Occurrence of hematopoietic neoplasms in Virginia oysters (Crassostrea virginica).

Occurrence of Hematopoietic Neoplasms in Virginia Oysters (Crassostrea v;rg;n;ca J 1 E. Michael Frierman and J. D. Andrews

2, 3

SUMMARY-A neoplastic disease of the hematopoietic system of oysters was found in tray-cultured native and laboratorybred populations and was associated with mortal'ities during field monitoring. The disease, characterized by nuclear abnormalities and intensive proliferation of hemocytes, was fi·rst observed at the Virginia Institute of Marine Science in 1963. Cases rarely occurred during 14 years (1959-1973 inclusive) of regular sampling and monitoring of thousands of oysters. However, two groups exhibited a high susceptibility to this disease in a program of intensive inbreeding to achieve genetic resistance to oyster pathogens.-J Natl Cancer Inst 56: 319324,1976.

The three predominant pathogens of oysters in Virginia are the haplosporidians M inchinia costalis and l\,f. nelsoni and the protistan Dermocystidium marinum (1-3). M. costalis is found mostly in the high-salinity seaside embayments of Virginia, Maryland, and Delaware from Cape Henry to Cape Henlopen. It causes intensive mortalities during May-June (4). M. nelsoni and D. marinum have wide geographic ranges and kill oysters in the Chesapeake and Delaware Bays where salinities exceed 150/00. These diseases have inflicted devastating, widespread mortalities on Virginia oyster populations for several decades, and large areas of public and rented oyster grounds have been abandoned (5-9). Extensive tray monitoring of oysters for diseases (10) and intensive sampling have provided 70 instances of a rare neoplasm similar to that described by Farley (11) and Farley and Sparks (12). MATERIALS AND METHODS

Histology.-Neoplasms were first observed on stained slide preparations at Virginia Institute of Marine Science (VIMS) by Andrews in oysters fixed in formalin, acetic acid, and alcohol. Transverse sections through dead and live oysters were stained with Harris' hematoxylin and eosin Y (13). These methods were used for all tumors diagnosed. Field studies.-Oysters used as controls for the monitoring of diseases in Virginia were mostly "disease free" groups from low-salinity areas of the James River, where only rare instances of known diseases have been found. These wild or native oysters were imported as field controls for laboratory-bred, disease-resistant lots and for assessment of disease prevalences in Virginia estuaries. Oysters were held in iron trays lined with galvanized wire mesh and suspended from the bottom by 12-inch legs. A zinc bar anode was attached to each oyster tray to reduce corrosion and to extend the life of the tray liners. Initially, 400-1,000 or more oysters were placed in each tray. Trays, distributed in major rivers to monitor M. nelsoni activity that occurs mostly in salinities between 15 and 250/00 (5-7), were checked about every 3 weeks during warm weather when disease activity was most intense, and monthly during cold seasons when changes were slower. The oysters were counted and cleaned of fouling organisms. Samples of 25 live oysters were regularly prepared histologically and examined to assess occurrence of diseases and to document sea-

sonality of disease activity (5). Dead oysters, both boxes (no meat) and gapers (meat present), were removed and gaper tissue was processed. Primarily, we studied live oysters histologically after the field epizootiology of M. nelsoni became known, and examined the gapers to confirm causes of mortalities. Trayed groups of known origin and history were monitored regularly from 1 to 10 years, with duration dependent on survival of oysters and the objectives of experiments. Laboratory techniques.-An underwater suspensionweighing technique was used for measurement of growth as indicated by shell deposition (14). This method, with control of time and source of oyster imports, permitted detection of sick oysters as evidenced by weekly lack of weight increase. Oysters were numbered individually and cleaned of fouling organisms before each weighing. RESULTS Description of Neoplasms

The first cases found at VIMS were called DutchWashington disease after Haskin,4 who noted oysters from various locations with sinuses congested with blood cells. Microscopic studies have never revealed a disease agent. Wide geographic occurrence with vague seasonality reduced the possibility of localized environmental causes. The disease, believed to be a neoplasm of the hematopoietic system (11, 12, 15-18),5 had the following histologic and cytologic characteristics: 1) atypical hyaline hemocytes characterized by enlarged nuclei and an unusual abundance of mitotic figures; 2) intensive proliferation of atypical hemocytes resulting in a highly invasive infiltration of connective tissues; 3) connective tissues disrupted by hemocyte invasion and sinuses congested with atypical hemocytes; and 4) wide range of hemocyte cell sizes, with rapid proliferation and differentiation (figs. 1-8). Field Monitoring of Oyster Trays

An extensive oyster-breeding program was initiated at VIMS in 1964 to produce genetically superior strains. Many experimental inbred lots were laboratory bred and field monitored with intensive selection. Two lots, P58 and PI04, were quite susceptible to hematopoietic neoplasms. Thirty-one hematopoietic neoplasms were diagnosed in 369 oysters from the 2 susceptible groups. Thirty-nine neoplasms were recorded from over 51,000 oysters examined from 1964 to 1973 in other progeny and native imports in trays (table 1). In the York River, native and laboratory-bred oysters have shown similar prevalences (0.06%; table 1) in moniReceived April 30, 1975; accepted September 4, 1975. Virginia Institute of Marine Science, Gloucester Point, Va. 23062. 3 We thank Dr. John C. Harshbarger, Smithsonian Institution, Washington, D.C.; Dr. Clyde J. Dawe, National Cancer Institute, Bethesda, Md.; and C. Austin Farley, National Marine Fisheries Service, Oxford, Md., for their assistance in identifying the diseases. 4 Haskin HH: Personal communication. 5 Harshbarger JG: Personal communication. 1

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JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 56, NO.2, FEBRUARY 1976

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320

FRIERMAN AND ANDREWS

TABLE l.-occurrence of hematopoietic neoplasms in progeny and native oysters in trays, 1964.-1973 a Live oysters Neoplasms

Tray oysters Number examined Progeny Susceptible P-58 P-104 Nonsusceptible Natives York River James River Piankatank River Rappahannock River Mobjack Bay Eastern Shore Seaside Bayside o

Gapers Neoplasms

Number of cases

Percent diseased

Number examined

Number of cases

Percent diseased

_ _ _

244 101 26,911

8 6 16

3.28 5.94 0.06

13 11 1,248

8 9 4

61.54 81.82 0.32

_ _ _ _ _

9,511 3,496 988 1,548 1,757

6

3 3

1,425 110

o o

0.06 0.09 0.30 0.00 0.00

9 419

7 0 0 0 0

0.49 0.00 0.00 0.00 0.00

_ _

3,717 421

o

0.00 0.00

121 92

0 0

0.00 0.00

o

o

Most laboratory-bred progeny were monitored in the York River.

tored groupS. Hematopoietic neoplasms occurred in 12.5% of all groups from 1964 to 1974. One or more cases appeared in 11.9% of 134 native and 13.2% of 106 laboratory~bred groups. Only P58 and PI04 were epi;. zootic (table 1). Lot PI04 best illustrated that hematopoietic neoplasms caused mortalities in oysters. The large percentage of gapers with neoplasms during July 1973 corresponded with the high mortalities and the high percentage of liveoyster infections in the absence of other diseases (textfig. 1). Tray P58 had concurrent hematopoietic neoplasms and M. nelsoni infections in live samples, and the presence of the two diseases precluded assignment of specific mortalities. Pathogenic diseases did not appear to affect prevalences of neoplasms occurring concurrently. Neoplasms in laboratory-bred oysters have appeared as early as 217 days from spawning and as late as 880 days. A case was diagnosed in a native oyster about 8 years of age. In the susceptible groups, neoplasms appeared at an age of 1 year and caused severe mortalities. The P58 cases were scattered from August 1969 through August GAPERS/NEOPLASMS LIVE/NEOPLASMS X

,

,

1/1

I/O

9/8

30/1

25/5

21/0

l

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15

II:

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Q.

.... Z ~

II:

~

10

....'f""""I~-r-----,----r--__r--r__-__r-___,

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APR

MAY

JUN

JUL

AUG

SEP

OCT

NOV

DEC

1973

I.-Death rates from hematopoietic neoplasms in tray PI04 in 1973. Cases in gapers and live oysters are shown with sampling dates designated by arrows. D. marinum caused deaths after August.

TABLE 2.-ChrQnologic list of hematopoietic neoplasms Date sampled 30 Aug 18 Aug 27 Oct 28Sep 5 Oct 24 Jul 14 Aug 17 Aug 3 Oct 9 Oct 15 Oct 16 Oct 15 Jul 18 Jul 7 Aug 7 Aug 8 Aug 25 Sep 27 Oct 24 Nov 18 Dec 28 Jan 24 Feb 6 Jul 6 Jul 27 Jul 28 Jul 14 Aug 14 Aug 20 Aug 21 Aug 14 Jul 1 Sep 24 Sep 11 Jul 10 Aug 13 Sep 24 May 6 Jul 6 Jul 6 Jul 11 Jul 11 Jul 19 Oct 2 Nov 30 Nov 30 Nov

63 64 65 66 66 67 67 67 67 67 68 68 69 69 69 69 69 69 69 69 69 70 70 70 70 70 70 70 70 70 70 71 71 71 72 72 72 73 73 73 73 73 73 73 73 73 73

Tray lots a Y16 Y19 P3 RMI P9 J17 P21 P38 P21 Y37 14-67 P53 P57 W69 P45 P45 P58 P58 P50 P58 P58 P58 P21 P58 Y60 P58 Y60 Y60 Y64 P58 Y61 PK5 J28 Y71 PK7 Y73 PK8 Y74 P104 P104 P87 P103 P100 J32 PlOO P104 P104

Live (L) Number or of cases gaper (G) G G L G G L L L L L L L L L L G L L L L G G G G G G G L G G G L L L L L L L L G G L L L L L G

1 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 2 2 1 4 1 2 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 5 8 1 3 1 1 1 1 1

Generation and source James River James River F I , Mobjack Bay James River F I , Mobjack Bay James River F l , James River F I seaside F I , James River James River F I , James River F I , Long Island, N. F I , Potomac River James River F 2, P10 F 2 , PlO F I , seaside F I , seaside F I , York River F I , seaside F I , seaside F I , seaside F I, James River F I , seaside Manokin River, M F I , seaside Manokin River, M Manokin River, M James River F I , seaside James River James River James River James River James River James River James River James River F 4 , P80 F 4, P80 F I , James River F I , James River F 4, P73 James River F 4, P73 F 4, P80 F 4 , P80

TEXT-FIGURE


G P = Progeny lots bred in laboratory; Y = York River controls; RM =Mobj Bay controls; J = James River controls; PK =Piankatank River controls; 14-6 progeny at nursery pond; and W69 =weighed lot.

321

HEMATOPOIETIC NEOPLASMS IN VIRGINIA OYSTERS

1970, whereas PI04 neoplasms occurred mostly in July 1973, with 2 in November 1973 (table 2). A group of 100 James River native oysters was brought to VIMS April I, 1969 for routine underwater weighing. Oyster #26 showed normal weight gain until July 10, 77° 00'

~

!

!

I

I'I

76° 00'

75°00'

-

t .• l

!t

when shell deposition diminished. On July 17, 1969, the oyster had lost weight and was found to have a hematopoietic neoplasm. The geographic distribution of the disease in Virginia estuaries included the James, York, Piankatank, and

"';'

r;.J

'

39° 00'

39° 00'

38° 00'

38 00'

37° 00'

o

10

37° 00'

20 NAUTICAL

MILES

77° 00'

75° 00'

2.-Map showing: • James River disease-free area; • tray stations with hematopoietic neopl1tsms; 0 tray stations without hematopoietic neoplasms.

TEXT-FIGURE


322


Ware Rivers. No cases were found from the high-salinity bayside or seaside areas of the Eastern Shore of Virginia or the low-salinity James River seed area (text-fig. 2). Water temperatures at which hematopoietic neoplasms occurred ranged from 27.9 0 to 1.1 0 C. Cases have been found 9 months of the year, with most appearing from July through November in salinity ranging from 10 to 22%0. In oysters kept in trays, heavy metals analysis indicated an above-normal level of zinc resulting from galvanized tray parts (19) 20). However, at this time no relationship to hematopoietic neoplasms in oysters can be ascribed to zinc. DISCUSSION

Prevalence of hematopoietic neoplasms in live oysters and mortality rates of oysters and gapers in trayed groups provide field support of pathologic evidence that these neoplasms cause deaths of oysters. The disease may be more frequent in rare genetic combinations of inbred oysters. Higher incidence in only 2 groups suggests this, since over 100 inbred laboratory groups exhibited approximately the same low incidence of neoplasms as imported native oysters. Progeny of P104 have been bred for further studies. Susceptible genetic races may provide excellent materials for studies of neoplasms in mollusks. REFERENCES

(1) WOOD JL, ANDREWS JD: Haplosporidium costale n. sp. (Sporozoa) associated with a disease of Virginia oysters. Science 136:710-711, 1962 (2) HASKIN HH, STAUBER LA, MACKIN JG: Minchinia nelsoni n. sp. (Haplosporida, Haplosporidiidae): Causative agent of the Delaware Bay oyster epizootic. Science 153:1414-1416, 1966 (3) MACKIN JG, OWEN HM, COLLIER A: Prelimifiary note on the occurrence of a new protistan parasite, Dermocystidium marinum n. sp. in Crassostrea virginica (Gmelin). Science 111:328-329, 1950 (4) ANDREWS JD, WOOD JL, HaESE HD: Oyster mortality studies in Virginia. III. Epizootiology of a disease caused by Haplosporidium costale. J Insect Pathol 4:327-343, 1962


(5) ANDREWS JD: Oyster mortality studies in Virginia. V. Ep zootiology of MSX, protistan pathogen of oysters. Ecolog 47:19-31, 1966 (6) - - - : Interaction of two diseases of oysters in natur waters. Proc Nat! Shellfish Assoc 57:38-49, 1967b (7) - - - : Oyster mortality studies in Virginia. VI. History an distribution of Minchinia nelsoni, a pathogen of oysters, i Virginia. Chesapeake Sci 8:1-13, 1967a (8) - - - : Oyster mortality studies in Virginia. VII. Review 4 epizootiology and origin of Minchinia nelsoni. Proc Na Shellfish Assoc 58:23-36, 1968 (9) ANDREWS JD, HEWATT WG: Oyster mortality studies in Vi ginia. II. The fungus disease caused by DermocystidiUJ marinum in oysters of Chesapeake Bay. Ecol Monograpl 27:1-26, 1957 (10) HEWATT WG, ANDREWS JD: Oyster mortality studies in Vi ginia. 1. Mortalities of oysters in trays at Gloucester Poin York River. Texas J Sci 6:121-133, 1954 (11) FARLEY CA: Probable neoplastic disease of the hematopoiet system in oysters (Crassostrea virginica and Crassostn gigas). Natl Cancer Inst Monogr 31:541-555, 1969 (12) FARLEY CA, SPARKS AK: Proliferative disease of hemocyte enddthelial cells, and connective tissue cells in mollus1 Bibl Haematol 36:610-617,1970 (13) DRUNY RA, WALLINGTON EA: Hematoxylin and eosin stainil method. In Carleton's Histological Technique (Cadet( HM, ed.), 4th ed. London, Oxford Univ Press, 1967, I 129-130 (14) ANDREWS JD: Measurement of shell growth in oysters 1 weighing in water. Proc Natl Shellfish Assoc 52:1-11, 196 (15) COUCH JA: An unusual lesion in the man tIe of an Americ; oyster Crassostrea virginica. Nat! Cancer Inst Mono 31:557-562, 1969 (16) FARLEY CA: Sarcomatoid proliferative disease in a wild pop lation of blue mussels (Mytilus edulis). J Nat! Cancer Ir 43:509-516, 1969 (17) PAULEY CB: A critical review '0£ neoplasms and tumor-Ii lesions in mollusks. Nat! Cancer Inst Monogr 31 :509-5 ! 1969 (18) WOLF PH: Neoplastic growth in two Sydney rock oyste Crassostrea co m mercialis (Iredale and Roughley). N: Cancer Inst Monogr 31:563-573, 1969 (19) HUGGETT RJ, CROSS FA, BENDER ME: Copper and zinc oysters and sediments from two coastal plain estuaries: Proceedings of the Symposium on Mineral Cycling Southeastern Ecosystems. Augusta, Ga, May 1974. In pre (20) HUGGETT RJ, BENDER ME, SLONE HD: Utilizing metal c( centration relationships in the Eastern oyster (Crassost1 virginica) to detect heavy metal pollution. Water Res 7:45 460, 1973

FIGURE

I.-Transverse section of healthy C. virginica showing connective tissue, digestive diverticula, and hemolymph sinus.

X

100

2.-Transverse section of live oyster with hematopoietic neoplasm. Note infiltration of connective tissue by abnormal hyaline hemocytes. X 100

FIGURE

FIGURE

3.-Neoplastic invasion of connective tissue of live oyster.

FIGURE

4.-Gaper with hematopoietic neoplasm. Note hemolymph sinus congested with masses of atypical hemocytes. X 200



X

200

323

FIGURE

50-Massive congestion of sinus by abnormal hemocytes in live oyster. X 200

FIGURE

6o-Abnormal hemocytes in sinus (a), connective tissue (b)o Note enlarged, densely staining nuclei. X 1,750

FIGURES

7, S.-Abnormal hyaline hemocytes. Arrows indicate mitotic figures. X 1,750

324



Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica).

Microbiomes of American Oysters (Crassostrea virginica) Harvested from Two Sites in the Chesapeake Bay.

Genetic by environmental variation but no local adaptation in oysters (Crassostrea virginica).

Occurrence and chemical structure of nonmethylene-interrupted dienoic fatty acids in American oyster Crassostrea virginica.

Transcriptome of American oysters, Crassostrea virginica, in response to bacterial challenge: insights into potential mechanisms of disease resistance.

Survey for protozoan parasites in Eastern oysters (Crassostrea virginica) from the Gulf of Maine using PCR-based assays.

Surveillance of Enteric Viruses and Microbial Indicators in the Eastern Oysters (Crassostrea virginica) and Harvest Waters along Louisiana Gulf Coast.

Identification of potential general markers of disease resistance in American oysters, Crassostrea virginica through gene expression studies.

Static tank depuration and chronic short-term experimental contamination of Eastern oysters (Crassostrea virginica) with Giardia duodenalis cysts.

Physiological response and resilience of early life-stage Eastern oysters (Crassostrea virginica) to past, present and future ocean acidification.

Presence of Octopamine and an Octopamine Receptor in Crassostrea virginica.

Cryopreservation of spermatozoa of the American oyster, Crassostrea virginica Gmelin.

Accumulation of cadium by the American oyster, Crassostrea virginica.

Chemotactic attraction of Crassostrea virginica hemolymph cells to Staphylococcus lactus.

Spatial genetic features of eastern oysters (Crassostrea virginica Gmelin) in the Gulf of Mexico: northward movement of a secondary contact zone.

Influence of Salmonella enterica Serovar Typhimurium ssrB on Colonization of Eastern Oysters (Crassostrea virginica) as Revealed by a Promoter Probe Screen.

Role of bacteria in bioaccumulation of mercury in the oyster Crassostrea virginica.

Transcriptomic analysis of candidate osmoregulatory genes in the eastern oyster Crassostrea virginica.

A microbial spoilage profile of half shell Pacific oysters (Crassostrea gigas) and Sydney rock oysters (Saccostrea glomerata).

Uptake and loss of inorganic mercury in the eastern oyster (Crassostrea virginica).

Growth and Survival of the American Oyster Crassostrea virginica in Jamaica Bay, New York.

The Presence of Histamine and a Histamine Receptor in the Bivalve Mollusc, Crassostrea virginica.

Beta-glucuronidase in the serum and hemolymph cells of Mercenaria mercenaria and Crassostrea virginica (Mollusca: Pelecypoda).

Comparative transcriptome analysis of two oysters, Crassostrea gigas and Crassostrea hongkongensis provides insights into adaptation to hypo-osmotic conditions.