Freshwater Mussel (Elliptio complanata) as a Sentinel Species: Vitellogenin and Steroid Receptors1, The

SYNOPSIS.

Freshwater mussels, Elliptic complanata were collected from a reference and pollutant-impacted pond on Cape Cod, MA. Glutathione-S-transferase (GST) activity was measured in gill, hepatopancreas and foot. In addition, content of seven heavy metals were measured in whole bodies. GST activity was significantly elevated in hepatopancreas and foot, as was whole body cadmium level in animals from the contaminated site suggesting that these animals have been exposed to organic and inorganic contaminants. Sodium dodecyl acrylamide gel electrophoresis (SDS-PAGE) analysis showed putative vitellogenins with molecular weight 180 and 205 kDa bands only in the ovary. In non-denatured gel electrophoresis ovarian extracts revealed two higher molecular weight bands at 550 and 700 kDa, which were reproductive stage specific. Western blotting of SDS-PAGE and non-denatured gels using the anti-scallop yolk-protein antibody confirmed the presence of cross-reacting bands of the same molecular weights in the ovary but not other tissues. Although several experiments involving steroid hormone exposure were done, no significant changes in vitellogenin protein levels were observed. However, using an anti-human ERβ antibody, ERβ positive bands were observed both in female foot, and the ovary. No cross reactivity with the antibody was observed in hepatopancreas. Additional studies are required to resolve questions of vitellogenin regulation and the role of (xeno)estrogens in bivalve molluscs.

INTRODUCTION

For a number of years we have studied the possible impact of xenobiotic contamination from a Superfund site, the Massachusetts Military Reservation (MMR), on local biota, and possibly humans. Contaminated ground water plumes which intersect with freshwater ponds, and probably impact the quality of residential well water, have been well described as to source, and flow of many contaminants (Air Force Center for Environmental Excellence AFCEE, 2003). We have described reproductive impairment and evidence of xenobiotic exposure in a sub-population of freshwater turtles (Chrysemys picta; Rie et al., 2001; Kitana et al, 2003, 2004; Rie et al, 2005) in this area. Given the long-lived nature of these turtles, and low population number, we have developed other sentinel species for field and laboratory studies (see paper by Novillo et al, 2004; this symposium, for work on C. elegans). Here we report studies of Elliptic) complanata, a species endemic to the fresh water ponds on Cape Cod in the vicinity of the MMR. This species is suitable as a model for both laboratory and field work because of its life history and exposure to the fresh water aquatic environment; as a filter feeder and bottom dweller it is likely to be exposed to all xenobiotics in the water and sediment.

Combining "sentinel species" with specific biomarkers provides important biological information on the potential impact of xenobiotics on the health of organisms and ecosystems (Van der Oost et al., 1997). Various biomarkers have been measured in mollusks and specifically in E. complanata, such as vitellin-like proteins, metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) (Gagne et al., 2001a, 2004; Hoarau et al., 2004). In this study, we report on glutathione S-transferase (GST) activity and vitellogenin-like proteins of E. complanata as biomarkers of exposure to contaminant mixtures in the field, and our attempts to date to develop yolk protein synthesis as a bioindicator of xenoestrogen exposure in this species

MATERIALS AND METHODS

Collection sites and maintenance of mussels

Freshwater mussels were collected from Johns Pond and Masphee Pond on Cape Cod, MA. The two ponds are located in the area surrounding the MMR and are used for human recreation. For our studies Masphee Pond is considered to be a potentially non-impacted site, and Johns Pond is considered to be a site impacted by intersecting contaminated ground water plumes. E. complanata gametes develop during winter and early spring (the vitellogenic period), and then are released between late spring and early summer. Zygotes develop within the mantle cavity and glochidia larvae are released in the early summer. Mussels from these ponds were collected by hand between early spring and late fall and returned to the laboratory. All mussels were maintained in an environmental chamber with an aeration system at 16°C and a 12 hr light/12 hr dark light cycle in the Department of Biology Aquarium Facility at Boston University. Mussels were kept in dechlorinated water and fed every other day with cultured green algae (Selenastrum capricornutum). All mussels were allowed to depurate at least for 24 hours in the aquarium before being used for experiments or assays.

Histology

The whole bodies of mussels at the vitellogenic stage were fixed in 10% neutral buffered formalin for 72 hours, and dehydrated through increasing concentrations of ethanol and embedded in paraffin. Serial sections (thickness, 5 µm) were cut, and stained with hematoxylin and eosin.