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The course and mortality of a hematopoietic neoplasm in the soft-shell clam, Mya arenaria
JOURNAL
OF
INVERTEBRATE
The Course KEITH Department
PATHOLOGY
39,
and Mortality Soft-Shell R. COOPER,* of Aquaculture
149-
(1982)
157
of a Hematopoietic Clam, Mya arenarial S. BROWN,
ROBERT Science Kingston,
Neoplasm
in the
AND PEI W. CHANG
and Pathology, University Rhode Island 02881
of Rhode
Island.
Received January 12, 1981; accepted June 29, 1981 Results from two experiments containing approximately 280 Mya arenaria indicated that significantly higher (P < 0.05) mortality occurred within the neoplastic clam population than in the nonneoplastic clam population. Using an in vivo blood cytological technique, five levels of severity were established. The levels were based on the number of neoplastic cells in the circulation with level 1 as the lowest severity and level 5 as the highest severity. Neoplastic clams that were diagnosed as the lowest level had higher survival rates (60%) than those clams diagnosed as the highest level (0%). The hematopoietic neoplasm in M. arenaria followed one of three courses: (1) in approximately 50% of the neoplastic clams the disease progressed to a higher severity and resulted in death: (2) in approximately 40% of the neoplastic clams the disease was chronic, i.e., remained at a stable level: and (3) in approximately 10% of the neoplastic clams the disease diminished in severity or disappeared entirely. In addition, 10% of the clams diagnosed as nonneoplastic at the beginning of the experiments were neoplastic by the termination of the experiment. The contraction of the disease may have been de novo or by stimulation of latent infections. The prevalence of the neoplasm in clams collected from an epizootic area followed a biphasic seasonal pattern. The highest prevalences occurred in October, November, and in May. The hematopoietic neoplasm in M. arenaria was also age and species specific. KEY WORDS: Mya arenaria: hematopoietic neoplasm, course, mortality, seasons, prevalence.
INTRODUCTION
A causal relationship between molluscan neoplasms and death has been demonstrated for Macoma balthica (Christensen et al., 1974) and for Crassostrea virginica (Frierman, 1976). A hematopoietic neoplasm in Mya arenaria was believed to be malignant based on cytologic characteristics and the increased death rate among neoplastic clams (Brown et al., 1977). This report is an extension of the previous work. Laboratory experiments were designed to address two questions: (1) were
neoplastic clams at a higher risk of death than nonneoplastic clams and (2) did the neoplasm always progress and result in death? Feral population studies were designed to examine the progression of the disease and the seasonal and age effects. All of these factors are important in understanding the epizootiology and the effect of neoplasia on a clam population. Our results corroborate the findings of Brown et al. (1977) and raise questions concerning the sequelae of the disease. MATERIALS
AND METHODS
Bleeding and collection of hemolymph.
’ This study was supported in part by a grant from the American Petroleum Institute. Contribution No. 1973 of the Rhode Island Agricultural Experiment Station. p To whom all correspondence and reprint requests should be addressed. Current address: Keith R. Cooper, Department of Pharmacology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pa. 19107.
Hemolymph from the posterior adductor muscle was drawn into a l-ml tuberculin syringe with a 26-gauge needle. Blood cytological techniques were routinely used to diagnose the presence of neoplastic cells in the hemolymph and their level of severity of the neoplasm (Cooper, 1979). Hemo149 0022-2011/82/020149-09$01.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
150
COOPER,
BROWN,
cytes were diagnosed for neoplasia using phase-contrast optics on fresh hemolymph samples and brightfield microscopy on methanol-Giemsa-stained blood smears. The level of disease was based on the number of circulating neoplastic cells in a O.l-ml hemolymph sample. Five levels were established based on the number of neoplastic cells in the blood with level 1 as the lowest severity and level 5 as the highest severity. The limits were arbitrarily set based on a logarithmic progression. Level 1 clams had s lo4 neoplastic cells/ml, while level 5 clams had ~10’ neoplastic cells/ml (Cooper, 1979). Histopathological procedures. All the clams were shucked and examined for gross lesions. Clam tissues were preserved in Dietrich’s solution (Armed Forces Institute of Pathology Manual, 3rd edition, p. 4, formol acetic acid). A longitudinal section of the siphon and a cross section, from the heart to the foot were dehydrated in alcohol and xylene, embedded in paraffin, sectioned at 6 pm, and stained with hematoxylin and eosin.
AND
CHANG
tissues processed for histology. This study will be referred to as experiment E-l within the text of this paper. Experiment E-2 was designed to duplicate E-l and to more closely examine both the progression and the remission of the disease. The clams were collected from Allen Harbor, Rhode Island, on September 28, 1978. Clams were bled and diagnosed on two separate occasions. Only clams with the same diagnosis for the two bleedings were used for the experiment. This minimized the probability of a wrong diagnosis. Thirty clams were classified as having a 1 or 2 level of severity of neoplasia and 31 clams were diagnosed at a 3 level. After 3 and 6 months in the flowing-seawater system, the clams were again bled and diagnosed. Experiment E-2 was terminated on March 26, 1979 (6 months), and all surviving clams were processed for histopathology. Field experiments. M. arenaria from Allen Harbor, Rhode Island, were collected on a monthly basis for a period of 20 months, beginning in July of 1977 and endLaboratory experiments. M. arenaria ing in March of 1979. Before being prowere collected from intertidal zones located cessed for histopathologic examination, the at Allen Harbor, Rhode Island. Ninety-one clams were allowed to depurate sediments clams were collected on September 27, accumulated during collection. Approxi1977. Following a single bleeding, 36 clams mately 1700 clams were diagnosed by hiswere diagnosed neoplastic. Over a lo- topathological method, while the remaining 1800 were diagnosed by the blood cytologimonth period, October 1977 to July 1978, the animals were maintained in a flowingcal techniques (see Materials and Methods, seawater system and monitored for mortalBleeding and collection of hemolymph). ity twice weekly. The temperature in the During the spring of 1977 a large clam set holding tanks for June and July was at or occurred within the study area. Collection near the maximal lethal tolerable temperaof juvenile clams from this spawning began ture for M. arenaria and caused the termiSeptember 1977 and continued until Ocnation of the planned 1Zmonth study at 10 tober 1978. The established population of months. Clams were bled at monthly interclams present prior to the spring of 1977 vals to determine changes in the disease se- was distinguished from the imported verity. Clams that died were examined juvenile clams by their size and vertical histopathologically. To determine the pos- distribution in the sediment. In this paper sible effect of multiple bleedings on the the established population of clams will be clams an additional 100 clams were not bled referred to as the adult population. and were monitored twice weekly for morFive other species of bivalve mollusks tality. At the termination of the study all present at Allen Harbor were also examsurviving clams were sacrificed and their ined for neoplasms: Mercenaria mer-
COURSE OF HEMATOPOIETIC
cenaria (N = 97), Macoma balthica (N = lOl), Geukensis dimusus (N = llO), Petricola pholadifoimis (N = 7), and Ensis minor (N = 3). The x2 square test for sig-
nificance was used to analyze the data. RESULTS Laboratory
Experiments
In experiments E-l and E-2, significantly higher mortality (x2 P < 0.05) occurred in the neoplastic clams than in the nonneoplastic clams (Fig. 1). The survival curves could be divided into two phases: (1) an initial steep phase and (2) a plateau period. The E-l survival curve had a second steep E-l
60
I OII
OC~.LIOV.DCC. 1977
, 0
Oct.
Jan.
Feb. E-2
,
Nov.Oec. 1978
,
,
Jan.
Feb.
TIME
IOlr.Apr.my li78
,
,
Itm.Apr. 1979
Jun.
Jul.
, my
(monthm)
FIG. 1. Percentage of clams surviving over time for experiments E-l and E-2. E-l began 9-27-77 and terminated 7-27-78. E-2 began 9-28-78 and terminated 3-26-79. In experiment E-l June and July temperatures in the holding tanks approached maximum tolerable temperature levels for Mya orennrin and accounted for the high mortality during that time. Neoplastic clams of all levels of severity, 0; neoplastic clams of levels of severity 1 and 2, A; neoplastic clams of levels of severity 3, n ; nonneoplastic clams, 0.
NEOPLASM
IN Myo
151
phase which occurred during June and July. We believe that this second phase resulted from elevated water temperatures and not as a result of any disease process. This hypothesis is based on the parallel appearance of the survival curves for neoplastic and nonneoplastic animals during this phase (Fig. 1, E-l), and the inability to correlate any visible lesions with the deaths of the nonneoplastic clams. Because of this unusually large losses of clams during the months of June and July, a second study (E-2) was designed to continue for 6 months during which time the temperature in the holding tanks remained at acceptable levels. As reported by Brown et al. (1977) .we found no effect of bleeding on the survival of clams. The majority of neoplastic clam deaths occurred during the first 2 months of both experiments. The clams that died during this period were those clams at the highest levels of severity and those in which the neoplasm had progressed and affected vital organ systems. Deaths of these animals are believed to result from displacement of normal organ tissue with neoplastic cells. In E-l and E-2, 16 of the 29 neoplastic clams that died and 8 of the 16 nonneoplastic clams that died were necropsied and examined histologically. An advanced hematopoietic neoplasm was found in all 16 of the blood diagnosed neoplastic clams, while the 8 nonneoplastic clams showed no significant lesion. Clams diagnosed by the blood cytological technique as having a low disease severity had higher survival rates than clams at more severe levels; level 1 neoplastic clams had 60% survival rate; level 2 neoplastic clams had 54% survival rate; level 3 neoplastic clams had 25% survival rate, and levels 4 and 5 neoplastic clams had 0% survival rate (Table 1, E-l). The life expectancy of a clam under these experimental conditions was s 3 months for clams diagnosed at a level 5 and ~6.5 months for clams diagnosed at a levels 3 and 4 (Table 1, footnote a). Following the steep phase on the survival
152
COOPER, BROWN, TABLE MORTALITY OF NEOPLASTICAND
AND CHANG 1 NONNEOPLASTIC
CLAMS
Number of clams Experiment E-lb
E-2’
Status
of clams
Nonneoplastic Neoplastic Level 1 Level 2 Level 3 Level 4 Level 5 Neoplastic (cumulative) Unknown’ Nonneoplastic Neoplastic Level 1 and 2 Level 3 Neoplastic (cumulative)
Initial condition
Mortality”
Percentage of survival
55
16
71
10 13 4 6 3 36 100
4 6 3 6 3 21 31”
60 54 25 0 0 41 69
30
0
100
31 31 62
1 7 8
96 77 87
a The average time to death for nonneoplastic clams was 9.4 months, neoplastic clams at levels 1 and 2 (combined) was 7.7 months, at levels 3 and 4 (combined) was 6.5 months, and level 5 was 3 months. DExperiment E-l began on Sept. 27, 1977 and terminated July 27, 1978. V Clam status was unknown because they were not bled. ’ Monthly bleeding had no apparent effect on mortality when compared to clams not bled (x2, P > 0.05). (I Experiment E-2 began on Sept. 28, 1978 and terminated March 26, 1979.
curve during October, November, and December is a plateau period in which few if any deaths occurred (Fig. 1). The surviving neoplastic clams were those clams diagnosed at the lower levels of the disease, i.e., levels 1, 2, and 3 which had not progressed to a lethal stage. At these levels the hematopoietic neoplasm followed one of three courses: (1) remained at a stable level, (2) progressed to a higher level, or (3) diminished in severity and/or disappeared. In experiment E-l, 44% (16/36) of the neoplastic clams increased in severity, 44% (16/36) of the neoplastic clams remained at the same level, and 12% (4/36) of the neoplastic clams had apparent remission of the disease (Table 2, E-l). Remission occurred at the low severity levels. In experiment E-2, 38% (23/61) of the neoplastic animals had remained at the same level, 43% (26/61) of the neoplastic animals had progressed to a higher level, and 20% (12/61) of the neoplastic animals had undergone apparent remission (Table 2, E-2). At 3 months, none of the level 3 clams had undergone remis-
sion, but at 6 months, two clams showed no signs of neoplastic cells either in fresh blood or in tissue section. It is also of interest, that 7 of the 31 clams initially diagnosed as level 3 were at level 1 by the termination of the experiment. All cases of remission were corroborated by tissue diagnosis. Also, 10% of the clams diagnosed as negative at the beginning of the experiment were positive by the termination of the experiment (Table 2). Feral Population Study
Seasonal variations in the prevalence of the hematopoietic neoplasm were observed in the adult M. arenaria collected from Allen Harbor, Rhode Island. The disease occurred throughout the year. The lowest percentage of neoplasia was 20% during the months of May, July, and August 1978 (Table 3). The highest prevalences occurred in the fall (43%) and late spring (43%). Following the peak in the fall, the percentage of neoplasia declined until February when the prevalence began to rise. After the sec-
COURSE
OF
HEMATOPOIETIC
NEOPLASM
TABLE COURSE
IN
2
OF DISEASE
IN CLAMS
Number of clams Experiment E-l”
E-2”
Status of clams
153
Myu
Progression of disease
At start of experiment
Same level
Remission
Nonneoplastic Neoplastic Level 1 Level 2 Level 3 Level 4 Level 5 Neoplastic (cumulative)
5.5
50
10 13 4 6 3 36
6 3 1 3 3 16
Nonneoplastic Neoplastic Levels 1 and 2 Level 3 Neoplastic (cumulative)
30
27
-
31 30 61
10 13’ 23
10 2 12
Increased 5
2 2 0 0 0 4
2 8 3 3 16 3 11 15 26
” Experiment E-l began on Sept. 27, 1977 and terminated on July 27, 1978. D Experiment E-2 began on Sept. 28, 1978 and terminated on March 26, 1979. C Of the 13 neoplastic clams that remained at level 3, 7 of them actually decreased in severity to level 1 at the end of the experiment, indicating that given more time they might have undergone remission.
TABLE PREVALENCE
Date of collection
3
OF HEMATOPOIETIC NEOPLASIA IN FERAL CLAMS FROM ALLEN HARBOR, AT DIFFERENT MONTHS, IN THE YEARS 1977- 1979
No. of clams collected
Percentage neoplastic
Percentage at each level of severity 1
2
3
4
5
RHODE
Sum percentages of neoplasia levels of severity 4 and 5
1977 July 2 Aug 27 Sept 27 Ott 27 Nov 27 Dee 10
61 98 143 150 41 136
28 19 23 43 39 40
36 25 16 42 30 18 18 26 31 20 25 27.5
32 7 32 10 13 26 21 21 13 18 10 27.5
0 0 13 14 18 10
7 10 39 35 36 37.5
1978 Feb 23 April 10 May 11 June 1 July 20 Aug 25 Sept 28 Nov 5 Nov 28
100 108 158 315 327 157 474 30 189
22 43 20 22 20 20 33 37 43
14 19 20 32 60 30 45 19 30
18 18 10 9 20 25 1.5 19 12
23 23 30 23 0 5 6 0 18
27 28 20 13 0 10 9 35 28
50 51 50 33 0 15 15 35 46
1979 Jan 9 Feb 28 March 28
100 176 181
31 24 34
42 10 12 25 29 15
16 13 12 9 18 6
19 42 32
32 51 38
18 12 20 23 20 30 25 27 12
ISLAND,
154
COOPER,
BROWN,
AND
CHANG
ond peak the prevalence again declined and plastic clams of levels 4 and 5 are at the remained at approximately 20% during the highest point, approximately 50%, the summer months. These trends were con- water temperature reaches the lowest point sistent from year to year (Fig. 2). (>5”C). It appears that low water temperaThe severity of the neoplasms followed a ture is associated with an increase in the seasonal pattern. By summing the per- severity of the neoplasia resulting in a centages of neoplastic clams of levels 4 and larger percentage of levels 4 and 5 neoplas5 at different months of the year and com- tic clams and high water temperature paring the results with the water temperacauses a drop in percentage neoplasia ture, an interesting pattern emerged (Fig. 2, among the levels 4 and 5 clams. Table 3). During July and August when the Neoplastic juvenile clams were found percentage of neoplastic clams of levels 4 throughout the experimental period (Fig. and 5 (7 and 10%; 0 and 15%) are at the 3). The percentage of neoplasia varied from lowest ebb, the water temperature, how- 3 to 12% in the first 9 months of sampling. lower prevalences of neoever, reaches the highest of the year (20°C). Significantly plasia for the first year were observed in the During February, March, April, May, and November when the percentage of neo- juvenile clams than in the adult clams (x2 P < 0.05). There was no biphasic pattern as was present in the adult clams. However, beginning in July, increases in the prevalence of neoplasia occurred (Fig. 3). The highest recorded prevalence was in September 1978 (30%). The corresponding peak in the adult population did not occur until December. The severity levels of the individual neoplastic cases are recorded in 00 Table 4. In the first 9 months there were two cases at a 5 severity level, while five cases were observed in the last 3 months. No neoplasms were found in any of the other five molluscan species sampled from the Allen Harbor area. DISCUSSION
FIG. 2. Percentage of levels of severity, (B) sum severity levels 4 and 5, and temperature. M. arrrmria Harbor, R.I. Two additional and 0, from Brown (1977) age of neoplasia.
neoplasia: (A) sum of all of percentage neoplasia of (C) average monthly water were collected from Allen points, as indicated by 0 were added to the percent-
Epizootic neoplasia has been reported to occur in populations of six molluscan genera: Ostrea. Crassostrea, Mytilus, Mercenaria, Mya, and Macoma, with a wide geographical distribution (Alderman, 1977; Balouet, pers. commun.; Brown et al., 1977; Christensen et al., 1974; Farley, 1969a,b; Farley and Sparks, 1970; Frierman, 1976; Mix, 1975; Yevich and Barszcz, 1976). The neoplasms became apparent in animals collected in September or October and continued through the winter and spring (Farley, 1976). A biphasic increase in prevalence was reported to occur in populations of Macoma balthica (Christensen et
COURSE OF HEMATOPOIETIC
NEOPLASM
155
IN Mya
**.dJ 0
O‘d
b,
II
I
Oct.
Nov.
Dec.
-0”
I
Jan.
I
Feb.
I
Mar.Apr.
I
I
May
I
Jun.
1977
I
Jul.
I
I
I
Aug.Sept.Oct.Nov.
I
I
Dec.
1978 TIME
(months)
FIG. 3. Prevalence of neoplasia in juvenile clams and adult clams collected at the same time and location, Allen Harbor, Rhode Island. Juvenile clams 0: adult clams 0.
al., 1974). We found that a similar seasonal pattern occurred in a hematopoietic neoplasm of adult M. arenaria (Fig. 2). However, the neoplastic condition was present year-round and did not disappear during certain times of the year, as in the case with neoplasms of the other five genera. The highest percentage of neoplasms in M. arenariu occurred in October-November (43%) and in April (43%). Before and after these peak periods the prevalence leveled off to 20% (Fig. 2). The high percentage of neoplasms occurred again in September 28-January 9 of 1978/1979 (Table 3). TABLE SEASONAL
Date of collection 9-27-77 10-27-77 11-21-17 12-10-77 2-26-78 4-10-78 5-11-78 7-l-78 7-20-78 9-6-78 9-28-78 10-28-78
M. arenaria less than a year of age had lower prevalences of neoplasia and did not demonstrate the seasonal variation observed in the adult population (Table 4). However, the juvenile clam population upon reaching the summer of their second year had a similar percentage of neoplastic individuals at severities comparable to the adult population. Frierman (1976) had reported a similar finding in laboratory-reared Crassostrea virginica. Neoplasms appeared in them at 1 year of age with severe mortality. Therefore, juvenile Mya could be a source of new susceptibles. 4
VARIATION OF HEMATOPOIETIC NEOPLASIA IN JUVENILE CLAMS FROM ALLEN HARBOR,RHODE ISLAND
Number of clams Examined 57 57 60 30 60 30 30 30 30 30 30 30
Disease level
Neoplastic 3 2 7 1 2 3 3 2 6 9 8 5
1
2
1 I I 1 1 2 1 2 4 3 4 2
2
3
4
5 1
3
1 1 1 1
3 2
1
2 2 1 1
1 1 2
Percentage neoplastic 5 4 12 4 4 10 10 7 20 30 26 20
156
COOPER,
BROWN,
There are three major observations made in this study concerning the fate of neoplastic clams. These are: (1) the hematopoietic neoplasm, reaching a level of severity of 3 or greater, becomes a progressive disease and results in death in the majority of the cases, (2) the hematopoietic neoplasm can also be a chronic disease in that it remains at a stable level for up to 10 months, (3) at low levels of severity (~3) the hematopoietic neoplasm can diminish in severity and show remission. The cyclic nature of the disease is believed to be due to a complex interaction between the abovementioned factors and the recruitment of susceptible individuals. The decreases in prevalence in May, June, December, and January can be explained by a combination of factors, such as the deaths of animals with severe neoplasms and the remission of animals which had low severity levels. The steady increase in the severity of diseased animals throughout the winter would indicate the progression of the disease. The decrease in the severity of the disease during July and August could be due to the high water temperature (Fig. 2) or to an influx of low-severity individuals into the neoplastic clam population. This influx of low-severity individuals could be a result of new susceptibles coming into the population. The chronic nature and the possible latency of the disease has not been studied. Their presence could be used to explain the presence of neoplasms year-round, In the laboratory experiments the percentage of clams at each degree of severity reflected what was observed in the feral population. The neoplasm was confined only to M. arenaria even though M. balthica and M. tnercenaria, two species of clams which have been reported to have neoplasms, occupied the same location. The seasonal variation in the percentage of neoplasms and the host specificity have suggested to several authors a possible infectious etiologic agent (Brown et al., 1977; Farley, 1969a; Sparks, 1972). Our laboratory has recently isolated a retrovirus-like particle
AND
CHANG
from neoplastic clams and has conducted preliminary transmission studies which support the viral etiology hypothesis (Oprandy et al., 1981; Chang et al., 1980; Cooper, 1979). Two possible physiological factors could be responsible for stimulating the onset of neoplasia. These factors are spawning and/or water temperature. The highest prevalence of neoplasm occurs during spawning and at water temperatures between 5” and 10”. Both of these factors should be examined more closely in the future by comparing neoplastic M. arenaria populations north and south of Cape Cod. The temperature of the water is different and spawning occurs only once in clams north of the Cape as opposed to twice on the South Shore (Pfitzenmeyer, 1972). Questions that require future investigation are: do clams that undergo remission remain refractile to the disease? If so, what factors are responsible for this protection? Are hematopoietic neoplastic cells in Mya true neoplastic cells as seen in vertebrate animals or just hyperplastic cells that undergo frequent remission? And finally, what factors are responsible for the remission, and the seasonal occurrence of hematopoietic neoplasm in A4ya arenariu? Though water temperature and spawning appears to be partially responsible for the remission and seasonal occurrence of the neoplasm, control laboratory experiments are needed to provide confirmation. REFERENCES D. J., VAN BANNING, P., AND PEREZCOLOMER, A. 1977. Two European oysters (Ostrea edulis) mortalities associated with an abnormal haemocytic condition. Aquaculturr. 10, 335-340. BROWN, R. S., WOLKE. R. E., SAILA, S. B., AND C. BROWN, 1977. Prevalence of neoplasia in 10 New England populations of the soft-shelled clam (Mya
ALDERMAN.
nrrnaria).
Am.
N. Y. Acnd.
Sci..
298, 522-534.
P. W., OPRANDY, J. J., BROWN, R. S., DAWSON, G. T., APPELDORN, R. S., AND YATES, V. J. 1980. “Viral Etiology of a Transmissible Hematopoietic Type of Molluscan Neoplasia.” Proceedings. 80th Annual Meeting, Amer. Sot. Microbial., p. 208.
CHANG.
COURSE OF HEMATOPOIETIC CHRISTENSEN, D. J., FARLEY, C. A., AND KERN, F. G. 1974. Epizootic neoplasms in the clam, Macoma balthicu (L) from the Chesapeake Bay. J. Nat. Cant Inst., 52, 1739-1749. COOPER, K. R. 1979. “The Hematopoietic Neoplasm in the Commercially Important Bivalve Mollusk Mya arenarin (Linne).” Ph.D. dissertation, University of Rhode Island. FARLEY, C. A. 1969a. Probably neoplastic disease of the hematopoietic system in oysters. Crassostrea virginica and Crassostrea gigas. Nat. Cancer Inst. Monogr., 31, 541-555. FARLEY, C. A. 1969b. Sarcomatid proliferative disease in a wild population of blue mussels (Mytilus edulis). .I. Nat. Cancer Inst., 43, 509-516. FARLEY, C. A. 1976. Proliferative disorders in bivalve mollusks. Mar. Fish. Rev., 38, 30-33. FARLEY, C. A. SPARKS, A. K. 1970. Proliferative disease of hemocytes, Haernatol., 36, 610-617. FRIERMAN, E. M. 1976. Occurrence of hematopoietic
NEOPLASM neoplasms ginica).
Mar.
IN
in Virginia Fish.
157
MYQ
Rev.,
oysters (Crassostrea
vir-
38, 34-36.
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. Invertebr. Pathol., 38, 45-51. MIX, M. C. 1975. Proliferative characteristics of atypical cells in native oysters (Ostrea /urida) from Yaquina Bay, Oregon. J. lnvertebr. Patho/., 16, 289-298. PFITZENMEYER, H. I. 1972. Tentative outline for inventory of molluscs; Mya arenaria (soft-shell clam). Chesapeake Sci. Suppl., 13, 182- 184. SPARKS, A. K. 1972. “Invertebrate Pathology, Noncommunicable Disease.” Academic Press, New York. YEVICH, P. P., AND BARSCZC, D. A. 1976. Gonadal and hematopoietic neoplasms in Mya arenaria. Mar. Fish.
Rev.
38, 42-43.
OF
INVERTEBRATE
The Course KEITH Department
PATHOLOGY
39,
and Mortality Soft-Shell R. COOPER,* of Aquaculture
149-
(1982)
157
of a Hematopoietic Clam, Mya arenarial S. BROWN,
ROBERT Science Kingston,
Neoplasm
in the
AND PEI W. CHANG
and Pathology, University Rhode Island 02881
of Rhode
Island.
Received January 12, 1981; accepted June 29, 1981 Results from two experiments containing approximately 280 Mya arenaria indicated that significantly higher (P < 0.05) mortality occurred within the neoplastic clam population than in the nonneoplastic clam population. Using an in vivo blood cytological technique, five levels of severity were established. The levels were based on the number of neoplastic cells in the circulation with level 1 as the lowest severity and level 5 as the highest severity. Neoplastic clams that were diagnosed as the lowest level had higher survival rates (60%) than those clams diagnosed as the highest level (0%). The hematopoietic neoplasm in M. arenaria followed one of three courses: (1) in approximately 50% of the neoplastic clams the disease progressed to a higher severity and resulted in death: (2) in approximately 40% of the neoplastic clams the disease was chronic, i.e., remained at a stable level: and (3) in approximately 10% of the neoplastic clams the disease diminished in severity or disappeared entirely. In addition, 10% of the clams diagnosed as nonneoplastic at the beginning of the experiments were neoplastic by the termination of the experiment. The contraction of the disease may have been de novo or by stimulation of latent infections. The prevalence of the neoplasm in clams collected from an epizootic area followed a biphasic seasonal pattern. The highest prevalences occurred in October, November, and in May. The hematopoietic neoplasm in M. arenaria was also age and species specific. KEY WORDS: Mya arenaria: hematopoietic neoplasm, course, mortality, seasons, prevalence.
INTRODUCTION
A causal relationship between molluscan neoplasms and death has been demonstrated for Macoma balthica (Christensen et al., 1974) and for Crassostrea virginica (Frierman, 1976). A hematopoietic neoplasm in Mya arenaria was believed to be malignant based on cytologic characteristics and the increased death rate among neoplastic clams (Brown et al., 1977). This report is an extension of the previous work. Laboratory experiments were designed to address two questions: (1) were
neoplastic clams at a higher risk of death than nonneoplastic clams and (2) did the neoplasm always progress and result in death? Feral population studies were designed to examine the progression of the disease and the seasonal and age effects. All of these factors are important in understanding the epizootiology and the effect of neoplasia on a clam population. Our results corroborate the findings of Brown et al. (1977) and raise questions concerning the sequelae of the disease. MATERIALS
AND METHODS
Bleeding and collection of hemolymph.
’ This study was supported in part by a grant from the American Petroleum Institute. Contribution No. 1973 of the Rhode Island Agricultural Experiment Station. p To whom all correspondence and reprint requests should be addressed. Current address: Keith R. Cooper, Department of Pharmacology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pa. 19107.
Hemolymph from the posterior adductor muscle was drawn into a l-ml tuberculin syringe with a 26-gauge needle. Blood cytological techniques were routinely used to diagnose the presence of neoplastic cells in the hemolymph and their level of severity of the neoplasm (Cooper, 1979). Hemo149 0022-2011/82/020149-09$01.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
150
COOPER,
BROWN,
cytes were diagnosed for neoplasia using phase-contrast optics on fresh hemolymph samples and brightfield microscopy on methanol-Giemsa-stained blood smears. The level of disease was based on the number of circulating neoplastic cells in a O.l-ml hemolymph sample. Five levels were established based on the number of neoplastic cells in the blood with level 1 as the lowest severity and level 5 as the highest severity. The limits were arbitrarily set based on a logarithmic progression. Level 1 clams had s lo4 neoplastic cells/ml, while level 5 clams had ~10’ neoplastic cells/ml (Cooper, 1979). Histopathological procedures. All the clams were shucked and examined for gross lesions. Clam tissues were preserved in Dietrich’s solution (Armed Forces Institute of Pathology Manual, 3rd edition, p. 4, formol acetic acid). A longitudinal section of the siphon and a cross section, from the heart to the foot were dehydrated in alcohol and xylene, embedded in paraffin, sectioned at 6 pm, and stained with hematoxylin and eosin.
AND
CHANG
tissues processed for histology. This study will be referred to as experiment E-l within the text of this paper. Experiment E-2 was designed to duplicate E-l and to more closely examine both the progression and the remission of the disease. The clams were collected from Allen Harbor, Rhode Island, on September 28, 1978. Clams were bled and diagnosed on two separate occasions. Only clams with the same diagnosis for the two bleedings were used for the experiment. This minimized the probability of a wrong diagnosis. Thirty clams were classified as having a 1 or 2 level of severity of neoplasia and 31 clams were diagnosed at a 3 level. After 3 and 6 months in the flowing-seawater system, the clams were again bled and diagnosed. Experiment E-2 was terminated on March 26, 1979 (6 months), and all surviving clams were processed for histopathology. Field experiments. M. arenaria from Allen Harbor, Rhode Island, were collected on a monthly basis for a period of 20 months, beginning in July of 1977 and endLaboratory experiments. M. arenaria ing in March of 1979. Before being prowere collected from intertidal zones located cessed for histopathologic examination, the at Allen Harbor, Rhode Island. Ninety-one clams were allowed to depurate sediments clams were collected on September 27, accumulated during collection. Approxi1977. Following a single bleeding, 36 clams mately 1700 clams were diagnosed by hiswere diagnosed neoplastic. Over a lo- topathological method, while the remaining 1800 were diagnosed by the blood cytologimonth period, October 1977 to July 1978, the animals were maintained in a flowingcal techniques (see Materials and Methods, seawater system and monitored for mortalBleeding and collection of hemolymph). ity twice weekly. The temperature in the During the spring of 1977 a large clam set holding tanks for June and July was at or occurred within the study area. Collection near the maximal lethal tolerable temperaof juvenile clams from this spawning began ture for M. arenaria and caused the termiSeptember 1977 and continued until Ocnation of the planned 1Zmonth study at 10 tober 1978. The established population of months. Clams were bled at monthly interclams present prior to the spring of 1977 vals to determine changes in the disease se- was distinguished from the imported verity. Clams that died were examined juvenile clams by their size and vertical histopathologically. To determine the pos- distribution in the sediment. In this paper sible effect of multiple bleedings on the the established population of clams will be clams an additional 100 clams were not bled referred to as the adult population. and were monitored twice weekly for morFive other species of bivalve mollusks tality. At the termination of the study all present at Allen Harbor were also examsurviving clams were sacrificed and their ined for neoplasms: Mercenaria mer-
COURSE OF HEMATOPOIETIC
cenaria (N = 97), Macoma balthica (N = lOl), Geukensis dimusus (N = llO), Petricola pholadifoimis (N = 7), and Ensis minor (N = 3). The x2 square test for sig-
nificance was used to analyze the data. RESULTS Laboratory
Experiments
In experiments E-l and E-2, significantly higher mortality (x2 P < 0.05) occurred in the neoplastic clams than in the nonneoplastic clams (Fig. 1). The survival curves could be divided into two phases: (1) an initial steep phase and (2) a plateau period. The E-l survival curve had a second steep E-l
60
I OII
OC~.LIOV.DCC. 1977
, 0
Oct.
Jan.
Feb. E-2
,
Nov.Oec. 1978
,
,
Jan.
Feb.
TIME
IOlr.Apr.my li78
,
,
Itm.Apr. 1979
Jun.
Jul.
, my
(monthm)
FIG. 1. Percentage of clams surviving over time for experiments E-l and E-2. E-l began 9-27-77 and terminated 7-27-78. E-2 began 9-28-78 and terminated 3-26-79. In experiment E-l June and July temperatures in the holding tanks approached maximum tolerable temperature levels for Mya orennrin and accounted for the high mortality during that time. Neoplastic clams of all levels of severity, 0; neoplastic clams of levels of severity 1 and 2, A; neoplastic clams of levels of severity 3, n ; nonneoplastic clams, 0.
NEOPLASM
IN Myo
151
phase which occurred during June and July. We believe that this second phase resulted from elevated water temperatures and not as a result of any disease process. This hypothesis is based on the parallel appearance of the survival curves for neoplastic and nonneoplastic animals during this phase (Fig. 1, E-l), and the inability to correlate any visible lesions with the deaths of the nonneoplastic clams. Because of this unusually large losses of clams during the months of June and July, a second study (E-2) was designed to continue for 6 months during which time the temperature in the holding tanks remained at acceptable levels. As reported by Brown et al. (1977) .we found no effect of bleeding on the survival of clams. The majority of neoplastic clam deaths occurred during the first 2 months of both experiments. The clams that died during this period were those clams at the highest levels of severity and those in which the neoplasm had progressed and affected vital organ systems. Deaths of these animals are believed to result from displacement of normal organ tissue with neoplastic cells. In E-l and E-2, 16 of the 29 neoplastic clams that died and 8 of the 16 nonneoplastic clams that died were necropsied and examined histologically. An advanced hematopoietic neoplasm was found in all 16 of the blood diagnosed neoplastic clams, while the 8 nonneoplastic clams showed no significant lesion. Clams diagnosed by the blood cytological technique as having a low disease severity had higher survival rates than clams at more severe levels; level 1 neoplastic clams had 60% survival rate; level 2 neoplastic clams had 54% survival rate; level 3 neoplastic clams had 25% survival rate, and levels 4 and 5 neoplastic clams had 0% survival rate (Table 1, E-l). The life expectancy of a clam under these experimental conditions was s 3 months for clams diagnosed at a level 5 and ~6.5 months for clams diagnosed at a levels 3 and 4 (Table 1, footnote a). Following the steep phase on the survival
152
COOPER, BROWN, TABLE MORTALITY OF NEOPLASTICAND
AND CHANG 1 NONNEOPLASTIC
CLAMS
Number of clams Experiment E-lb
E-2’
Status
of clams
Nonneoplastic Neoplastic Level 1 Level 2 Level 3 Level 4 Level 5 Neoplastic (cumulative) Unknown’ Nonneoplastic Neoplastic Level 1 and 2 Level 3 Neoplastic (cumulative)
Initial condition
Mortality”
Percentage of survival
55
16
71
10 13 4 6 3 36 100
4 6 3 6 3 21 31”
60 54 25 0 0 41 69
30
0
100
31 31 62
1 7 8
96 77 87
a The average time to death for nonneoplastic clams was 9.4 months, neoplastic clams at levels 1 and 2 (combined) was 7.7 months, at levels 3 and 4 (combined) was 6.5 months, and level 5 was 3 months. DExperiment E-l began on Sept. 27, 1977 and terminated July 27, 1978. V Clam status was unknown because they were not bled. ’ Monthly bleeding had no apparent effect on mortality when compared to clams not bled (x2, P > 0.05). (I Experiment E-2 began on Sept. 28, 1978 and terminated March 26, 1979.
curve during October, November, and December is a plateau period in which few if any deaths occurred (Fig. 1). The surviving neoplastic clams were those clams diagnosed at the lower levels of the disease, i.e., levels 1, 2, and 3 which had not progressed to a lethal stage. At these levels the hematopoietic neoplasm followed one of three courses: (1) remained at a stable level, (2) progressed to a higher level, or (3) diminished in severity and/or disappeared. In experiment E-l, 44% (16/36) of the neoplastic clams increased in severity, 44% (16/36) of the neoplastic clams remained at the same level, and 12% (4/36) of the neoplastic clams had apparent remission of the disease (Table 2, E-l). Remission occurred at the low severity levels. In experiment E-2, 38% (23/61) of the neoplastic animals had remained at the same level, 43% (26/61) of the neoplastic animals had progressed to a higher level, and 20% (12/61) of the neoplastic animals had undergone apparent remission (Table 2, E-2). At 3 months, none of the level 3 clams had undergone remis-
sion, but at 6 months, two clams showed no signs of neoplastic cells either in fresh blood or in tissue section. It is also of interest, that 7 of the 31 clams initially diagnosed as level 3 were at level 1 by the termination of the experiment. All cases of remission were corroborated by tissue diagnosis. Also, 10% of the clams diagnosed as negative at the beginning of the experiment were positive by the termination of the experiment (Table 2). Feral Population Study
Seasonal variations in the prevalence of the hematopoietic neoplasm were observed in the adult M. arenaria collected from Allen Harbor, Rhode Island. The disease occurred throughout the year. The lowest percentage of neoplasia was 20% during the months of May, July, and August 1978 (Table 3). The highest prevalences occurred in the fall (43%) and late spring (43%). Following the peak in the fall, the percentage of neoplasia declined until February when the prevalence began to rise. After the sec-
COURSE
OF
HEMATOPOIETIC
NEOPLASM
TABLE COURSE
IN
2
OF DISEASE
IN CLAMS
Number of clams Experiment E-l”
E-2”
Status of clams
153
Myu
Progression of disease
At start of experiment
Same level
Remission
Nonneoplastic Neoplastic Level 1 Level 2 Level 3 Level 4 Level 5 Neoplastic (cumulative)
5.5
50
10 13 4 6 3 36
6 3 1 3 3 16
Nonneoplastic Neoplastic Levels 1 and 2 Level 3 Neoplastic (cumulative)
30
27
-
31 30 61
10 13’ 23
10 2 12
Increased 5
2 2 0 0 0 4
2 8 3 3 16 3 11 15 26
” Experiment E-l began on Sept. 27, 1977 and terminated on July 27, 1978. D Experiment E-2 began on Sept. 28, 1978 and terminated on March 26, 1979. C Of the 13 neoplastic clams that remained at level 3, 7 of them actually decreased in severity to level 1 at the end of the experiment, indicating that given more time they might have undergone remission.
TABLE PREVALENCE
Date of collection
3
OF HEMATOPOIETIC NEOPLASIA IN FERAL CLAMS FROM ALLEN HARBOR, AT DIFFERENT MONTHS, IN THE YEARS 1977- 1979
No. of clams collected
Percentage neoplastic
Percentage at each level of severity 1
2
3
4
5
RHODE
Sum percentages of neoplasia levels of severity 4 and 5
1977 July 2 Aug 27 Sept 27 Ott 27 Nov 27 Dee 10
61 98 143 150 41 136
28 19 23 43 39 40
36 25 16 42 30 18 18 26 31 20 25 27.5
32 7 32 10 13 26 21 21 13 18 10 27.5
0 0 13 14 18 10
7 10 39 35 36 37.5
1978 Feb 23 April 10 May 11 June 1 July 20 Aug 25 Sept 28 Nov 5 Nov 28
100 108 158 315 327 157 474 30 189
22 43 20 22 20 20 33 37 43
14 19 20 32 60 30 45 19 30
18 18 10 9 20 25 1.5 19 12
23 23 30 23 0 5 6 0 18
27 28 20 13 0 10 9 35 28
50 51 50 33 0 15 15 35 46
1979 Jan 9 Feb 28 March 28
100 176 181
31 24 34
42 10 12 25 29 15
16 13 12 9 18 6
19 42 32
32 51 38
18 12 20 23 20 30 25 27 12
ISLAND,
154
COOPER,
BROWN,
AND
CHANG
ond peak the prevalence again declined and plastic clams of levels 4 and 5 are at the remained at approximately 20% during the highest point, approximately 50%, the summer months. These trends were con- water temperature reaches the lowest point sistent from year to year (Fig. 2). (>5”C). It appears that low water temperaThe severity of the neoplasms followed a ture is associated with an increase in the seasonal pattern. By summing the per- severity of the neoplasia resulting in a centages of neoplastic clams of levels 4 and larger percentage of levels 4 and 5 neoplas5 at different months of the year and com- tic clams and high water temperature paring the results with the water temperacauses a drop in percentage neoplasia ture, an interesting pattern emerged (Fig. 2, among the levels 4 and 5 clams. Table 3). During July and August when the Neoplastic juvenile clams were found percentage of neoplastic clams of levels 4 throughout the experimental period (Fig. and 5 (7 and 10%; 0 and 15%) are at the 3). The percentage of neoplasia varied from lowest ebb, the water temperature, how- 3 to 12% in the first 9 months of sampling. lower prevalences of neoever, reaches the highest of the year (20°C). Significantly plasia for the first year were observed in the During February, March, April, May, and November when the percentage of neo- juvenile clams than in the adult clams (x2 P < 0.05). There was no biphasic pattern as was present in the adult clams. However, beginning in July, increases in the prevalence of neoplasia occurred (Fig. 3). The highest recorded prevalence was in September 1978 (30%). The corresponding peak in the adult population did not occur until December. The severity levels of the individual neoplastic cases are recorded in 00 Table 4. In the first 9 months there were two cases at a 5 severity level, while five cases were observed in the last 3 months. No neoplasms were found in any of the other five molluscan species sampled from the Allen Harbor area. DISCUSSION
FIG. 2. Percentage of levels of severity, (B) sum severity levels 4 and 5, and temperature. M. arrrmria Harbor, R.I. Two additional and 0, from Brown (1977) age of neoplasia.
neoplasia: (A) sum of all of percentage neoplasia of (C) average monthly water were collected from Allen points, as indicated by 0 were added to the percent-
Epizootic neoplasia has been reported to occur in populations of six molluscan genera: Ostrea. Crassostrea, Mytilus, Mercenaria, Mya, and Macoma, with a wide geographical distribution (Alderman, 1977; Balouet, pers. commun.; Brown et al., 1977; Christensen et al., 1974; Farley, 1969a,b; Farley and Sparks, 1970; Frierman, 1976; Mix, 1975; Yevich and Barszcz, 1976). The neoplasms became apparent in animals collected in September or October and continued through the winter and spring (Farley, 1976). A biphasic increase in prevalence was reported to occur in populations of Macoma balthica (Christensen et
COURSE OF HEMATOPOIETIC
NEOPLASM
155
IN Mya
**.dJ 0
O‘d
b,
II
I
Oct.
Nov.
Dec.
-0”
I
Jan.
I
Feb.
I
Mar.Apr.
I
I
May
I
Jun.
1977
I
Jul.
I
I
I
Aug.Sept.Oct.Nov.
I
I
Dec.
1978 TIME
(months)
FIG. 3. Prevalence of neoplasia in juvenile clams and adult clams collected at the same time and location, Allen Harbor, Rhode Island. Juvenile clams 0: adult clams 0.
al., 1974). We found that a similar seasonal pattern occurred in a hematopoietic neoplasm of adult M. arenaria (Fig. 2). However, the neoplastic condition was present year-round and did not disappear during certain times of the year, as in the case with neoplasms of the other five genera. The highest percentage of neoplasms in M. arenariu occurred in October-November (43%) and in April (43%). Before and after these peak periods the prevalence leveled off to 20% (Fig. 2). The high percentage of neoplasms occurred again in September 28-January 9 of 1978/1979 (Table 3). TABLE SEASONAL
Date of collection 9-27-77 10-27-77 11-21-17 12-10-77 2-26-78 4-10-78 5-11-78 7-l-78 7-20-78 9-6-78 9-28-78 10-28-78
M. arenaria less than a year of age had lower prevalences of neoplasia and did not demonstrate the seasonal variation observed in the adult population (Table 4). However, the juvenile clam population upon reaching the summer of their second year had a similar percentage of neoplastic individuals at severities comparable to the adult population. Frierman (1976) had reported a similar finding in laboratory-reared Crassostrea virginica. Neoplasms appeared in them at 1 year of age with severe mortality. Therefore, juvenile Mya could be a source of new susceptibles. 4
VARIATION OF HEMATOPOIETIC NEOPLASIA IN JUVENILE CLAMS FROM ALLEN HARBOR,RHODE ISLAND
Number of clams Examined 57 57 60 30 60 30 30 30 30 30 30 30
Disease level
Neoplastic 3 2 7 1 2 3 3 2 6 9 8 5
1
2
1 I I 1 1 2 1 2 4 3 4 2
2
3
4
5 1
3
1 1 1 1
3 2
1
2 2 1 1
1 1 2
Percentage neoplastic 5 4 12 4 4 10 10 7 20 30 26 20
156
COOPER,
BROWN,
There are three major observations made in this study concerning the fate of neoplastic clams. These are: (1) the hematopoietic neoplasm, reaching a level of severity of 3 or greater, becomes a progressive disease and results in death in the majority of the cases, (2) the hematopoietic neoplasm can also be a chronic disease in that it remains at a stable level for up to 10 months, (3) at low levels of severity (~3) the hematopoietic neoplasm can diminish in severity and show remission. The cyclic nature of the disease is believed to be due to a complex interaction between the abovementioned factors and the recruitment of susceptible individuals. The decreases in prevalence in May, June, December, and January can be explained by a combination of factors, such as the deaths of animals with severe neoplasms and the remission of animals which had low severity levels. The steady increase in the severity of diseased animals throughout the winter would indicate the progression of the disease. The decrease in the severity of the disease during July and August could be due to the high water temperature (Fig. 2) or to an influx of low-severity individuals into the neoplastic clam population. This influx of low-severity individuals could be a result of new susceptibles coming into the population. The chronic nature and the possible latency of the disease has not been studied. Their presence could be used to explain the presence of neoplasms year-round, In the laboratory experiments the percentage of clams at each degree of severity reflected what was observed in the feral population. The neoplasm was confined only to M. arenaria even though M. balthica and M. tnercenaria, two species of clams which have been reported to have neoplasms, occupied the same location. The seasonal variation in the percentage of neoplasms and the host specificity have suggested to several authors a possible infectious etiologic agent (Brown et al., 1977; Farley, 1969a; Sparks, 1972). Our laboratory has recently isolated a retrovirus-like particle
AND
CHANG
from neoplastic clams and has conducted preliminary transmission studies which support the viral etiology hypothesis (Oprandy et al., 1981; Chang et al., 1980; Cooper, 1979). Two possible physiological factors could be responsible for stimulating the onset of neoplasia. These factors are spawning and/or water temperature. The highest prevalence of neoplasm occurs during spawning and at water temperatures between 5” and 10”. Both of these factors should be examined more closely in the future by comparing neoplastic M. arenaria populations north and south of Cape Cod. The temperature of the water is different and spawning occurs only once in clams north of the Cape as opposed to twice on the South Shore (Pfitzenmeyer, 1972). Questions that require future investigation are: do clams that undergo remission remain refractile to the disease? If so, what factors are responsible for this protection? Are hematopoietic neoplastic cells in Mya true neoplastic cells as seen in vertebrate animals or just hyperplastic cells that undergo frequent remission? And finally, what factors are responsible for the remission, and the seasonal occurrence of hematopoietic neoplasm in A4ya arenariu? Though water temperature and spawning appears to be partially responsible for the remission and seasonal occurrence of the neoplasm, control laboratory experiments are needed to provide confirmation. REFERENCES D. J., VAN BANNING, P., AND PEREZCOLOMER, A. 1977. Two European oysters (Ostrea edulis) mortalities associated with an abnormal haemocytic condition. Aquaculturr. 10, 335-340. BROWN, R. S., WOLKE. R. E., SAILA, S. B., AND C. BROWN, 1977. Prevalence of neoplasia in 10 New England populations of the soft-shelled clam (Mya
ALDERMAN.
nrrnaria).
Am.
N. Y. Acnd.
Sci..
298, 522-534.
P. W., OPRANDY, J. J., BROWN, R. S., DAWSON, G. T., APPELDORN, R. S., AND YATES, V. J. 1980. “Viral Etiology of a Transmissible Hematopoietic Type of Molluscan Neoplasia.” Proceedings. 80th Annual Meeting, Amer. Sot. Microbial., p. 208.
CHANG.
COURSE OF HEMATOPOIETIC CHRISTENSEN, D. J., FARLEY, C. A., AND KERN, F. G. 1974. Epizootic neoplasms in the clam, Macoma balthicu (L) from the Chesapeake Bay. J. Nat. Cant Inst., 52, 1739-1749. COOPER, K. R. 1979. “The Hematopoietic Neoplasm in the Commercially Important Bivalve Mollusk Mya arenarin (Linne).” Ph.D. dissertation, University of Rhode Island. FARLEY, C. A. 1969a. Probably neoplastic disease of the hematopoietic system in oysters. Crassostrea virginica and Crassostrea gigas. Nat. Cancer Inst. Monogr., 31, 541-555. FARLEY, C. A. 1969b. Sarcomatid proliferative disease in a wild population of blue mussels (Mytilus edulis). .I. Nat. Cancer Inst., 43, 509-516. FARLEY, C. A. 1976. Proliferative disorders in bivalve mollusks. Mar. Fish. Rev., 38, 30-33. FARLEY, C. A. SPARKS, A. K. 1970. Proliferative disease of hemocytes, Haernatol., 36, 610-617. FRIERMAN, E. M. 1976. Occurrence of hematopoietic
NEOPLASM neoplasms ginica).
Mar.
IN
in Virginia Fish.
157
MYQ
Rev.,
oysters (Crassostrea
vir-
38, 34-36.
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. Invertebr. Pathol., 38, 45-51. MIX, M. C. 1975. Proliferative characteristics of atypical cells in native oysters (Ostrea /urida) from Yaquina Bay, Oregon. J. lnvertebr. Patho/., 16, 289-298. PFITZENMEYER, H. I. 1972. Tentative outline for inventory of molluscs; Mya arenaria (soft-shell clam). Chesapeake Sci. Suppl., 13, 182- 184. SPARKS, A. K. 1972. “Invertebrate Pathology, Noncommunicable Disease.” Academic Press, New York. YEVICH, P. P., AND BARSCZC, D. A. 1976. Gonadal and hematopoietic neoplasms in Mya arenaria. Mar. Fish.
Rev.
38, 42-43.