. However, emphasis is placed on within-coast comparisons.

Finally, we note that large-scale climate processes acting through variations in temperature and rainfall (salinity) may play an important role in establishing many of the observed parasite/pathology distributional patterns over broad geographic regions (Kim and Powell, in press). Because the process of contaminant accumulation is normally temperature-dependent [START_REF] Reid | Interactive e ects of temperature and pollutant stress[END_REF][START_REF] Kennedy | E ects of temperature on xenobiotic metabolism[END_REF] and because contaminant source loadings and local concentrations are

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Final xDraft { September 11, 2007 21 a ected by rainfall and freshwater in ow [START_REF] Wilson | Spatial and temporal distributions of contaminant body burden and disease in Gulf of Mexico oyster populations: The role of local and large-scale climatic controls[END_REF][START_REF] Kim | The geographic distribution of population health and contaminant body burden in Gulf of Mexico oysters[END_REF], trends in the physiology of contaminant accumulation could be similar to trends in the occurrence of parasitism (e.g., [START_REF] Wenzlo | A survey of heavy metals in the surf clam, Spisula solidissima, and the ocean quahog, Arctica islandica, of the mid-Atlantic coast of the United States[END_REF] without any inherent interaction between the two. Accordingly, we focus on distinctive patterns that, by their uniqueness, seem unlikely to be explained in so simple a fashion.

Trends in Correlations: The Contaminant Perspective Trends in Correlations: The Contaminant Perspective Trends in Correlations: The Contaminant Perspective Di erent contaminants seldom occur in isolation in the environment but are present as mixtures with other organic and inorganic contaminants [START_REF] Moore | Responses of the mussel Mytilus edulis to copper and phenanthrene: Interactive e ects[END_REF][START_REF] Hagopian-Schlekat | Acute toxicity of ve sediment-associated metals, individually and in a mixture, to the estuarine meiobenthic harpacticoid copepod Amphiascus tenuiremis[END_REF]. [START_REF] Murphy | Assessment of the potential for toxic interactions among environmental pollutants[END_REF] suggested that identi ed biological responses to chemical stress are unlikely to have been caused by a single contaminant (see also [START_REF] Ringwood | Cellular responses of oysters, Crassostrea virginica, to metal-contaminated sediments[END_REF]. Synergies are commonly reported from controlled multi-contaminant experiments [START_REF] Hagopian-Schlekat | Acute toxicity of ve sediment-associated metals, individually and in a mixture, to the estuarine meiobenthic harpacticoid copepod Amphiascus tenuiremis[END_REF][START_REF] Chu | Synergistic toxicity of multiple heavy metals ACCEPTED MANUSCRIPT Final xDraft[END_REF]. PCA analyses of 1995-1998 Mussel Watch data resolved ve groups of contaminants (Table 1). In both mussels and oysters, these ve included one dominated by PAHs, one dominated by pesticides, and three dominated by metals. Most of the common contaminants fell on one or the ve primary PCA axes. The most signi cant exception was naphthalene that, among the ve most common PAHs, did not load on any of the primary contaminant PCA axes. Metals produced a much more complex picture of spatial trends in body burden than did either the pesticides or PAHs. Furthermore, certain similarities existed in the metal groupings between the two taxa. Ag and Zn fell on the same PCA axis in both PCAs. Mirex and Pb fell on the same PCA axis in both PCAs. Cu and Hg did likewise, as did Zn, Ni, and Cd.

Contrasted to the relative simplicity of contaminant trends was the diversity exposed by the PCAs for parasites and pathologies. Very few similarities between the sentinel bivalve taxa existed. This was due in part to the di erent taxa parasitizing these bivalves. Cestodes and nematodes were present only in oysters. Trematode metacercariae were vastly more abundant in mussels, as were major pathologies. Further examples abound. Thus, the relationship between parasites/pathologies and contaminants can be expected to be di erent between taxa despite the similarity in contaminant pattern. Final xDraft { September 11, 2007 22 Despite the complexity of the biology, signi cant contaminant versus parasite/pathology di erences occurred much more frequently than expected by chance when the two suites of PCA factors were compared. Nevertheless, when the contributors to the PCA factors were compared individually, not all individual variable pairs in each signi cant factor showed correlations in a posteriori Spearman's Rank tests (Tables 3456), even though the overall comparison between factor scores reached high levels of signi cance (often P 0:001). In some cases, none of them did! Thus, the analysis suggests that combined e ects of many contaminants and parasites may be important, leading to complex biological-contaminant interactions with synergiies both of biological and chemical origin.

Overall, correlations between parasites/pathologies and contaminants were more frequent with metals, frequent with pesticides, and less frequent with PAHs in mussels. In oysters, correlations with pesticides and metals were about equally frequent, but correlations with PAHs were still rare. Most associations may be due to inherently di erent contaminant loads in species and coasts, but even at the lowest spatial scale (i.e. East-coast M. edulis, Gulf-coast oysters), the metals (and pesticides in oysters) produced most of the signi cant correlations. Inasmuch as metals contribute a large portion of the analytes examined in this study, this outcome might be anticipated. However, the frequency accrues not from this source, but from the diversity of metal groups in the PCA, of which three were identi ed in each taxon, and the common occurrence of signi cant Spearman's Rank correlations with individual metals contributing to these metal-dominated PCA factors. Negative and positive correlations occurred with about the same frequency, both in oysters and in mytilids, once the suspect gregarines were excluded for the mytilids. For metals, important positive associations were recognized between Pb, Hg, and Cu and trematode sporocysts in mytilids. Negative correlations occurred between a number of metals and trematode metacercariae, as well as both types of pathologies in mytilids. For the oysters, positive correlations involved principally Pb, with Cd and Ni contributing most of the negative ones. The positive relationship between major pathologies and Pb, and the negative relationship between tissue pathologies and both Cd and Ni occurred in both mytilid and oyster datasets Final xDraft { September 11, 2007 23 Correlations with pesticides and PCBs, though less common than with the metals in mussels, were relatively frequent in both taxa and more common in oysters than in mytilids. The majority of associations related to pesticides were negative ones. In mytilids, positive correlations were limited to the PCBs. None existed in the oysters. Negative correlations occurred with mirex for the mytilids and with a variety of pesticides and the PCBs in oysters. A negative correlation between cestodes from East-coast oysters with total hexachlorocyclohexane (HCH), a synthetic pesticide that does not occur naturally in the environment, was identi ed by [START_REF] Kim | Parasites of sentinel bivalves in the NOAA Status and Trends Program: Distribution and relationship to contaminant body burden[END_REF]. In this study, all potential relationships of cestodes in oysters with manufactured organic chemicals (i.e. total DDT, aldrin, dieldrin, lindane, and total PCBs) were negative (Tables 5 and6), suggesting that locations with higher infection intensity of cestodes tended to co-occur with lower body burden of these contaminants.

PAHs rarely showed signi cant relationships with parasites/pathologies in this study. [START_REF] Powell | In uence of parasitism in controlling the health, reproduction and PAH body burden of petroleum seep mussels[END_REF] reported similarly that parasites observed in petroleum seep mussels were nevertheless rarely signi cantly associated with PAH body burdens.

Lipophilic organic contaminants (e.g., PAHs) are easily depurated by spawning as they are enriched in gametic tissue [START_REF] Jovanovich | Seasonal variation in uptake and depuration of anthracene by the brackish water clam Rangia cuneata[END_REF][START_REF] Ellis | Sources of local variation in polynuclear aromatic hydrocarbon and pesticide body burden in oysters (Crassostrea virginica) from Galveston Bay, Texas[END_REF]. [START_REF] Ellis | Sources of local variation in polynuclear aromatic hydrocarbon and pesticide body burden in oysters (Crassostrea virginica) from Galveston Bay, Texas[END_REF] noted that pesticides are concentrated in eggs but not in sperm, so that depuration through spawning would in uence only a portion of the bivalve population. Metals are less in uenced by spawning (e.g., [START_REF] Cunningham | Factors a ecting the accumulation and removal of mercury from tissues of the American oyster Crassostrea virginica[END_REF][START_REF] Greig | Trace metal content in the American oyster[END_REF] than organics, and thus metals tend to stay in the tissues. This may be the reason why the majority of associations identi ed in this study came from metals, as they are present in higher concentrations over longer periods, and so can more strongly in uence the biology of both host and parasite. Pesticides fell in between as anticipated by their apparently more limited depuration through spawning [START_REF] Ellis | Sources of local variation in polynuclear aromatic hydrocarbon and pesticide body burden in oysters (Crassostrea virginica) from Galveston Bay, Texas[END_REF]. PAHs, most easily depurated through spawning, were rarely characterized by signi cant correlations.

The inverse ranking of spawning as a depuration mechanism and the frequency of signi cant correlation are gripping and argue for a much closer look at this key linkage in explaining spatial trends in monitoring studies. The same ranking would Final xDraft { September 11, 2007 24 promote a greater and longer-term interaction between parasites/pathologies and metals than PAHs, as is observed. The infrequency of signi cant e ects between Se and parasites/pathologies is potentially noteworthy in this regard as this metal may be lost through spawning [START_REF] Baldwin | Spatial and temporal variation of selenium concentration in ve species of intertidal molluscs from Jervis Bay, Australia[END_REF]. However, one cannot exclude the di erential in e ects levels that tend to be much lower for pesticides than PAHs [START_REF] Long | Incidence of ACCEPTED MANUSCRIPT Final xDraft { September 11, 2007 38 adverse biological e ects within ranges of chemical concentrations in marine and estuarine sediments[END_REF] as contributing to the intermediary nature of the pesticides. PAHs may simply be less toxic and therefore less in uential in a ecting the occurrence of parasites/pathologies.

Trends in Correlations: The Biological Perspective Trends in Correlations: The Biological Perspective Trends in Correlations: The Biological Perspective Of the 23 signi cant correlations involving parasites, after discounting the gregarine relationships in mytilids and the rare parasite group that combines a variety of taxa, only 3 involve single-celled eukaryotes or prokaryotes (Tables 3 and5). This is signi cantly fewer than expected by chance (p = 0:01; P < 0:0001). The remaining involve multi-cellular eukaryotes and nearly all of them either cestodes, trematode sporocysts, or trematode metacercariae. Some of the frequently signi cant relationships observed with tissue pathologies, particularly the hemocytic in ltration in oysters, probably accrue also in this way, as these worms, with the exception of the trematode sporocysts, are often observed to locally increase hemocyte concentration in bivalves [START_REF] Cheng | The coracidium of the cestode Tylocephalum and the migration and fate of this parasite in the American oyster, Crassostrea virginica[END_REF][START_REF] Cheng | The occurrence and resorption of Tylocephalum metacestodes in the clam Tapes semidecussata[END_REF][START_REF] Kim | Surfclam histopathology survey along the Delmarva mortality line[END_REF]. The few correlations for single-celled parasites all involve proliferating protozoa (P. marinus) or protozoa reaching high body burdens through transmission (the gregarines). With two exceptions, the parasites involved in signi cant correlations with contaminants are not known to do great damage to the host, the exception being P. marinus and the trematode sporocysts. The non-random distribution of signi cant correlations and the tendency for the larger or more numerous parasites to be involved argue that unequal sequestration of contaminants between host and parasite tissue is a potential mediator. Negative and positive correlations occur with about equal frequency, suggesting that, if present, the principal di erential may be either a lower or higher body burden in parasitic tissue. This expectation di ers from that expected from [START_REF] Sures | Parasites as accumulation indicators of heavy metal pollution[END_REF].

One interesting tendency was for correlations with pathologies to be principally with the mytilids. The few in oysters always co-occurred with cestodes and are

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Final xDraft { September 11, 2007 25 likely derived from hemocytic in ltration near cestode cysts. With the mytilids, pathologies, major and tissue (mostly granulocytomas in this case), were common and correlations widespread among the metal, pesticide, and PAH contaminants.

A posteriori correlations did not occur more frequently than expected by chance in East-coast oysters and West-coast mussels. The ready explanation of multiple taxa on the West coast compromising the analysis is belied by the case of oysters on the East coast. These two coastline stretches are unique in having the widest temperature range. Each contains signi cant provincial boundaries [START_REF] Hall | Shallow-water marine climates and molluscan provinces[END_REF][START_REF] Valentine | Numerical analysis of marine molluscan ranges on the extratropical northeastern Paci c shelf[END_REF]. The in uence of the large temperature range on parasite distributional pattern potentially minimizes the possibility that correlations with contaminants can be identi ed. Shorter coastline stretches bounded by provincial boundaries might be investigated advantageously.

Noteworthy Speci c Relationships Noteworthy Speci c Relationships Noteworthy Speci c Relationships [START_REF] Couch | Prospective study of infectious and noninfectious diseases in oysters and shes in three Gulf of Mexico estuaries[END_REF] found that industrialized sites had a tendency to show a higher frequency of oysters parasitized or diseased, suggesting that pollution stress can be a major contributing factor in the occurrence of disease in animals from degraded habitats. [START_REF] Villalba | Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the R ias of Galicia (NW Spain)[END_REF] noticed a higher parasite load in mussels from areas of higher levels of pollution in Spain. Other studies have suggested possible relationships between certain parasites or disease conditions in marine bivalves and pollution by identifying chemical contaminants as causative factors for detrimental biological e ects. [START_REF] Kraak | The e ect of the parasite Phyllodistomum macrocotyle (Trematoda) on heavy metal concentrations in the freshwater mussel Dreissena polymorpha[END_REF] found that organisms weakened by parasitism contained higher levels of some metals, for example. From all of these studies, one might anticipate that positive correlations between parasite body burden and contaminant body burden would be the norm. Nevertheless, in this study, positive correlations were no more common than negative ones. A strong tendency existed for the correlations to be of equivalent trend in both mytilid and oyster taxa. Thus, correlations with Cd and Ni were negative in both taxa. Positive correlations occurred in both taxa for Pb.

Exposure to contaminants can increase the bivalve host's susceptibility to disease and thus enhance disease infections [START_REF] Chu | Relationship between pollution and susceptibility to infectious disease in the Eastern oyster, Crassostrea virginica[END_REF][START_REF] Chu | Laboratory investigations of susceptibility, infectivity and transmission of Perkinsus marinus in oysters[END_REF][START_REF] Mcdowell | The e ects of lipophilic organic contaminants on reproductive physiology and disease processes in marine bivalve molluscs[END_REF]. This study identi ed two positive associations with

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Final xDraft { September 11, 2007 26 P. marinus, for Pb and Se. No correlations were negative. [START_REF] Guth | Potentiation of zinc stress caused by parasitic infection of snails[END_REF] showed that snails parasitized by trematodes were more susceptible to Zn than unparasitized snails. In contrast, Zn entered into a number of signi cant correlations with trematodes in this study; all were negative. From a previous Mussel Watch Program by the EPA, [START_REF] Yevich | Histopathology as a monitor for marine pollution. Results of histopathological examinations of the animals collected for the US 1976 Mussel Watch Program[END_REF] reported correlations between locations with high parasitism and locations high in PCBs. [START_REF] Kim | Parasites of sentinel bivalves in the NOAA Status and Trends Program: Distribution and relationship to contaminant body burden[END_REF] also reported that trematode sporocysts in M. edulis were positively correlated with body burdens of total PCBs. Evidence to support these observations from this study includes the fact that total PCB levels in mytilids were positively associated with infections by trematode sporocysts. [START_REF] Barszcz | Chronic e ects of three crude oils on oysters suspended in estuarine ponds[END_REF] reported that oysters exposed to crude oils had an increased intensity of infection, showing that contaminant stress plays a role in increased parasitism. [START_REF] Ne | Histopathologic and biochemical responses in arctic marine bivalve molluscs exposed to experimentally spilled oil[END_REF] also noted a relationship between parasitism and oil exposure. In this study, relationships with PAHs were rare; the only noteworthy exception was the relationships elucidated in West-coast mussels between a variety of PAHs and pathologies. [START_REF] Barszcz | Chronic e ects of three crude oils on oysters suspended in estuarine ponds[END_REF] also noted that bivalves with reduced resistance to infection due to higher contaminant body burdens are more likely to become hosts to opportunistic parasites and that contaminant exposure may help parasites already present in hosts to proliferate. [START_REF] Laird | Microecological factors in oyster epizootics[END_REF] forwarded a similar hypothesis. Our analysis of rare parasites, anticipated to follow this trend, yielded few signi cant correlations, though all were positive.

Lowe and [START_REF] Lowe | The cytology and occurrence of granulocytomas in mussels[END_REF] showed that areas of high pollution have a high incidence of granulocytomas characterized by an in ammatory cellular condition in mytilid mussels from England and Wales. Con rmatory data come from [START_REF] Villalba | Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the R ias of Galicia (NW Spain)[END_REF][START_REF] Sv Ardh | Bacteria, granulocytoma and trematode metacercariae in the digestive gland of Mytilus edulis: Seasonal and interpopulation variation[END_REF][START_REF] Sv Ardh | Incidence of hemocytes and parasites in coastal populations of blue mussels (Mytilus edulis){testing correlations with area, season, and distance to industrial plants[END_REF]. In this study, tissue pathologies, principally granulocytomas, in mytilids were more common and abundant in East-coast M. edulis, especially from Massachusetts to Delaware, than in West-coast mytilids. Contaminant concentrations are also high in these animals, on the average [START_REF] O'connor | Results from the NOAA National Status and Trends Program on distribution and e ects of chemical contamination in the coastal and estuarine United States[END_REF][START_REF] O'connor | National distribution of chemical concentrations in mussels and oysters in the USA[END_REF]. With the exception of Cd, highly signi cant correlations between tissue pathologies and contaminants were positive in mytilids. This included metals (Pb, Hg) and PCBs. Final xDraft { September 11, 2007 27 Ceroid bodies or brown cells are thought to have detoxi cation abilities [START_REF] Zaroogian | The role of the red gland in Mercenaria mercenaria in detoxi cation[END_REF][START_REF] Zaroogian | E ect of selected inhibitors on cadmium, nickel, and benzo(a)pyrene uptake into brown cells of Mercenaria mercenaria[END_REF]. Cd, Ni, and certain PAHs are implicated [START_REF] Zaroogian | E ect of selected inhibitors on cadmium, nickel, and benzo(a)pyrene uptake into brown cells of Mercenaria mercenaria[END_REF]. Interestingly, correlations with ceroid bodies were always positive, with one exception in mytilids. These positive correlations occurred solely in oysters and involved Zn and certain PAHs. Digestive gland atrophy has been shown to be responsive to environmental stress in mytilids and other bivalves [START_REF] Widdows | Responses of Mytilus edulis to the water-accumulated fraction of North Sea oil[END_REF][START_REF] Axiak | Petroleum hydrocarbons in the marine bivalve Venus verrucosa: Accumulation and cellular responses[END_REF][START_REF] Hinton | Histopathologic biomarkers[END_REF][START_REF] Da Ros | Preliminary results of a eld study on some stress-related parameters in Tapes philippinarum naturally infected by the protozoan Perkinsus sp[END_REF], particularly metals. In this study, all signi cant correlations were observed in mytilids and all were positive. Their absence in oysters is possibly explained by the relationship of this tissue condition and nutritional status in oysters [START_REF] Winstead | Digestive tubule atrophy in eastern oysters, Crassostrea virginica (Gmelin, 1791), exposed to salinity and starvation stress[END_REF]but see Gold-Bouchot et al., 1995).

Conclusions

Estuarine and coastal marine waters receive many kinds of organic and inorganic contaminants that potentially have adverse e ects on biota living in these habitats and possibly on humans who consume them. Sessile bivalve mollusks can be used e ectively as bioindicators to monitor the status and trends of coastal water quality. Whether parasites/pathologies can be used as biomarkers is still unclear, but some observations are noteworthy. The distribution of parasites/pathologies within taxa and along coasts is su ciently complex that a universal relationship with contaminants is an unachievable goal. Relationships may be present at smaller spatial scales, however, and within taxon.

Several studies have suggested parasites as potential biomarkers of pollution. Some focus on particular parasite types [START_REF] Thulin | Can sh parasites be used to monitor pollution?[END_REF]; others on broader indicies of parasite species richness and diversity [START_REF] Schmidt | Parasites of ounder (Platichthys esus L.) from the German Bight, North Sea, and their potential use in biological e ects monitoring. C. Pollution e ects on the parasite community and a comparison to biomarker responses[END_REF]. In this study, the gregarines, the most common parasites in oysters exclusive of the disease-causing P. marinus, were responsible for a large fraction of the relationships with contaminants found in Gulf-coast oysters. These were exclusively inversely associated with pesticides, suggesting an underlying relationship. Cestodes, another common parasite, were exclusively negatively associated with metals and pesticides. The possibility that some parasites such as the gregarines and cestodes in oysters might be useful biomarkers is indicated by this study. That the common parasites are most important is particularly critical to establish biomarkers of general useful-Final xDraft { September 11, 2007 28 ness. The trend for common parasites to display correlations with contaminants is also present in mussels, where trematode sporocysts and metacercariae contributed most signi cant correlations, and nearly exclusively with metals. In contrast to the promise shown by the metals, and by pesticides in oysters, PAH biomarkers are unlikely to be found among the parasites. Correlations with PAHs were remarkably rare. The ease of depuration of PAHs through spawning o ers a likely explanation.

Interactions between di erent types of contaminants present as mixtures in estuarine and coastal waters must be common. Not all individual parasite/pathologycontaminant pairs in each signi cant PCA factor pair showed signi cant correlations, although the overall comparison between PCA factors loaded by multiple biological and contaminant variables reached high levels of signi cance. Thus, this study re-enforces the contention that combined e ects of many contaminants and parasites may be important, and emphasizes the complexity of parasite-contaminant interactions, considering the other biological and environmental variables involved. For this reason, parasites are unlikely to be responsive to single contaminants, but they are likely to respond to classes of contaminants and the number of such classes is relatively few; ve in the case of mytilids and oysters in this study. Thus, these results also support continued evaluation of parasite/contaminant interactions.

As parasitism can in uence the sensitivity of host bivalves to contaminant exposure and a ect the spawning of animals as a means of contaminant loss, and as parasites may respond directly to contaminants and in uence host body burden directly, the study of parasites and pathologies should be included in biomonitoring and toxicity studies as is now the case in the Mussel Watch Program. The supposition that increased parasitism and increased contaminant body burden should co-occur was not uniformly supported, however. A surprising number of signi cant correlations between contaminants and parasites/pathologies, about half of all correlations, were negative, suggesting that some contaminants may negatively a ect parasites more than their hosts. The importance of parasites in community diversity is attracting increasing attention [START_REF] Hawkins | Global patterns of parasitoid assemblage size[END_REF][START_REF] Sousa | Can models of soft-sediment community structure be complete without parasites[END_REF][START_REF] Thomas | Parasitism and ecology of wetlands: A review[END_REF][START_REF] Rohde | Ecology and biogeography of marine parasites[END_REF]. As parasites are potentially more sensitive than hosts to environmental disturbance, the possibility that contaminants may reduce the abundance of some parasites is deserving of attention. More multidisciplinary Final xDraft { September 11, 2007 29 studies on the interactive biological in uences of environmental stressors (e.g., multiple contaminants) on marine organisms, both hosts and parasites, in estuaries and coastal waters are required to further elucidate these relationships. Final xDraft { September 11, 2007 46 Legends for Figures 1 3 1 7 2 1 2 5 2 9 3 3 3 7 4 1 4 5 4 9 5 3 5 7 6 1 6 5 6 9 7 3 7 7 8 1 8 5 8 9 9 3 9 7 1 0 1 1 0 5 1 0 9 1 1 3 1 1 7 1 2 1 1 2 5 Bay ContamBayAve.dat Cd

  Fig.1

  As, Cd, Cr, Cu, Fe, Hg, Ni, Pb, Se and Zn); the ve PAHs normally present in highest concentration (naphthalene, phenanthrene, uoranthene, pyrene and chrysene) and the sum of 24 PAH compounds (total PAHs), as de ned by[START_REF] Jackson | Galveston Bay: Temporal changes in the concentrations of trace organic contaminants in National Status and Trends oysters (1986-1994)[END_REF]; the sum of concentrations of DDT and its metabolites, DDE and DDD (total DDTs); four chlorinated pesticides other than DDT (aldrin, dieldrin, lindane and mirex); the sum of concentrations of all chlordanes (total chlordanes) see[START_REF] Jackson | Galveston Bay: Temporal changes in the concentrations of trace organic contaminants in National Status and Trends oysters (1986-1994)[END_REF] for constituents] and the sum of concentrations of 18 individual PCB congeners (total PCBs) see[START_REF] Jackson | Galveston Bay: Temporal changes in the concentrations of trace organic contaminants in National Status and Trends oysters (1986-1994)[END_REF] for constituents].

	ACCEPTED MANUSCRIPT
	Final xDraft { September 11, 2007 10
	(Ag,

Table 1 .

 1 Bay 62; Gulf-coast oyster sites,Bays 63-93; West-coast mytilid Final xDraft { September 11, 2007 47 sites, Bays 94-125; Hawaii, Bay 126. Data for Bays 37 and 38 not included. Figure7. Bay-mean body burdens of aldrin, dieldrin and lindane. Vertical bars mark sectional boundaries. Great Lakes/Hudson River dreissenid sites, Bays 1-11; Factor load analysis for the four PCAs. PCAs were conducted separately for mytilids and oysters. For each taxon, a parasite/pathology PCA and a contaminant PCA were run. Variables assigned had factor loads 0.5 on the indicated factor axis.

	Figure 1. Bay-mean body burdens of Ag, As and Cd. Vertical bars mark sectional
	boundaries. Great Lakes/Hudson River dreissenid sites, Bays 1-11; East-coast
	mytilid sites, Bays 12-36; East-coast oyster sites, Bays 37-61; Puerto Rico, Bay
	62; Gulf-coast oyster sites, Bays 63-93; West-coast mytilid sites, Bays 94-125;
	Hawaii, Bay 126. Data for Bays 37 and 38 not included.
	Figure 2. Bay-mean body burdens of Cr, Cu and Fe. Vertical bars mark sectional
	boundaries. Great Lakes/Hudson River dreissenid sites, Bays 1-11; East-coast
	mytilid sites, Bays 12-36; East-coast oyster sites, Bays 37-61; Puerto Rico, Bay
	62; Gulf-coast oyster sites, Bays 63-93; West-coast mytilid sites, Bays 94-125;
	Hawaii, Bay 126. Data for Bays 37 and 38 not included.
	Figure 3. Bay-mean body burdens of Hg, Ni and Pb. Vertical bars mark sectional
	boundaries. Great Lakes/Hudson River dreissenid sites, Bays 1-11; East-coast
	mytilid sites, Bays 12-36; East-coast oyster sites, Bays 37-61; Puerto Rico, Bay
	62; Gulf-coast oyster sites, Bays 63-93; West-coast mytilid sites, Bays 94-125;
	Hawaii, Bay 126. Data for Bays 37 and 38 not included.
	Figure 4. Bay-mean body burdens of Se, Zn and naphthalene. Vertical bars mark
	sectional boundaries. Great Lakes/Hudson River dreissenid sites, Bays 1-11;
	East-coast mytilid sites, Bays 12-36; East-coast oyster sites, Bays 37-61; Puerto
	Rico, Bay 62; Gulf-coast oyster sites, Bays 63-93; West-coast mytilid sites, Bays
	94-125; Hawaii, Bay 126. Data for Bays 37 and 38 not included.
	Figure 5. Bay-mean body burdens of phenanthrene, uoranthene and pyrene. Vertical
	bars mark sectional boundaries. Great Lakes/Hudson River dreissenid sites,
	Bays 1-11; East-coast mytilid sites, Bays 12-36; East-coast oyster sites, Bays
	37-61; Puerto Rico, Bay 62; Gulf-coast oyster sites, Bays 63-93; West-coast
	mytilid sites, Bays 94-125; Hawaii, Bay 126. Data for Bays 37 and 38 not
	included.
	Figure 6. Bay-mean body burdens of chrysene, total PAHs and total DDTs. Vertical bars
	mark sectional boundaries. Great Lakes/Hudson River dreissenid sites, Bays
	1-11; East-coast mytilid sites, Bays 12-36; East-coast oyster sites, Bays 37-61;
	Puerto Rico,

Table 2 .

 2 Results of ANOVAs comparing contaminant (C) factors with para- site/pathology (P) factors derived from PCA analysis of mytilids and oysters. Factor loads are de ned in Table 1. Only comparisons signi cant at 0:05 are reported.

		Parasite/
		Pathology Contaminant Signi cance
	Taxon All Mytilids	Factor P1	Factor C2	Level 0.027
		P1	C5	0.015
		P2	C2	0.0001
		P2	C4	0.0015
		P3	C1	0.0046
		P4	C4	0.0046
	East Coast only	P3	C1	0.0056
		P3	C5	0.0049
		P4	C4	0.033
	West Coast only	P2	C1	0.024
	All Oysters	P1	C5	0.0018
		P2	C5	0.007
		P4	C1	0.0029
		P4	C2	0.0041
		P4	C3	0.0015
	East Coast only	P3	C1	0.028
		P3	C5	0.017
	Gulf Coast only	P2	C5	0.0097
		P3	C3	0.026
		P4	C1	0.0001
		P4	C2	0.001
		P4	C3	0.023

Table 3 .

 3 Signi cance levels for signi cant ( = 0:05) Spearman's Rank correlation tests on parasite/pathology-contaminant pairs found signi cant in ANOVA, for all mytilids. Parentheses indicate negative correlations. Factor pairs are de ned in Table1. Signi cance levels are provided in Table2. Results of subsequent binomial tests to determine if the correlation is based solely on the co-occurrence of low values are given in the righthand column by a non-signi cant result.

	Spearman's Binomial
	Signi cance Signi cance

Table 3

 3

	. continued		Spearman's Binomial
				Signi cance Signi cance
	Factor Pair Contaminant Parasite/Pathology C2-P2 Cu trematode sporocyst	Level 0.0011	Level 0.025
		Hg	major pathology	0.036
		Hg	tissue pathology	0.0004	0.05
		Hg	trematode sporocyst	0.0001	0.005
		Pb	major pathology	0.003
		Pb	tissue pathology	0.0001	0.0005
		Pb	trematode sporocyst	0.0001	0.005
		mirex	tissue pathology	0.02	0.005
		total PCB	major pathology	0.0001	0.005
		total PCB	tissue pathology	0.0001	0.005
	C4-P2	total PCB trematode sporocyst Cd major pathology	0.0002 (0.0001)	0.01
		Cd	tissue pathology	(0.0001)	0.005
		Cd	trematode sporocyst (0.0001)	0.02
		Cr	major pathology	(0.013)
		Cr	tissue pathology	(0.013)
		Fe	tissue pathology	(0.014)
		Ni	tissue pathology	(0.022)
		Zn	major pathology	(0.0005)
	C1-P3	Zn uoranthene	tissue pathology rare parasite	(0.044) 0.047
		pyrene	rare parasite	0.025
	C4-P4	total PAH Cd	rare parasite trematode metacercaria (0.0001) 0.027	0.005
		Cr	trematode metacercaria (0.0004)	0.05
		Fe	trematode metacercaria (0.0001)
		Fe	ceroid body	(0.0001)	0.01
		Fe	digestive gland atrophy 0.035
		Ni	trematode metacercaria (0.0001)	0.005
		Zn	trematode metacercaria (0.0013)	0.02

Table 4 .

 4 Signi cance levels for signi cant ( = 0:05) Spearman's Rank correlation tests on parasite/pathology-contaminant pairs found signi cant in ANOVA, for mytilids on the East and West coast. Parentheses indicate negative correlations. Factor pairs are de ned in Table 1. Signi cance levels are provided in Table 2. Results of subsequent binomial tests to determine if the correlation is based solely on the co-occurrence of low values are given in the righthand column by a nonsigni cant result.

		East Coast
			Spearman's Binomial
			Signi cance Signi cance
	Factor Pair Contaminant Parasite/Pathology C5-P3 Ag rare parasite C4-P4 Cr trematode metacercariae (0.0008) Level (0.0069)	Level 0.05
	Cr	digestive gland atrophy 0.0004	0.05
	Fe	trematode metacercariae (0.0001)	0.05
	Fe	ceroid body	(0.0005)
	Fe	digestive gland atrophy 0.0038	0.05
	Ni	trematode metacercaria (0.0001)	0.05
	Ni	digestive gland atrophy 0.028
	Zn	trematode metacercaria (0.0063)
		West Coast
			Spearman's Binomial
			Signi cance Signi cance
	Factor Pair Contaminant Parasite/Pathology Level C1-P2 phenanthrene major pathology 0.0001	Level 0.02
	uoranthene major pathology 0.0001	0.02
	pyrene	major pathology 0.0001	0.005
	chrysene major pathology 0.0001
	total PAH major pathology 0.0001

Table 5 .

 5 Signi cance levels for signi cant ( = 0:05) Spearman's correlation tests on parasite/pathology-contaminant pairs found signi cant in ANOVA, for all oysters. Parentheses indicate negative correlations. Factor pairs are de ned in Table1. Signi cance levels are provided in Table2. Results of subsequent binomial tests to determine if the correlation is based solely on the co-occurrence of low values are given in the righthand column by a non-signi cant result.

	Spearman's Binomial
	Signi cance Signi cance

Table 6 .

 6 Signi cance levels for signi cant ( = 0:05) Spearman's Rank correlation tests on parasite/pathology-contaminant pairs found signi cant in ANOVA, for Gulf oysters. Parentheses indicate negative correlations. Factor pairs are de ned in Table1. Signi cance levels are provided in Table2. Results of subsequent binomial tests to determine if the correlation is based solely on the co-occurrence of low values are given in the righthand column by a non-signi cant result.

	Spearman's Binomial
	Signi cance Signi cance

Acknowledgments

We thank the Status and Trends eld and laboratory teams at Texas A&M University and TDI-Brooks International who collected the animals for histopathological analysis. The Status and Trends program is supported through contracts with the U.S. Department of Commerce, National Oceanic and Atmospheric Administration, National Ocean Service.