Several cases of disseminated neoplasia in mussels Mytilus edulis (L.) in western Long Island Sound.ABSTRACT Eight cases of disseminated neoplasia were found among 540 specimens of Mytilus edulis collected from an intertidal beach in Connecticut in western Long Island Sound. This was unusual, because disseminated neoplasia is very tare in M. edulis bur causes epizootic mortalities in another mussel species, M. trossulus. According to histology, mussels showed a continuum of disease progression, from early stages with a few anaplastic cells around the stomach epithelium to more advanced cases with multiple foci of neoplastic cells in the tissues, finally to terminal cases with profuse infiltration of neoplastic cells in all tissues. Flowcytometric hemocyte analyses were performed to compare immune functions between neoplastic and healthy mussels. Circulating hemocytes from the neoplastic mussels showed significantly less phagocytosis and significantly more apoptosis than hemocytes from the healthy mussels. Hemocyte cell density measured by flow cytometry increased in the hemolymph with progression of the disease on histological sections. In situ hybridization was performed to detect apoptosis also on paraffin sections. There were more apoptotic neoplastic cells in the early stages of the disease than in the later stages. These observations suggest the need for further studies on apoptosis-regulating genes to explain differences in susceptibility to neoplasia of different Mytilus species, and the role of apoptosis in the progression of disseminated neoplasia.
KEY WORDS: apoptosis, flow-cytometry, Long Island Sound, Mytilus edulis, neoplasia
Disseminated neoplasia, a malignant proliferation of circulating cells, has been reported in 15 species of marine bivalves with a world-wide distribution (Peters 1988). The disease is characterized by large, anaplastic cells in hemolymph vessels and sinuses, and in visceral-mass, muscle, and mantle tissues (Barber 2004). These cells are polyploid (Elston et al. 1990, Moore et al. 1991), have large pleomorphic nuclei with one or more prominent nucleoli, and have a high nucleus-to-cytoplasm ratio. Mitotic figures are common in these cells. Anaplastic cells divide, whereas the number of healthy hemocytes decreases. This condition has also been called sarcomatoid proliferative disease (Farley 1969), proliferative atypical hemocytic condition (Lowe & Moore 1978), epizootic sarcoma (Farley et al. 1986), sarcomatous neoplasia (Brousseau 1987), transmissible sarcoma (Farley et al. 1991), and systemic neoplasia (Moore et al. 1991). The disease causes significant mortalities and decrease in market harvest in economically-important species such as mussels, clams, and cockles (Elston et al. 1992).
Mussels, Mytilus spp., include several species: M. edulis, M. trossulus, M. galloprovinciales, M. chilensis, M. coruscus, M. californianus, M. platensis, M. planulatus, and M. desolationis (Gosling 1992). Prior to the use of electrophoresis, taxonomy was based on shell morphology, a characteristic that varies with numerous environmental factors. Consequently, there is confusion in the literature concerning occurrence of disseminated neoplasia in particular mussel species. The following Mytilus species have been reported to be afflicted with disseminated neoplasia: M. edulis, M. trossulus, M. galloprovincialis, and M. chilensis. Prevalences vary greatly, and genetics are considered to play a role in the susceptibility of the mussels to the disease (Fuentes et al. 2002). Disseminated neoplasia reaches epizootic prevalences and causes significant mortalities in M. trossulus in Washington, OR, and British Columbia on the west coast of North America (Elston et al. 1992). Disseminated neoplasia has also been observed in M. trossulus from the Baltic Sea (Rasmussen 1986, Sunila 1987), and at Nakhodka Bay, close to Vladivostok, Russia, on the Pacific coast (Usheva & Frolova 2000). In South America, disseminated neoplasia was reported in M. chilensis at 2.4% of 572 specimens sampled in Chiloe Island, Chile (Campalans et al. 1998).
Mytilus edulis and M. galloprovincialis display low prevalences of disseminated neoplasia, and the disease is not associated with mortalities (Elston et al. 1992). In M. galloprovincialis, cases of disseminated neoplasia were reported in the Mediterranean Sea, Italy (Tiscar et al. 1990, Zizzo et al. 1991), in the east Atlantic Ocean in Galicia, Spain (Figueras et al. 1991, Villalba et al. 1997, Fuentes et al. 2002), in California, USA (Elston et al. 1992), and in the Black Sea, Russia (Ciocan & Sunila 2005). Disseminated neoplasia has been detected at a low prevalence in M. edulis in the Irish Sea (0.73%, Green & Alderman 1983) and in the North Sea (1.6%, Lowe & Moore 1978). Hillman et al. (1992) reported seven cases of disseminated neoplasia in Massachusetts, New York, and Connecticut among over 8,000 mussels studied during six years of the Mussel Watch Program. The two cases reported in Connecticut were sampled from Housatonic River.
On the east coast of the USA, there are two species of blue mussels, Mytilus edulis and M. trossulus. In northern Canada and Alaska, blue mussel populations are composed mainly of M. trossulus, but in Newfoundland, Nova Scotia, and Maine, M. edulis and M. trossulus occur sympatrically (Penney & Hart 1999, Rawson et al. 2001). In Long Island Sound, where the mussels were collected for the current study, the only reported mussel present is M. edulis (McDonald et al. 1991).
We found several cases of disseminated neoplasia in mussels, M. edulis, at one site in Long Island Sound. Many neoplastic cells appeared to be mitotic or apoptotic, and the TUNEL assay was performed on tissue sections to demonstrate the proliferation/cell death cycle. Flowcytometric analysis of hemocytes was performed to study differences in circulating cells between neoplastic and healthy mussels in cell density, phagocytosis, reactive oxygen species (ROS), and apoptosis.
MATERIALS AND METHODS
Blue mussels, Mytilus edulis (44.1-77.2 rum shell length), were collected from Westcott Cove, Stamford, CT, USA, from an intertidal beach on the north shore of Long Island Sound in May and June of 2007. A total of 540 mussels were diagnosed for disease with histopathology, for hemocyte immune functions with flow cytometry, and 5 ncoplastic specimens for apoptosis with in situ hybridization.
A 4-mm cross-section of each mussel, including digestive diverticula, gills, mantle, kidneys, plicate membranes, and the byssus gland, was dissected and fixed in Davidson's fixative for 48 h at 4[degrees]C. The tissues were then rinsed in 50% ethanol in filtered seawater, and transferred to 70% ethanol. Samples were dehydrated and embedded in paraffin. After processing, 5-[micro]m sections were stained using a hematoxylin-eosin staining procedure (Howard et al. 2004) and examined under a light microscope. Neoplastic mussels were categorized according to the severity of the disease from 1-8, with 1 being a very early case and 8 representing a terminal case.
In situ Hybridization
Five neoplastic mussels were studied to detect apoptosis in tissues. A new set of sections was mounted on positively-charged slides for apoptosis detection using TUNEL, terminal deoxynucleotidyl transferase (TdT) mediatcd dUTP nick end labeling assay with Apoptag[R] (Chemicon International). This method is based on the specific binding of digoxigeninlabeled nucleotides to the exposed 3'OH ends of DNA fragments by TdT. An antidioxigcn antibody conjugated to peroxidase binds to the digoxigcnin-labeled nucleotides, and the reaction can be observed after treatment with a peroxidase substrate that creates a brown stain on histological sections (Gavrieli et al. 1992). The slides were washed in xylene twice for 5 min each and dehydrated. Preparations were washed with PBS for 5 min and digested with Proteinase-K (20 [micro]g [mL.sup.-1]) for 15 min at room temperature. Samples were then treated with 0.5% (v:v) Triton X-100 for 10 min. Afterwards, the slides were quenched in 3% hydrogen peroxidc in PBS for 5 min at room temperature, washed in deionized water twice for 5 min each time, and treated with Apoptag[R] equilibrium buffer. The slides were then incubated with TdT enzyme and nuclcotidcs (1 h, +37[degrees]C). After incubation, the reaction was stopped with Apoptag[R] stop/wash buffer (10 min), and antidioxigenin peroxidase conjugate was applied to the slide (30 min, room temperature). After four washes in PBS, 2 min each, the stain was developed with a peroxidase substrate. Cells were counterstained in a 0.5% (w:v) methyl green solution for 8 min at room temperature. The preparations were dehydrated in butanol and mounted. The positive control was a histological section of a normal, postweaning female rat mammary gland, included in the Apoptag[R] kit. Two negative controls were used: in one PBS was substituted for TdT; in the other, the section was stained with peroxidase substrate without adding TdT or antibody conjugate to detect possible endogenous peroxidase activity.
For four of the five specimens studied, apoptotic index was counted based on 10 fields of 100 neoplastic cells each (total 1,000 cells per mussel). One of the five mussels showed too few neoplastic cells to be counted.
Hemolymph was withdrawn with a 21-gauge needle and a 1-mL syringe from the adductor muscle of each individual mussel and stored temporarily in an Eppendorf microcentrifuge tube on ice. Analyses were done on hemolymph extracted from each individual mussel. Cell density of circulating neoplastic cells and hemocytes (cells [mL.sup.-1]), and the following immunological functions were measured: percentage of circulating cells showing phagocytosis of multiple fluorescent beads, respiratoryburst response expressed as arbitrary units (Hegaret et al. 2003a, b), and the percentage of apoptotic circulating cells (Goedken et al. 2005a). Apoptosis was measured by Annexin-V, which recognizes externalized phosphatidyl serine, a membrane phospholipid restricted to the inner leaflet in normal cells but expressed on external surfaces of apoptotic cells. Propidium iodine (PI) was used in conjunction with Annexin-V to detect dead cells, because living and apoptotic cells exclude PI, but the membranes of necrotic cells are permeable to PI (Goedken et al. 2005a). For the hemolymph analyses, a FACScan flow-cytometer (BD Biosciences, San Jose, CA) was used.
Results for hemolymph analyses were checked for normality and analyzed statistically using the Mann-Whitney test to assess differences between control and neoplastic mussels. Simple Regression was used to relate cell density of circulating neoplastic cells and hemocytes with the different neoplastic stages of mussels. The statistical software used was Statgraphics Plus (Manugistics, Inc., Rockville, MD, USA).
Neoplastic Cells in Histological Sections
There were 8 specimens with disseminated neoplasia in the samples, representing a mean prevalence of 1.5% (8/540). These cases included different stages of proliferation from initial to terminal disease. When organized according to the stage, the first case was very early with only a few neoplastic cells in the connective tissue underlying the stomach epithelium. The second case had small isolated foci of neoplastic cells in the digestive diverticulum. The third case was advancing with small isolated foci of neoplastic cells in gills, plicate membranes, mantle, and between kidney tubules. The fourth case was advanced with larger foci of neoplastic cells in digestive diverticulum, gills, kidneys, plicate membranes, and mantle. The fifth case was very advanced with infiltration of gills, plicate membranes and mantle with large islets of neoplastic cells. The sixth case had all tissues invaded by neoplastic cells, but there were some gonadal follicles still producing gametes. The seventh and eighth cases represented terminal stages of the disease, with profuse invasion of neoplastic cells into all tissues.
In sinuses and hemolymph vessels, neoplastic cells appeared round (Fig. 1A). With the high cell densities in the terminal cases of neoplasia, however, the cells were irregularly-shaped and gathered within tissues in a puzzle-like manner (Fig. 1B). Neoplastic cells showed a high degree of anaplasia, high nucleus-to-cytoplasm ratio, usually one prominent nucleolus, partially condensed chromatin, and high mitotic index. Often, several mitotic figures were present in a single microscope field (Fig. 1B). On the hematoxyline-eosine stained sections, several neoplastic cells had morphological characteristics of apoptosis, that is, condensation of chromatin to the periphery of the nucleus and crescent-shaped chromatin (Fig. 1A, C). One case had formation of a tumor giant cell, another characteristic feature for anaplasia, among neoplastic cells in the connective tissue in the digestive diverticulum (Fig. 1D).
In situ Hybridization
In situ hybridization revealed a proportion of neoplastic cells with a positive reaction to Apoptag[R] (Fig. 2A). Peroxidase substrate precipitated as a brown stain within the nuclei of apoptotic cells and apoptotic bodies. Apoptotic index of mussels ranged from 3.8% in early cases of neoplasia to 0.2% in terminal neoplasia stages (Fig. 3). Most of the staining was concentrated along the nuclear membranes with some condensed staining within the nucleoplasm. Apoptotic cells that stained positively with Apoptag[R] still had morphological characteristics of neoplastic cells with large nuclei and scarce cytoplasm (Fig. 2B).
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Neoplastic mussels had a significantly-higher percentage of apoptotic hemocytes compared with the control mussels (P < 0.01) (Fig. 4). Mean values and standard errors of means were 7.63 [+ or-] 4.45% for the diseased mussels and 2.12 [+ or-] 1.86% for control mussels. Hemocyte phagocytosis was also significantly different for the two types of mussels (P < 0.01) being higher in the controls (Fig. 5). Mean values and standard errors of means were 56.18 [+ or-] 12.72% for control mussels and 37.98 [+ or-] 13.13% for neoplastic mussels. There were no significant differences between the two types of mussels for ROS (P > 0.05). Mean cell densities of circulating, neoplastic cells and hemocytes did not show statistically-significant differences between control and neoplastic mussels (P > 0.05) (Fig. 6); minimum values were similar for both types ofmussels, but maximum values diverged from 8.71 [10.sup.5] cell [mL.sup.-1] for control mussels to 14.89 [10.sup.5] cell [mL.sup.-1] for neoplastic mussels. The relationship between cell density and the stage of neoplasia in mussels was statistically significant (P < 0.05) when analyzed through Simple Regression; correlation coefficient was 0.72 (Fig. 7). Mussels with a more advanced stage of neoplasia contained a higher cell density of circulating cells.
Acquisition of several blue mussels from the same population with disseminated neoplasia offered a rare opportunity to study cellular changes associated with progression of the disease. We focused especially on apoptosis, because apoptosis has been described as a cell mechanism to prevent the proliferation of malignant neoplasia in vertebrates (Kliche & Hoffken 1999, Elmore 2007). To our knowledge, this is the first report of increased apoptosis in cells of a bivalve with disseminated neoplasia.
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Apoptosis is a regulated pathway of cell death, also called programmed cell death or cell suicide. Apoptosis is preceded by production of endonucleases that cleave the DNA into fragments of 180-200 base pairs or multiples thereof (Cohen et al. 1992). The nucleus condenses, cytoplasm shrinks, and the cell membrane blebs (zeiosis). Blebbing leads to the formation of apoptotic bodies that are composed of membrane-bound cytoplasm, nuclear fragments, and organelles. The apoptotic bodies are engulfed by phagocytosis immediately by neighboring cells without eliciting an inflammatory response (Cohen et al. 1992, White 1996).
Farley (1969) mentioned "increased sarcomatoid cell pyknosis" in disseminated neoplasia of mussels that possibly referred to apoptotic chromatin condensation. Apoptosis was not recognized as a distinct pathway of cell death before 1970 (Kerr et al. 1972), and is often referred as "condensation necrosis" in older literature (Cohen et al. 1992). Elston et al. (1988a) traced the progression of disseminated neoplasia in individual M. trossulus. From the beginning of the disease, 50% of the mussels progressed to advanced disease, most of which died; 20% showed advancement of the disease but went into remission, and 25% remained disease free. Remission included "the destruction of neoplastic cells" but morphological characteristics of moribund neoplastic cells were not specified. It is possible that remission as observed by Elston et al. (1988a) was based on an apoptotic cell-death pathway.
In this study, two different methods were used to detect apoptosis: Annexin-V with flow cytometry and Apoptag[R] in tissue sections. Annexin-V detects an early stage of apoptosis (externalized phosphatidyl serine); Apoptag[R] detects an end product of apoptosis (DNA fragments). There were more apoptotic, circulating cells in the neoplastic mussels compared with the healthy mussels. Apoptotic index in tissue sections ranged from 0.2% to 3.8% and suggested a declining trend with progression of neoplasia. Sunila (1991) also reported a decrease in mitotic index of neoplastic cells in the clam Mya arenaria during the course of the disease. Obviously, the larger the proliferative pool of replicating cells, the faster the progression of the disease. It is possible that more neoplastic cells leave the pool of dividing cells in the beginning of the disease through apoptosis; thus, there is more mitosis and apoptosis in early stages of the disease.
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Several papers have reported apoptosis in bivalve tissues. Sunila and LaBanca (2003) reported apoptosis in different infectious diseases of C. virginica, and Da Silva et al. (2006) found apoptotic hemocytes in gill lesions of Ostrea edulis. Goedken et al. (2005a) studied the effects of salinity and temperature on apoptosis of C. virginica hemocytes, and the differences in apoptosis between C. virginica and C. gigas hemocytes after challenge with Perkinsus marinus (Goedken et al. 2005b). Apoptosis was induced in Crassostrea gigas hemocytes with noradrenaline by Lacoste et al. (2002) and by Arg-Gly-Asp peptides by Terahara et al. (2003), in C. virginica hemocytes with cadmium by Sokolova et al. (2004), and in M. galloprovincialis gills with trin-butyltin by Micic et al. (2001).
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Monoclonal antibodies against neoplastic cells of M. trossulus were produced by Noel et al. (1991). The antibodies reacted with normal hemocytes and neoplastic cells, indicating common antigens, and that the likely cell of origin to the neoplasia is a hemocyte. Several articles demonstrate that disseminated neoplasia in bivalves is transmissible between individuals, suggesting an infective etiology (Twomey & Mulcahy 1988, Appeldoorn et al. 1984, Collins & Mulcahy 2003). Elston et al. (1988b) inoculated disease-free M. trossulus with intact hemolymph, or homogenized hemolymph from mussels with disseminated neoplasia. Both groups of mussels developed the disease, suggesting viral transmission of the disease. Reverse transcriptase (RT), an enzyme common for all retroviruses, was detected in neoplastic cells but not in healthy hemocytes of Mya arenaria (Medina et al. 1993, House et al. 1998). More recently, Romalde et al. (2007) detected reverse transcriptase in cockles, Cerastoderma edule by two different methods: a commercial RetroSys[TM] RT activity kit, and a product-enhanced RT (PERT) assay. They also provided an electron micrograph of 120 nm retrovirus-like particles.
Many studies have addressed the potential role of pollution in initiating disseminated neoplasia (Elston et al. 1992, Barber 2004). Findings are, however, contradictory in that no induction of neoplasia by contaminants was demonstrated in other studies. It is possible, however, that some pollutants may exacerbate pre-existing virai infections. In the present paper, eight cases of neoplasia were discovered at one site in western Long Island Sound. Sunila et al. (2004) failed to detect neoplasia among 360 mussels, M. edulis, during a one-year study at a site 12 miles east of the Stamford beach where neoplastic mussels were found in the present study. In Long Island Sound, there is a decreasing west-to-east pollution cline in heavy metals in sediments (Mecray & Buchholtzten Brink 2000), organic pollutants in sediments, and concentrations of both in blue mussel tissues (Turgeon & O'Connor 1991). Also, there is an allele frequency cline in M. edulis in Long Island Sound. [Lap.sup.94] (leucine aminopeptidase) progressively declines from west to east on the northern and southern shores (Gosling 1992). This locus, however, has no discriminating power to differentiate between the different Mytilus species or subspecies, as there is well-documented evidence for the selective effect of salinity or pollution on Lap allele frequencies in Mytilus (Gosling 1992).
Part of the response to neoplasia in bivalves is the proliferation of anaplastic-appearing cells in the semiopen vascular system (Elston et al. 1992). No flow cytometry studies have been done previously to demonstrate the increase in cell density in the hemolymph of neoplastic mussels during disease progression. Cooper et al. (1982) staged the severity of neoplasm in Mya arenaria according to number of neoplastic cells present in the hemolymph. Our findings show a relationship between cell density and the stage of neoplasia; the more severe stage of neoplasia on a histological section, the higher the density of neoplastic cells and hemocytes in circulation. In the present study, there was significantly less phagocytosis in circulating cells of neoplastic mussels compared with healthy mussels (Fig. 5). Kent et al. (1989) exposed Mytilus edulis with hemic neoplasia to yeast cells for phagocytosis studies and performed in vivo bacterial-clearance experiments. These authors concluded that mussels with advanced hemic neoplasia have compromised immune systems. Moreover, Beckmann et al. (1992) studied the surface receptors of M. arenaria hemocytes with hemic neoplasia and exposed these hemocytes to yeast cells; phagocytosis did not occur in the neoplastic hemocytes, because they were not able to adhere to and engulf the yeast cells. No significant differences were found for ROS when comparing neoplastic and control hemocytes. According to the authors' knowledge, there are no previous reports of immune parameters in bivalves with disseminated neoplasia using flow cytometry.
Many genes regulating tumor proliferation are highly conserved throughout the animal kingdom. The tumor suppressor gene p53 regulates the cell cycle and prevents neoplastic transformation. Loss of p53 function can lead to unchecked cell growth and contribute to tumor pathogenesis. A p53 homologue has been described in association with disseminated neoplasia in Mya arenaria by Kelley et al. (2001), and in M. edulis by Ciocan and Rotchell (2005). Walker et al. (2006) also reported sequestration of p53 protein complexed with mortalin (a Hsp70 family member) to the cytoplasm of neoplastic cells in M. arenaria, but not in normal hemocytes. Ciocan et al. (2006) reported induced expression of ras, an oncogene involved in cell-growth signaling cascades, in neoplastic M. trossulus cells.
Presence of apoptotic neoplastic cells, and their decrease during disease progression, indicates that proliferation of disseminated neoplasia in mussels is genetically regulated. It is possible that the high susceptibility of M. trossulus, low prevalences observed in M. edulis, and tare observations of disseminated neoplasia in M. galloprovincialis, are connected to apoptosis-regulating genes. Thus, studies to identify apoptosis-regulating genes (blc-2, ced, p53) in mussels are warranted to explore reasons for geographic distribution of the disease and the differences in susceptibility between different Mytilus species.
The senior author was supported by a grant from the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA) and partially financed by the RTA04-023 INIA research project. The following Milford Laboratory staff provided much-appreciated technical assistance: Gary H. Wikfors, Helene Hegaret, Jennifer H. Alix, April N. Croxton, Mark S. Dixon, Diane Kapareiko, Yaqin Li, Shannon L. Meseck, and Barry C. Smith. The senior author thanks Montserrat Ramon for her constant support.
Appeldoorn, R. S., C. W. Brown, R. S. Brown, P. W. Chang, K. R. Cooper, E. Lorda, S. Saila, H. Walker & R. E. Wolke. 1984. Field and laboratory studies to define the occurrence of neoplasia in the soft-shell clam, Mya arenaria. American Petroleum Institute, Washington, DC. 201 pp.
Barber, B. J. 2004. Neoplastic diseases of commercially important marine bivalves. Aquat. Liv. Res. 17:449-466.
Beckmann, N., M. P. Morse & C. M. Moore. 1992. Comparative study of phagocytosis in normal and diseased hemocytes of the bivalve mollusc Mya arenaria. J. Invert. Pathol. 59:124-132.
Brousseau, D. J. 1987. Seasonal aspects of sarcomatous neoplasia in Mya arenaria (soft-shell claro) from Long Island Sound. J. Invert. Pathol. 50:269-276.
Campalans, M., M. Gonzales & P. Rojas. 1998. Neoplasia in Mytilus chilensis cultivated in Chiloe Island (Chile). Bull. Eur. Ass. Fish Pathol. 18:93-95.
Ciocan, C. M. & I. Sunila. 2005. Disseminated neoplasia in the blue mussels, Mytilus galloprovincialis, from the Black Sea, Romania. Mar. Pollut. Buli. 50:1335-1339.
Ciocan, C. M. & J. M. Rotchell. 2005. Conservation of cancer genes in the marine invertebrate Mytilus edulis. Environ. Sci. Technol. 39:3029-3033.
Ciocan, C. M., J. D. Moore & J. M. Rotchell. 2006. The role of ras gene in the development of haemic neoplasia in Mytilus trossulus. Mar. Environ. Res. 62:S147-S150.
Cohen, J. J., R. C. Duke & V. A. Fadok. 1992. Apoptosis and programmed cell death in immunity. Annu. Rev. Immunol. 10:267-293.
Collins, C. M. & M. F. Mulcahy. 2003. Cell-free transmission of a haemic neoplasm in the cockle Cerastoderma edule. Dis. Aquat. Org. 54:61- 67.
Cooper, K. R., R. S. Brown & P. W. Chang. 1982. The course and mortality of a hematopoietic neoplasm in the soft-shell clam, Mya arenaria. J. Invert. Pathol. 39:149-157.
Da Silva, P. M., A. Villalba & I. Sunila. 2006. Branchial lesions associated with abundant apoptotic cells in oysters Ostrea edulis of Galicia (NW Spain). Dis. Aquat. Org. 70:129-137.
Elmore, S. 2007. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35:495-516.
Elston, R. A., M. L. Kent & A. S. Drum. 1988a. Progression, lethality and remission of hemic neoplasia in the bay mussel Mytilus edulis. Dis. Aquat. Org. 4:135-142.
Elston, R., M. L. Kent & A. S. Drum. 1988b. Transmission of hemic neoplasia in the bay mussel, Mytilus edulis, using whole cells and cell homogenate. Dev. Comp. Immunol. 12:719-727.
Elston, R. A., A. S. Drum & S. K. Allen. 1990. Progressive development of circulating polyploid cells in Mytilus with hemic neoplasia. Dis. Aquat. Org. 8:51-59.
Elston, R. A., J. D. Moore & K. Brooks. 1992. Disseminated neoplasia of bivalve molluscs. Rev. Aquat. Sci. 6(5,6):405-466.
Farley, C. A. 1969. Sarcomatid proliferative disease in a wild population of blue mussel (Mytilus edulis). J. Natl. Cancer I. 43:509-516.
Farley, C. A., S. V. Otto & C. L. Reinisch. 1986. New occurrence of epizootic sarcoma in Chesapeake Bay soft shell clams, Mya arenaria. Fish. Bull. (Wash. D. C.) 84:851-857.
Farley, C. A., D. L. Plutschak & R. F. Scott. 1991. Epizootiology and distribution of transmitable sarcoma in Maryland softshell clams, Mya arenaria, 1984-1988. Environ. Health Perspect. 90:35-41.
Figueras, A. J., C. F. Jardon & J. R. Caldas. 1991. Diseases and parasites of rafted mussels (Mytilus galloprovincialis Lmk): Preliminary results. Aquaculture 99:17-33.
Fuentes, J., J. L. Lopez, E. Mosquera, J. Vazques, A. Villalba & G. Alvarez. 2002. Growth, mortality, pathological conditions and protein expression of Mytilus edulis and M. galloprovincialis crosses cultured in the Ria de Arousa (NW Spain). Aquaculture 213:233-251.
Gavrieli, Y., Y. Sherman & S. A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493-501.
Goedken, M., B. Morsey, I. Sunila, C. Dungan & S. De Guise. 2005a. The effects of temperature and salinity on apoptosis of Crassostrea virginica hemocytes and Perkinsus marinus. J. Shellfish Res. 24:177-183.
Goedken, M., B. Morsey, I. Sunila & S. De Guise. 2005b. Immunomodulation of Crassostrea gigas and Crassostrea virginica cellular defense mechanisms by Perkinsus marinus. J. Shellfish Res. 24:487-496.
Gosling, E. 1992. Genetics of Mytilus. In: E. Gosling, editor. The mussel Mytilus: Ecology, physiology, genetics and culture. Galway: Elsevier. pp. 309-382.
Green, M. & D. J. Alderman. 1983. Neoplasia in Mytilus edulis L. from United Kingdom waters. Aquaculture 30:1-10.
Hegaret, H., G. H. Wikfors & P. Soudant. 2003a. Flow-cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation I. Haemocyte types and morphology. J. Exp. Mar. Biol. Ecol. 293:237-248.
Hegaret, H., G. H. Wikfors & P. Soudant. 2003b. Flow-cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation II. Haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst. J. Exp. Mar. Biol. Ecol. 293:249-265.
Hiltman, R. E., R. A. Lordo, R. G. Menton, C. S. Peven, A. D. Uhler, E. Crecelius & W. G. Steinhauer. 1992. Relationship of environmental contaminants to occurrence of neoplasia in mussels (Mytilus edulis) from east and west coast mussel watch sites. P. Mar. Technol. Soc. 1:230-239.
House, M. L., C. H. Kim & P. W. Reno. 1998. Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity. Dis. Aquat. Org. 34:187-192.
Howard, D. W., E. J. Lewis, B. J. KeIler & C. S. Smith. 2004. Histological techniques for marine bivalve mollusks and crustaceans. NOAA Technical Memorandum NOS NCCOS 5:218.
Kelley, M. L., P. Winge, J. D. Heaney, R. E. Stephens, J. H. Farell, R. J. Van Beneden, C. L. Reinisch, M. P. Lesser & C. W. Walker. 2001. Expression of homologues for p53 and p73 in the softshell clam (Mya arenaria), a naturally-occurring model for human cancer. Oncogene 20:748-758.
Kent, M. L., R. A. Elston, M. T. Wilkinson & A. S. Drum. 1989. Impaired defense mechanisms in bay mussels, Mytilus edulis, with hemic neoplasia. J. Invert. Pathol. 53:378-386.
Kerr, J. F., A. H. Wyllie & A. R. Currie. 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26:239-257.
Kliche, K.-O. & K. Hoffken. 1999. The role of apoptosis in hematologic malignances and modulation of apoptosis as a new therapeutic approach. J. Cancer Res. Clin. Oncol. 125:226-231.
Lacoste, A., A. Cueff & S. A. Poulet. 2002. P35-sensitive caspases, MAP kinases and Rho modulate [beta]-adrenergic induction of apoptosis in mollusc immune cells. J. Cell Sci. 115:761-768.
Lowe, D. M. & M. N. Moore. 1978. Cytology and quantitative cytochemistry of a proliferative atypical hemocytic condition in Mytilus edulis (Bivalvia, Mollusca). J. Natl. Cancer L 60:1455-1459.
McDonald, J. H., R. Seed & R. K. Koehn. 1991. Allozymes and morphological characters of three species of Mytilus in the northern and southern hemispheres. Mar. Biol. 111:323-333.
Medina, D. J., G. E. Paquette, E. C. Sadasiv & P. W. Chang. 1993. Isolation of infectious particles having reverse transcriptase activity and producing hematopoietic neoplasia in Mya arenaria. J. Shellfish Res. 12:112-113.
Mecray, E. L. & M. R. Buchholtzten Brink. 2000. Contaminant distribution and accumulation in the surface sediments of Long Island Sound. J. Coast. Res. 16:575-590.
Micic, M., N. Bihari, Z. Labura, W. E. G. Muller & R. Batel. 2001. Induction of apoptosis in the blue mussel Mytitus galloprovincialis by tri-n-butylin chloride. Aquat. Toxicol. 55:61-73.
Moore, J. D., R. A. Elston, A. S. Drum & M. T. Wilkinson. 1991. Alternate pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus. J. Invert. Pathol. 58:231-243.
Noel, D., V. Boulo, D. Chagot, E. Mialhe, F. Paolucci, C. Clavies, E. Hervaud, R. Elston & H. Grizel. 1991. Preparation and characterization of monoclonal antibodies against neoplastic hemocytes of the bay mussel Mytilus edulis (Bivalvia). Dis. Aquat. Org. 10:51-58.
Penney, R. W. & M. J. Hart. 1999. Distribution, genetic, and morphometry of Mytilus edulis and M. trossulus within a mixed species zone. J. Shellfish Res. 18:367-374.
Peters, E. C. 1988. Recent investigations on the disseminated sarcomas of marine bivalve molluscs. Am. Fish. Soc. (Spec. Pub.) 18:74-92.
Rasmussen, L. P. D. 1986. Occurrence, prevalence and seasonality of neoplasia in the marine mussel Mytilus edulis from three sites in Denmark. J. Invert. Pathol. 48:117-123.
Rawson, P. D., S. Hayhurst & B. Vanscoyoc. 2001. Species composition of blue mussel populations in the northeastern Gulf of Maine. J. Shellfish Res. 20:31-38.
Romalde, J. L., M. L. Vilarino, R. Beaz, J. M. Rodriguez, S. Diaz, A. Villalba & M. J. Carballal. 2007. Evidence of retroviral etiology for disseminated neoplasia in cockles (Cerastoderma edule). J. Invert. Pathol. 94:95-101.
Sokolova, I. M., S. Evans & F. M. Hughes. 2004. Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition. J. Exp. Biol. 207:3369-3380.
Sunila, I. 1987. Histopathology of mussels (Mytilus edulis L.) from the Tvarminne area, Gulf of Finland (Baltic Sea). Ann. Zool. Fennici 24:55-69.
Sunila, I. 1991. Respiration of sarcoma cells from the soft-shell clam Mya arenaria L. under various conditions. J. Exp. Mar. Biol. Ecol. 150:19-29.
Sunila, I. & J. LaBanca. 2003. Apoptosis in the pathogenesis of infectious diseases of the eastern oyster Crassostrea virginica. Dis. Aquat. Org. 56:163-170.
Sunila, I., L. Williams, S. Russo & T. Getchis. 2004. Reproduction and pathology of blue mussels, Mytilus edulis (L.) in an experimental longline in Long Island Sound, Connecticut. J. Shellfish Res. 23:731-740.
Terahara, K., K. G. Takahashi & K. Mori. 2003. Apoptosis by RGD-containing peptides observed in hemocytes of the Pacific oyster, Crassastrea gigas. Der. Comp. Immunol. 27:521-528.
Tiscar, P. G., N. Zizzo, R. Compagnucci & D. Iaffaldano. 1990. Dati preliminary sulla patologia infettiva ed infestiva dei molluschi eduli tamellibranchi allevati o raccolti sui litorali Pugliesi. Atti Soc. It. Sc. Vet. XLIV:709-712.
Turgeon, D. D. & T. P. O'Connor. 1991. Long Island Sound: Distributions, trends, and effects of chemical contamination. Estuaries 14:279-289.
Twomey, E. & M. F. Mulcahy. 1988. Transmission of a sarcoma in the cockle Cerastoderma edule (Bivalvia:Mollusca) using cell transplants. Dev. Comp. Immunol. 12:195-200.
Usheva, L. N. & L. T. Frolova. 2000. Neoplasia in the connective tissue of the mussel Mytilus trossulus from polluted areas of Nakhodka Bay, Sea of Japan. Rus. J. Dev. Biol. 31:53-57.
Villalba, A., S. G. Mourelle, M. J. Carballal & C. Lopez. 1997. Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the Rias of Galicia (NW Spain). Dis. Aquat. Org. 31:127-139.
Walker, C., S. Bottger & B. Low. 2006. Mortalin-based cytoplasmic sequestration of p53 in a nonmammalian cancer model. Am. J. Pathol. 168:1526-1530.
White, E. 1996. Life, death, and the pursuit of apoptosis. Gene. Dev. 10:1-15.
Zizzo, N., P. G. Tiscar & A. Troncone. 1991. Neoplasia in mitili (Mytilus galloprovincialis). Bol. Soc. Ital. Patol. Ittica 7:19-21.
EVE GALIMANY (1) * AND INKE SUNILA (2)
(1) IRTA, Crta. Poble Nou s/n, St. Carles de la Rapita 43540, Spain; (2) State of Connecticut, Department of Agriculture, Bureau of Aquaculture, P.O. Box 97, Milford, Connecticut 06460
* Corresponding author: E-mail: firstname.lastname@example.org