Freshwater mussels (family Unionidae) are critically imperiled with an estimated 70% of species classified as endangered, threatened or of special concern.5 Many mussel species are declining so rapidly that their existence may come to depend on captive management and propagation. Unfortunately, when relocated or brought into captivity, a high proportion of these normally long-lived animals die within the first year.2 Health assessment has largely been limited to lethal methods.4 The development of nonlethal methods of hemolymph collection from mussels3 has facilitated progress in identifying cytological1 and chemical3 indicators of health. In a recent study,1 L-cysteine (25 mg/ml) was identified as a useful anticoagulant for maintaining hemocyte morphology, and hemocyte morphological characteristics of Quadrula quadrula were described. However, the development of health assessment methods has been slow, in part due to tremendous species variability, relatively low cellularity of hemolymph, and hemocyte sensitivity to common laboratory diluents and activation. The objective of this study was to determine the optimal conditions for processing of hemolymph in the field and laboratory to maintain cell morphology and viability for physiologic assays. Hemolymph was collected from nine Q. quadrula, three mussels per study. The viability of hemocytes was quantified using Trypan blue staining to determine the effects of temperature, pH, and exposure to anticoagulant during sample processing. In study 1, hemolymph was stored at room temperature or on ice, and cell viability was examined at 1, 4 and 6 h. Hemolymph viability declined from 84% at 4 h and to 73% at 6 h at room temperature, whereas viability of cells stored on ice declined more rapidly from 77.8% at 4 h to 59.15% at 6 h. In study 2, hemocytes were suspended at room temperature and the viability of untreated cells and cells exposed to the anticoagulant L-cysteine (25 mg/ml) was compared at 1, 4, 6, 10, and 24 h after collection. Viability of cells exposed to L-cysteine was lower at all time points compared to controls, whose viability remained > 90% throughout the study (p < 0.05). In study 3, the viability of hemocytes stored in L-cysteine at pH 6.5, 7.0, or 8.0 was examined at 1, 4, and 6 h. Cells stored in L-cysteine at a pH of 8.0 maintained the highest viability at each time point (80-73%) compared to those stored at a pH of 6.5 or 7.0 which ranged from 66-73% and 45-63%, respectively, for 6 h. Cell morphologies revealed that exposure of hemolymph to L-cysteine had marked effects on cell degranulation and crenation after 1 hour at all pH levels and such effects increased at 4 and 6 h. These results indicate that hemocytes of Q. quadrula maintain viability in vitro when stored untreated at room temperature for up to 24 h. However, there is a need to further optimize in vitro storage conditions to minimize cell coagulation and activation over time.
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2. Cope W.G., M.C. Hove, D.L. Waller, D.J. Hornbackh, M.R. Bartsch, L.A. Cunningham, H.L. Dunn and A.R. Kapuscinski. 2003. Evaluation of relocation of unionid mussels to in situ refugia. J Molluscan Studies 69: 27-34.
3. Volety A.K. 2008. Effects of salinity, heavy metals and pesticides on health and physiology of oysters in the Caloosahatchee Estuary, Florida. Ecotoxicology 17(7): 579-90.
4. Gustafson L.L., M.K. Stoskopf, A.E. Bogan, W. Showers, T.J. Kwak, S. Hanlon, and J.F. Levine. 2005. Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae). Dis Aquat Org 65: 159-165.
5. Burkard M.J., S.A. Leavell, R.B. Weiss, K. Kuehnl, K.H. Valentine, G.T. Watters, and B.A. Wolfe. Analysis and cytologic characterization of hemocytes from freshwater mussels (Quadrula spp.). Vet Clin Pathol, in press.