Abstract
Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge.
Highlights
Hemocytes of bivalve mollusks are capable of phagocytosis, encapsulation, and enzymatic digestion [1], and play a major role in the immune system and homeostasis [2]
Morphological and ultrastructural analyses of hemocytes Light-microscopy observation of C. rhizophorae hemocytes stained with May-Grunwald-Giemsa showed three main cell types: hemoblast-like cells, agranular cells, and granular cells (Figure 1)
Different approaches were used to examine the hypothesis that a single cell type could account for all the hemocyte morphologies that have been widely observed in bivalve hemolymph
Summary
Hemocytes of bivalve mollusks are capable of phagocytosis, encapsulation, and enzymatic digestion [1], and play a major role in the immune system and homeostasis [2] Their ability to transport proteins and substances such as heavy metals from organ to organ confers on them an important role in response to toxins [3,4]. Recent evidence from other invertebrates shows that hemocytes have stem cell-like behavior, giving rise to neurons in the cerebral ganglia [6,7], which suggests that these cells may be even more important than previously thought Despite their enormous potential for the study of physiological ecology, research on hemocytes has been impeded by the lack of a consensus on their classification. Flow cytometry has been applied to analyze the variations in hemocyte number and frequency; the strength of the conclusions in these studies is, in our opinion, limited by the small number of specimens [10,14,15]
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