Morphology and Mobility of Oyster Hemocytes: Evidence for Seasonal Variations
M.G. McCormick-Ray1, MS; T. Howard2, MD
Bivalve Hemocytes can recognize, locate, ingest, transport, and digest foreign particles, and they are active in phagocytosis, encapsulation, inflammation, and wound repair (Fisher, 1986). Hemocytes thus form a primary line of internal defense for bivalves. Mobility is an essential factor underlying these processes, allowing the cells to shuttle between anatomical compartments and to patrol the interface between the host organism and its environment. In C. virginica, the ability to move is generally associated with granulocytes (Cheng, 1975) but Parley (1968) for C. virginica and Ruddell (1969) for C. gigas reported that agranulocytes may also play an important role.
The classification of bivalve Hemocytes is rooted in controversy (Cheng, 1975, Fisher, 1986; Auffret, 1988) but most agree that granular and agranular Hemocytes can be readily recognized (Takatsaki, 1934; Liebman, 1946; Galtsoff, 1964; Narain, 1973; Feng, et al., 1971; Cheng, 1975, Renwrantz, et al., 1979; Fisher, 1986, Auffret, 1988). However, environmental factors and examination procedures may influence types of cells observed. And although locomotion is an essential characteristic of the phagocytic cell and the defensive process (Fisher, 1988), Hemocytes also perform other seasonal life history functions such as spawning, gametogenesis, growth, and energy reserve buildup (Bayne, 1976; McCormick-Ray, 1987) and locomotion may not be as essential. Thus we sought to determine whether dominant cell types and locomotion changed with season.
Hemocytes of Crassostrea virginica were video recorded and tracked to determine their locomotive rates and to assign these rates to Wright-stained morphological variants. From 24 oysters examined in January, February, March and May, 1571 hemocytes were video recorded, identified, and their rate of locomotion (ROL) measured. Granulocytes (3 types) and agranulocytes (1 lymphoid and 3 nonlymphoid types) were recognized . Focusing on 15 oysters in March and May, 20,318 hemocytes were counted from duplicate slides to verify the classification and to show that predominant hemocytes vary greatly between samples and among individual oysters, yet monthly population differences can be detected. The cell types identified here correspond to Cheng's (1981) morphological scheme of granular and nongranular hemocytes. Auffret (1988), using Cheng's scheme, described hyalinocytes as we did for lymphoids. Hyalinocytes and lymphoids have large nuclei, irregular shapes, and relatively reduced cytoplasm.
Measured rates of locomotion indicate that granulocytes moved significantly faster (3.3 um/min) than agranulocytes (0.7 um/min) because most (81 %) agranulocytes were not mobile. Of the mobile hemocytes, granulocytes were also significantly faster (4.8 vs. 3.5 um/min, P<0.0001), and basophilic granulocytes (BASOs) were the most active and abundant cell-type. Thus, our results show that granulocytes, especially the subpopulation BASOs, were the most numerous and mobile cell-types and may be the sentry cells of C. virginica. Their mobility may reflect their capabilities in phagocytosis (Fisher and Newell, 1986) since the molecular machinery necessary for phagocytosis and locomotion may be similar. Further, a portion of the granulocytes were not mobile, and the number of mobile cells decreased in May. This may reflect a seasonal change in function or ability to locomote.
Examination of monthly percentages of cells and ROL indicates that granulocyte dominance and ROL are not invariable. Granulocyte percentages of more than 60% in January, February, and March decreased to 32% in May, and granulocyte BASO dominance was reduced to 1 5% Further, percentages of mobile granulocytes decreased from greater than 65% in January, February, and March to 50% in May, and ROL decreased from greater than 2.8 um/min in these months to 2.2 um/min. in May. The fewer mobile hemocytes tracked in May had significantly (P<0.05) lower average ROL (4.0 um/min.) than those in January and March (4.7 um/min. each). Agranulocyte numbers increased in May due to an increase in nonlymphoid cells. The ROL for agranulocytes and the granulocyte subpopulation SGs decreased from January to May. This trend, however, was less certain for the other granulocytes BASOs and EOSs, suggesting that ROL for them, the most active cells, may be independent of the activity of the other cells. Agranulocytes were least often mobile, and the least mobile cell was the agranulocyte RF (round, flat), but it was not entirely immobile. Although the ROL for all hemocytes and the number of mobile cells decreased in May, the average ROL for mobile hemocytes was similar from January to March with a decrease in May. Thus, there was a change in May in the average ROL for all cells, and a decrease in the number of cells that were mobile.
In summary, general morphologic features and mobile behavior of oyster hemocytes show consistently definable characteristics under light microscopy. Although the results of this study do not indicate functional roles for the hemocytes, they do suggest morphologic diversity, especially in agranulocytes. New dominant cell-types may appear perhaps to fill a seasonal function. Although the ROL and the percentages of mobile, nonlymphoid agranulocytes changed little in comparison to mobile lymphoids over the 4 months, their increased percentage observed in May could relate to the life cycle of C. virginica in spring.
The response of oyster hemocytes to wounds and to foreign invasions is said to vary with the type of invader (Fisher, 1986). Farley (1968) has shown that agranular hemocytes predominate in response to the protozoan parasite Haplosporidian nelsoni while other investigators have found granular cells the most responsive to bacteria and viruses (Fisher, 1986).
Seasonality is an important factor in infection and mortality of Chesapeake Bay oysters by the pathogenic protozoan Haplosporidium nelsoni in the disease MSX (Andrews, 1984). Andrews has shown that the period of infectivity extends from mid-May to November. Our results have shown a drop in the numbers of granulocytes in May and an increase in the numbers of an agranulocyte. This may support Farley's finding that agranulocytes are the responsive cells in MSX infection. Fisher et al. (1989) also found a significant decrease in ROL in oyster mobile hemocytes between March and May. These changes in hemocyte types and ROL observed in the May oyster population may be related to physiological changes, coinciding with Andrews (unpublished) observations that oysters in Virginia begin developing gametes in May.
1. Andrews, J.D. Reproduction of oysters in Virginia. Unpublished manuscript. 1980.
2. Andrews, J.D. 1984. Epizootiology of diseases of oysters (Crassostrea virginica). Helgolander Meeresunters. 37:149-66.
3. Auffret, M. 1988. Bivalve hemocyte morphology. Am. Fish. Soc. Sp. Publ. 18, 169-177.
4. Bayne, B.L. (Ed). 1976. Marine mussels: Their ecology and physiology. Cambridge, Cambridge University Press. Pp. 1-506.
5. Cheng, T.C. 1975. Functional morphology and biochemistry of molluscan phagocytes. Ann. N.Y. Acad. Sci. 266, 343-379.
6. Cheng, T.C. 1981. Bivalves. In: "Invertebrate Blood Cells" (N.A. Ratcliff and A.F. Rowley, Eds.), pp. 233-300. Academic Press, New York.
7. Farley, C.A. 1968. Minchinia nelsoni (Haplosporidia) disease syndrome in the American oyster Crassostrea virginica. J. Protozool 15, 585-599.
8. Feng, S.Y., J.S. Feng, C.N. Burke, and L.H. Khairallah. 1971. Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca Pelecypoda) Z. Zellforsch. 120, 222-245.
9. Fisher, W.S. 1986. Structure and Functions of Oyster Hemocytes. In: "Immunity in Invertebrates" (M. Brehelin, Ed.), pp. 25-35. SpringerVerlag, Berlin Heidelberg.
10. Fisher, W.S. 1988. Environmental influence on host response. Am. Fish. Soc. Publ. 18, 225-237.
11. Fisher, W.S. and Roger I.E. Newell. 1986. Salinity effects on the activity of granular hemocytes of American oysters, Crassostrea virginica. Biol. Bull. 170,122-34
12. Galtsoff, P.S., 1964. The American oyster. U.S. Fish and Wildlife Service Fishery Bulletin 64, 1-480
13. Huffman, J.E. and M.R. Tripp. 1982. Cell types and hydrolytic enzymes of soft shell clam (Mya arenaria) hemocytes. J. Invertebra. Pathol. 40, 68-74.
14. Liebman, E. 1946. On trephocytes and trephocytosis: a study on the role of leucocytes in nutrition and growth. Growth 10, 291-330.
15. McCormick-Ray, M.G. 1987. Hemocytes of Mytilus edulis affected by Prudhoe Bay crude oil emulsion. Mar. Environ. Res. 22,107-122.
16. Narain, A.S. 1973. The amoebocytes of lamellibranch molluscs, with special reference to the circulating amoebodytes. Malacol. Rev. 6, 1-12.
17. Renwrantz, L.R., T.P. Yoshino, T.C. Cheng, K.R. Auld. 1979. Size determination of hemocytes from the American oyster, Crassostrea virginica, and the description of a phagocytosis mechanism. Jahrb Zool Abt Physiol Zoomorph 83, 1-12.
18. Ruddell, C.L. 1969. A cytological and histochemical study of wound repair in the Pacific oyster, Crassostrea gigas. PhD. Thesis, University of Washington, Seattle.
19. Takatsuki, S. I. 1934. On the nature and functions of the amoebodytes of Ostrea edulis. Quart. J. Microsc. Sci. 76, 379-431.