Seasonal aspects of sarcomatous neoplasia in Mya arenaria (soft-shell clam) from Long Island Sound

JOURNAL

OF INVERTEBRATE

Seasonal

PATHOLOGY

50,

269-276 (1987)

Aspects of Sarcomatous Neoplasia in Mya arenaria (Soft-Shell Clam) from Long Island Sound DIANE J. BROUSSEAU Department

of Biology,

Fairfield

University.

Fairfield,

Connecticut

06430

Received March 11, 1987; accepted June 22, 1987 Mya arenaria were collected monthly for 2.5 years from three populations in Long Island Sound. Histopathological examination revealed that 6.1% of the clams from Stonington, Connecticut, 12.9% of the clams from the Saugatuck River, Westport, Connecticut, and 12.7% of those from Old Mill Beach, Westport, also in Connecticut, had sarcomatous neoplasms. This is the first documented account of the occurrence of clam neoplasm in populations from this geographic area. Peak prevalences of 45, 59, and 60%. respectively, were found in clams from the three study sites. The prevalence of neoplasms in clams collected from three epizootic areas showed a pronounced seasonal pattern, with the highest incidences occurring in the late fall-winter of each year studied. The regular, seasonal occurrence of neoplasia in field populations does not support the hypothesis that pollution alone is the cause of the disorder. Q 1987 Academic PRESS. IK. KEY WORDS: Mya arenaria; sarcomatous neoplasia; Long Island Sound; prevalence; seasons.

(1977) described two types of neoplasms in clams from Maine. One, similar to that deThe frequency of reports in neoplasia in scribed by Farley (1969a), they called “hebivalve molluscs has increased dramatimatopoietic neoplasm” because of the gencally in the past 15 years. Since Farley eral similarity of neoplastic cells and hemo(1969a) first suggested that proliferative cytes, and because of its occurrence in disorders characterized by increased vascular spaces. Since the origin of these numbers of “leukemia-like” cells in the neoplastic cells has not been determined tissues and organs of these animals was a (Brown et al., 1977), and since some mononeoplasm, there have been reports of its clonal antibodies developed against neooccurrence in 11 species of bivalves: four plastic cells do not cross react with normal species of oysters (Wolf, 1969; Farley, hemocytes (Farley et al., 1986), Farley and 1969b; Frierman, 1976; Mix et al., 1977), co-workers prefer the term “sarcoma” for six species of clams (Barry and Yevich, this disease. The other neoplasm arises 1972; Christensen et al., 1974; Brown et from the germinal epithelium and is apparal., 1976; Farley, 1976; Mix et al., 1977), ently of gonadal origin. Interestingly, to and one species of mussel (Farley, 1969a, date gonadal neoplasms have not been 1976; Cosson-Mannevy et al., 1984). The found in southern New England in the general prevalence of the disease combined United States, whereas both types have with recent data indicating an increased been found in northern New England. prevalence of neoplasia in populations in For the most part, sampling for neoplasia Chesapeake Bay (Farley et al., 1986) is has been irregular and restricted to the last causing growing concern among those con- several years and to certain geographic nected with the shellfish industry. areas, making it difficult to determine One species reported to be particularly whether spatial and temporal patterns of susceptible to neoplastic disease is the neoplasia in M. arenaria exist. In those commercially valuable clam Mya arenaria studies carried out over a time period suffi(Brown et al., 1977). Yevich and Barszcz cient to provide evidence for a seasonal INTRODUCTION

269 0022-201 l/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All tights of reproduction in any form reserved.

270

DIANE .I. BROUSSEAU

FIG. 1. Map showing locations of the three study sites: Barn Island in Stonington. CT (A), Saugatuck River in Westport. CT (B), and Old Mill Beach in Westport, CT (C).

pattern of neoplasia, however, neoplasms appear to be most common during the winter months (Cooper et al., 1982; Farley et al., 1986). The purpose of this long-term study was to determine (1) if neoplasia exists in populations of the soft-shell clam, M. arenaria, in Long Island Sound, (2) the prevalence of such occurrences if they do exist, and (3) the annual and seasonal incidences of the condition. MATERIALS

AND METHODS

Monthly collections of M. arenaria were made from three intertidal mudflats, one located at Barn Island in Stonington (lat. 41”21’N; long. 71”53’W), the second in the Saugatuck River in Westport (lat. 41”06’N; long. 73”23’W), and the third at Old Mill Beach in Westport (lat. 41”07’N; long.

73”21’W), all in Connecticut (Fig. 1). Sampling at the Stonington site began in June 1983 and at the Westport sites in March 1984. The study was completed in December 1985. A total of 3963 clams, 19.0-94.5 mm shell length, were examined. In the laboratory, each clam was shucked and the visceral mass (gonad, digestive gland, and gastrointestinal tract) was removed and fixed in 10% buffered formalin. The M. arenaria tissues were then prepared histologically for examination according to the method of Brousseau (1978). Microscopical examination of the stained tissues was made to determine whether neoplastic disorders were present in clams from the three study sites. Diagnosis of histologic sections is reliable for the detection of severity levels of 0. I- 1.O neoplastic cell to hemocyte ratios (stages 3-5) (Farley et al., 1986).

FIG. 2. Gonadal tissue of a female Myu arenaria showing infiltration by neoplastic cells in connective tissue between the follicles. f, Follicle; o. oocyte; n, neoplastic cell. x 110. FIG. 3. Section through the intestine of A4ya urenuria showing extensive infiltration by atypical cells. n, Neoplastic cell. x 110. FIG. 4. Digestive gland of Myu urenariu showing infiltration of connective tissue between the tubules and ducts by neoplastic cells. dt, Digestive tubule; n, neoplastic cell. x 110. FIG. 5. Gonadal tissue of Mya urenuriu showing evidence of an advanced sarcomatoid condition. Note the reduced size of the gonadal follicle. f, Follicle. x I IO.

SEASONAL

ASPECTS OF NEOPLASIA

IN Mya

271

272

DIANE J. BROUSSEAU

TABLE 1 PREVALENCESOFSARCOMATOUSNEOPLASIAIN arenaria FROMTHREEPOPULATIONSIN LONGISLANDSOUND Site Barn Island, Stonington, CT Saugatuck River, Westport, CT Old Mill Beach, Westport. CT

Sample size

Number neoplastic

Mya

TABLE 2 INCDENCESOFSARCOMATOUSNEOPLASMSINTHE POPULATIONOF Mya arenaria FROMBARNISLAND, STONINGTON.CONNECTICUT

Percentage neoplastic

1621

99

6.1

1267

164

12.9

107.5

136

12.7

RESULTS

Histological examination revealed the presence of sarcomatous neoplastic cells in clams collected from each of the study sites. This condition is characterized by the infiltration and replacement of vesicular connective tissue in the visceral mass, by enlarged, atypical, mitotically active cells, usually round or oval in shape. The early lesions appear to develop multifocally as local neoplasms in connective tissue (Fig. 2). In animals with early lesions, normal gametogenesis appears to be occurring. As the disease state advances, infiltration of neoplastic cells extends throughout the connective tissues of the digestive tract, digestive gland, kidney, and gonad (Figs. 3, 4). In some cases, the disorder was extensive enough to cause reduction in the size and appearance of digestive tubules, as well as alteration and loss of germinal follicles in the gonad (Fig. 5). Such extensive invasion may eventually lead to loss of sexual characteristics and the arrest of gametogenic development. There was no evidence of gonadal neoplasia in the three populations of M. arenaria studied here, nor was there any evidence of gross disorders, such as tumors, present in any of the examined clams. Of the 3963 soft-shell clams examined histologically, 399 were diagnosed as neoplastic. This is the first published report of this disease occurring in M. arenaria from this geographic area. The prevalence of

Collection date June 7, 1983 June 13, 1983 June 24, 1983 July 12, 1983 July 23, 1983 August 8, 1983 August 24, 1983 September 9, 1983 October 7, 1983 November 6, 1983 December 4, 1983 January 18, 1984 February 15, 1984 March 14, 1984 April 14, 1984 May 17, 1984 June 8, 1984 July 12, 1984 August 21, 1984 September 26, 1984 October 21, 1984 November 21, 1984 December 20. 1984 January 20. 1985 February 20, 1985 March 21, 1985 April 19, 1985 May 5. 1985 June 3. 1985 July 16, 1985 August 15, 1985 September 3, 1985 October 14, 1985 October 21, 1985 November 8, 1985 December 14, 1985 Total

Sample size

Number neoplastic

% Neoplastic

30 45 36 30 19 28 24 30 42 30 28 29 33 30 39 65 197 29 29 29 30 29 29 30 29 30 200 98 48 29 30 59 29 70 29 30

1 1 4 0 0 0 2 0 4 12 8 13 9 3 3 3 5 0 0 0 2 3 5 0 1 0 9 0 2 0 0 0 6 1 1 1

3.3 2.2 11.1 0.0 0.0 0.0 8.3 0.0 9.5 40.0 28.6 44.8 27.3 10.0 7.7 4.6 2.5 0.0 0.0 0.0 6.7 10.3 17.2 0.0 3.4 0.0 4.5 0.0 4.2 0.0 0.0 0.0 20.7 1.4 3.4 3.3

1621

99

6.1

neoplasia, however, varied among the three sites. The Barn Island, Stonington population had an overall frequency of occurrence of 6.1% for the period June 1983 to December 1985, whereas the incidence of neoplasia from March 1984 to December 1985 in both Westport populations was approximately twice as high (Table 1). Peak prevalences of 45, 59, and 60% were re-

SEASONAL

ASPECTS

TABLE 3 INCIDENCES OF SARCOMATOUS NEOPLASIA IN THE POPULATION OF Mya arenaria FROM THE SAUGATUCK RIVER, WESTPORT, CONNECTICUT Collection date March 1, 1984 April 5, 1984 May 3, 1984 May 29, 1984 June 22. 1984 June 25. 1984 July 18, 1984 August 9, 1984 September 19. 1984 October 8, 1984 November 20, 1984 December 19. 1984 January 29. 1985 February 18, 1985 March 20. 1985 April 17, 1985 May 3. 1985 June 4, 1985 July 14, 1985 August 7. 1985 September 10. 1985 October 9, 1985 November 8, 1985 December 11, 1985 Total

OF NEOPLASIA

TABLE 4 INCIDENCES OF SARCOMATOUS NEOPLASIA IN THE POPULATION OF Mya arenaria FROM OLD MILL BEACH, WESTPORT, CONNECTICUT

%

Sample size

Number neoplastic

30 29 35 197 29 133 30 29 30 30 30 30 30 30 30 198 91 50 29 30 60 28 30 29

17 17 6 15 2 12 1 0 2 4 4 5 6 5 3 39 9 5 0 I 3 1 3 4

56.7 58.6 17.1 7.6 6.9 9.0 3.3 0.0 6.7 13.3 13.3 16.7 20.0 16.7 10.0 19.7 9.9 10.0 0.0 3.3 5.0 3.6 10.0 13.8

1267

164

12.9

273

IN Mya

Collection date

Neoplastic

Sample size

Number neoplastic

28 30 194 28 28 27 28 30 27 30 30 30 29 190 98 50 29 20 59 30 30 30

11 5 25 0 0 0 0 2 8 4 18 4 8 28 4 8 1 0 5 3 2 0

39.3 16.7 12.9 0.0 0.0 0.0 0.0 6.7 29.6 13.3 60.0 13.3 27.6 14.7 4.1 16.0 3.4 0.0 8.5 10.0 6.7 0.0

1075

136

12.7

March 24, 1984 April 25, 1984 May 24, 1984 June 22, 1984 July 18. 1984 August 10, 1984 September 18, 1984 October 21, 1984 November 19. 1984 December 18, 1984 January 19, 1985 February 17. 1985 March 19, 1985 April 18, 1985 May 7. 1985 June 6, 1985 July 13. 1985 August 7, 1985 September 10, 1985 October 9. 1985 November 7, 1985 December 1 I, 1985 Total

cases also reported year (Fig. 8). ported for the Barn Island, Saugatuck River, and Old Mill Beach sites, respectively (Tables 2, 3, 4). Both annual and seasonal variations in the prevalence of neoplasms in M. arenaria from the three populations were apparent. In the Stonington population, peak prevalence occurred in late fall-winter of each year studied. Incidences, however, were significantly higher during that period in 1983-1984 than in either fall-winter of 1984-1985 or fall of 1985 (Fig. 6). Similarly, neoplasia at the Saugatuck River site appeared to be most severe during the winter of 1984 (Fig. 7). At the Old Mill Beach site, on the other hand, the highest incidences of neoplasia were reported for the fall-winter of 1984-1985, despite the large number of

during

9% Neoplastic

the previous

DISCUSSION

Data on the prevalence of sarcomatous neoplasia in populations of M. arenaria indicate that it has reached epizootic levels (prevalence of > 1%) in some mid-Atlantic and New England states. Long-term periodic surveys of soft-shall clams in the Chesapeake Bay has shown that clam sarcomas first appeared in that area in 1979. Infrequent cases were reported from 1979 through most of 1983; in late 1983 prevalences of over 40% were detected (Farley, 1987). Cooper et al. (1982) reported prevalences of 20 to 40% in soft-shell clam populations in Allen Harbor, Rhode Island, sampled nearly monthly between July 1977 and March 1979. During a 1976 survey of

274

DIANE J. BROUSSEAU

100 90.. so-.

90

1983 Ii 1 49

52

30

42

30

28

70..

70

so-

1984 30

80

29 232

162

30

29 30

30

30

30

1

50.~ 40.. 30.-

90 100

Y L < 2 0 y

80.

1984

29

33

30 39

65 197 29

29 29

30

29

29

1

70. so-so+

70oo50 ‘lo-. 30..

.U”

go.8o..

1985 30

29

30

200

98

48

29

30

59

99

29

30

70.. 60 50 .’ 40

MONTHS

7. Monthly prevalences of hematopoietic neoplasia in the population of Mya arenuria from Saugatuck River, Westport, CT. Sample sizes are given at the top of the bar for each month. FIG.

30.~ 20 ‘.

FIG. 6. Monthly prevalences of hematopoietic neoplasia in the population of Mya arenaria from Barn Island, Stonington, CT. Sample sizes are given at the top of the bar for each month.

New England clams, Brown et al. (1977) found neoplasia in six populations in Rhode Island, one in Massachusetts and three in Maine. Prevalences as high as 64% were observed. In 1982 and 1983, prevalences of IO-90% were reported for M. arenaria from New Bedford Harbor, Massachusetts (Reinisch et al., 1984). High prevalences of the disorder in the three populations of M. arenaria studied here suggest epizootic levels of neoplasia in Long Island Sound. Monthly prevalences of up to 45% for the Stonington population and 60% for the two Westport ones are comparable to the highest incidences re-

ported for other locations, especially since the histological method used in this study is reported to be the most conservative of the diagnostic techniques currently in use (Farley et al., 1986). Although the prevalence of neoplasms in M. arenaria differ among sites in Long Island Sound, as they do elsewhere, the results of this study suggest there is a seasonal aspect to its occurrence. High prevalences of the condition, first noted in the late fall, persisted through the winter months and gradually subsided in early spring. In studies of M. arenariu populations from Rhode Island (Cooper et al., 1982) and the Chesapeake Bay (Farley, 1987) a similar pattern was reported. Farley (1969a) and Farley and Sparks (1970) reported that peak prevalence of hematopoietic neoplasms in the blue mussel, Mytilus edufis, occurred in December and that advanced cases were most common in the early winter. Christensen et al. (1974) indi-

SEASONAL

ASPECTS OF NEOPLASIA

FIG. 8. Monthly prevalences of hematopoietic neoplasia in the population of Mya arenaria from Old Mill Beach, Westport, CT. Sample sizes are given at the top of the bar for each month.

Recent studies of the clinical course of this disorder have demonstrated that under laboratory conditions, the disease progressed from early to advanced stages and resulted in the death of all sarcomatous clams in 5 months (Farley, 1987). The etiology of the observed condition, however, remains unresolved. Possible links to environmental pollution have been suggested (Yevich and Barszcz, 1977; Harshbarger, 1977; Brown et al., 1979; Reinisch et al., 1984); however, no direct correlation between prevalence of neoplasia and the type or degree of environmental pollution has been demonstrated. Attention has turned to the possibility that biologic agents are responsible for molluscan neoplasm. Brown (1980) demonstrated transmissibility by a water-borne agent, whereas experimental studies by Oprandy et al. (1981) and

275

Oprandy and Chang (1983) suggested that the agent was filterable. The regular, seasonal pattern of the disorder apparent in this study suggests a biologic agent; however, it is impossible to rule out physical or chemical agents or the involvement of adverse water quality. The seasonal pattern of occurrence observed in this study suggests areas for future investigations. Any one or a combination of the following hypotheses can be presented to explain the higher reported incidences of neoplasia in the winter months: (1) the disappearance of the causal/infective agent during the summer months, (2) remission of the disease as implied by Cooper et al. (1982), or (3) increased mortality of neoplastic individuals in the population as suggested by the laboratory studies conducted by Farley et al. (1986), resulting in the survival of resistant or noninfected clams. Further attempts to better understand these aspects of molluscan neoplasia and how they are operating in field populations are needed.

cated winter seasonality for a gill sarcoma described in the duck clam, Macoma balthica.

IN Mya

ACKNOWLEDGMENki I thank C. A. Farley for his critical review of the manuscript.

REFERENCES BARRY, M. M., AND YEVICH, P. P. 1972. Incidence of gonadal cancer in the quahog, Mercenaria mercenaria.

Oncogenesis,

26, 96-97.

BROUSSEAU. D. J. 1978. Spawning cycle, fecundity and recruitment in a population of soft-shell clam, Mya arenaria from Cape Ann, Massachusetts. Fish. Bull., 76, 155- 166. BROWN, R. S. 1980. The value of the multidisciplinary approach to research on marine pollution effects as evidenced in a three-year study to determine the etiology and pathogenesis of neoplasia in the soft-shell clam. Mya arenaria. Rapp. P. V. Rem. Cons. Inr. Explor. Mer, 179, 125-128. BROWN, R. S., WOLKE, R. E., BROWN, C. W., AND SAILA, S. B. 1979. Hydrocarbon pollution and the prevalence of neoplasia in New England soft-shell clams (Mya arenaria). In “Animals as Monitors of Environmental Pollutants,” pp. 41-51. Natl. Acad. Sci.. Washington, D.C.

276

DIANE

J. BROUSSEAU

BROWN, R. S., WOLKE, R. E., AND SAILA, S. B. 1976. A preliminary report on neoplasia in feral populations of the soft shell clam, Mya arenaria: Prevalence, histopathology and diagnosis. In “Proceedings First Int. Colloqu. Invertebr. Pathol.,” pp. 151-158. Queens Univ., Kingston, Ontario. BROWN, R. S., WOLKE, R. E., SAILA, S. B., AND BROWN, C. W. 1977. Prevalence of neoplasia in 10 New England populations of the soft-shell clam (Myu

arenaria).

Ann.

N.Y.

Acad.

Sri.,

298,

522-534. CHRISTENSEN, D. J., FARLEY, C. A., AND KERN, F. G. 1974. Epizootic neoplasms in the clam Macorna balthica (L.) from the Chesapeake Bay. J. Natl.

Cancer

Inst.,

52, 1739-

1749.

COOPER, K. R.. BROWN, R. S.. AND CHANG, P. W. 1982. The course and mortality of a hematopoietic neoplasm in the soft-shell clam, Mya arenaria. J. Invertebr.

Pathol.,

39, 149- 157.

COSSON-MANNEVY, M. A., WONG, C. S., AND CRETNEY. W. J. 1984. Putative neoplastic disorders in mussels (Mytilus edulis) from southern Vancouver Island waters, British Columbia. J. Invertebr. Pathol., 44, 151-160. FARLEY, C. A. 1969a. Sarcomatoid proliferative disease in a wild population of blue mussels Mytilus edulis. J. Nutl. Cancer Inst.. 43, 5099516. FARLEY, C. A. 1969b. Probable neoplastic disease of the hematopoietic system in oysters, Crassostrea virginica and Crassostrea gigas. Nail. Cancer Inst. Monogr.,

31, 541-555.

FARLEY, C. A. 1976. Proliferative disorders in bivalve molluscs. Mar. Fish. Rev., 38, 30-33. FARLEY, C. A. 1987. Selected aspects of neoplastic progression in mollusks. In “Progressive Stages of Malignant Neoplastic Growth,” Vol. 1, “Fundamental Aspects of Neoplastic Progression” (H. E. Kaiser, Ed.). Nijhoff, New York. FARLEY, C. A., Oreo. S. V.. AND REINISCH, C. L. 1986. New occurrence of epizootic sarcoma in

Chesapeake Bay soft-shell clams (Mya arenaria). Bull., 84, 851-857. FARLEY, C. A. AND SPARKS, A. K. 1970. Proliferative diseases of haemocytes, endothelial cells and connective tissue cells in molluscks. Bibi. Haemotol., 36, 610-617. FRIERMAN, E. M. 1976. Occurrence of hematopoietic neoplasms in Virginia oysters (Crassostrea virFish.

ginica).

Mar.

Fish.

Rev.,

38, 34-36.

HARSHBARGER, J. C., OTTO, S. V., AND CHANG, S. C. 1977. Proliferative disorders in Crassostrea virginica and Mya arenariu from Chesapeake Bay and intranuclear virus-like inclusions in Mya arenaria with germanomas from a Maine oil spill site. Haliotis.

8, 243-248.

MIX, M. C., PRIBBLE, H. J., RILEY, R. J.. AND ToMASOVIC. S. P. 1977. Neoplastic disease in bivalve molluscs from Oregon estuaries with emphasis on research on proliferative disorders in Yaquina Bay oysters. Ann. N. Y. Acad. Sci.. 298, 356-373. OPRANDY, J. J.. AND CHANG, l? W 1983. 5-Bromodeoxyuridine induction of hematopoietic neoplasia and retrovirus activation in the soft-shell clam, Mya arenaria. J. Invertebr. Pathol.. 42, 196-206. OPRANDY. J. J., CHANG, P. W., PRONOVOST, A. D., COOPER, K. R., BROWN, R. S., AND YATES, V. J. 1981. Isolation of a viral agent causing hematopoietic neoplasia in the soft-shell clam, Mya arenaria.

J. Imvertebr.

Pathol.,

38, 45-51.

REINISCH, C. L., CHARLES, A. M., AND STONE, A. M. 1984. Epizootic neoplasia in soft shell clams collected from New Bedford Harbor. Hazard. Waste, 1, 73-81. WOLF, P. H. 1969. Neoplastic growth in two Sydney rock oysters, Crassostrea commercialis. Natl. Cancer

Inst.

Monogr.,

31, 563-573.

YEVICH, P. P., AND BARSZCZ, C. A. 1977. Neoplasia in soft-shell clams (Mya arenariu) collected from oil-impacted sites. Ann. N.Y. Acad. Sci.. 298, 409-426.