Structural and functional characterisation of the blood cells of the bivalve mollusc,Scrobicularia plana

Light and electron microscopical studies were carried out in order to characterise the blood cells of the bivalve mollusc, Scrobicularia plana. Three types of haemocytes were recognised: eosinophilic granular haemocytes, basophilic granular haemocytes and basophilic agranular haemocytes. The eosinophilic granulocytes were vesicular and contained large granules whereas the basophilic granulocytes were found to contain small granules and glycogen 'lakes'. The basophilic agranular haemocytes were significantly smaller than the granular haemocytes and had a high nucleus to cytoplasm ratio. Functional characterisation of the blood cells identified activity for the lysosomal enzymes: acid phosphatase, beta-glucuronidase, non-specific esterase and arylsulphatase. There was also a weak staining reaction for phenoloxidase and peroxidase activities. Phagocytosis of Gram-positive bacteria was demonstrated by the haemocytes and antibacterial activity was shown by cell-free haemolymph. Assays to determine release of reactive oxygen species from the haemocytes did not detect any reactive oxygen generation.     

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda,Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes.Extensive hemocyte aggregates (''islets'') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.     

Hemocyte classification and differential counts in the dungeness crab, Cancer magister

The primary purposes of this research were to describe and classify the circulating hemocytes of Cancer magister and devise a method for making differential hemocyte counts for crustaceans. C. magister hemocytes were classified using two simple criteria: the presence or absence of cytoplasmic granules and staining characteristics of the granules, if present. Hyalinocytes (HC) were devoid of granules, intermediate granulocytes (IG) contained basophilic granules or a mixture of basophilic and acidophilic granules, and eosinophilic granulocytes (EG) contained large, acidophilic granules. Hemocyte renewal and a hypothetical maturation sequence of C. magister hemocytes are described and discussed. Differential counts revealed that granulocytes were more abundant than hyalinocytes. For 22 crabs, the mean percentage (and range) of each hemocyte class was: IG, 65.97 (57.50–73.80); EG, 17.76 (4.70–26.47); and HC, 16.25 (3.40–34.67). After additional data are collected and analyzed, the routine use of differential counts may prove to be a valuable method for monitoring the status and health of C. magister and perhaps other crustaceans as well.     

An electron microscope study of the circulating leucocytes of the marine mussel,Mytilus edulis

Circulating leucocytes of the mussel, Mytilus edulis, were studied by electron microscopy. Based on morphological criteria, the leucocytes were classified as agranular and granular leucocytes, dependent upon the presence or absence of specific granules in their cytoplasm. Furthermore, the existing literature is being critically revised, and it is suggested that agranular and granular leucocytes might belong to the same cell line.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Ultrastructure of salivary glands of Ornithodoros (Ornithodoros) moubata (Ixodoidea: Argasidae)

The paired salivary glands of unfed adult Ornithodoros (Ornithodoros) moubata are composed of type I (agranular) and type II (granular) alveoli. Type I alveoli consis of one large central cell surrounded by peripheral cells having the morphology of fluid-transporting epithelia. Type II alveoli contain granular and agranular cells; the former are comprised of morphologically distinct types of cells (a, b, and c) containing granules of different structures and chemical composition with respect to polysaccharide and protein. The agranular cells are the interstitial and cap cells. Golgi bodies and rough endoplasmic reticulum (RER) are found in all granular cells and apparently are involved in granule formation. No appreciable structural changes were observed in type I alveoli during or after feeding. Type c cell granules are released before granules from types a and b cells and may contain anticoagulant substances that promote the blood flow of the host during the tick feeding. Although the cap cells are not structurally affected by feeding, interstitial cells are developed into transporting epithelia.     

Microscopical studies on the hemocytes of bivalves and their phagocytic interaction with selected bacteria

Hemocytes represent one of the most important defense mechanisms against foreign material in Mollusca. The morphology, hematological parameters and behaviour of hemolymph cells were studied in the southern quahogMercenaria campechiensis, the eastern oysterCrassostrea virginica, and the blood arkAnadara ovalis challenged with the bacteriaVibrio vulnificus andV. anguillarum. Two general classes of hemocytes (granular and agranular) exist inC. virginica andM. campechiensis. In contrast,A. ovalis possesses 3 general classes (granular, agranular and erythrocytes). Three types of granules were identified by light microscopy. When hemolymph cells were studied by transmission electron microscopy, the cytoplasm of hemolymph cells was noted to contain many organelles, including electron dense granules. Both agranular and granular hemolymph cells were capable of colchicine-sensitive pseudopodial movement and spreading. The results indicate that marine bivalves possess hemolymph blood cells which may play a role in the internal defense paralleling mammalian phagocytes. The morphology of these cells, as determined by light, scanning and transmission electron microscopy, showed some similarity to mammalian-mononuclear phagocytes. The sub-cellular events of molluscan hemocyte phagocytosis ofV. vulnificus andV. anguillarum were studied by both scanning and transmission electron microscopy. The role of these cells and the factors which govern their behavior are of economic and public health importance.     

Response of eastern hemlock looper hemocytes to selected stages of Entomophthora egressa and other foreign particles

Indirect evidence for the natural existence of the free-protoplast stage of the fungus Entomophthora egressa in the eastern hemlock looper, Lambdina fiscellaria fiscellaria, is presented. The protoplasts were viable after 72 hr postinjection and subsequent development in the host produced conidia characteristic of E. egressa. The hemocytes studied (plasmatocytes, granular cells, and spherule cells) did not adhere to the protoplasts either in vivo or in vitro. Cells of Escherichia coli and sporangiospores of Absidia repens adhered to the granular cells in vitro. The granular cells adhered to the hyphae of Rhizopus nigricans in vitro. The spherule cells strongly adhered to the hyphae and hyphal bodies of E. egressa in vitro. The protoplasts, hyphae, and conidia of E. egressa and the hemocytes of L. fiscellaria fiscellaria adhered to positively charged DEAE-Sephadex beads and not to negatively charged CM-Sephadex beads. Aspects of active and passive strategies for protoplast evasion of host hemocytes are discussed with some emphasis on hemocyte-protoplast electrostatic repulsion and active secretion of hemocyte inhibitors by the protoplasts.     

New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes

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.     

Hemocytes of the palaemonids Macrobrachium rosenbergiiand M. acanthurus,and of the Penaeid Penaeus paulensis

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages. J. Morphol. 236:209–221, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructures and classification of circulating hemocytes in 9 botryllid ascidians (chordata: ascidiacea)

Ultrastructures of circulating hemocytes were studied in 9 botryllid ascidians. The hemocytes are classified into five types: hemoblasts, phagocytes, granulocytes, morula cells, and pigment cells. These five types are always found in the 9 species. They should represent the major hemocyte types of the circulating cells in the blood. Hemoblasts are small hemocytes having a high nucleus/cytoplasm ratio. There are few granular or vacuolar inclusions in the cytoplasm. Phagocytes have phagocytic activity and their shape is variable depending on the amount of engulfed materials. In granulocytes, shape and size of granules are different among the species. Morula cells are characterized by several vacuoles filled with electron dense materials. In pigment cells, the bulk of the cytoplasm is occupied by one or a few vacuoles containing pigment granules. We also described some other hemocyte types found in particular species. Furthermore, we encountered free oocytes circulating in the blood in two species, Botryllus primigenus and Botrylloides lentus.     

Categorization of hemocytes of three gastropod species Trachea vittata (Muller), Pila globosa (Swainson) and Indoplanorbis exustus (Dehays)

Light microscopic observations were made on the hemocytes of three gastropod species namely Trachea vittata, Indoplanorbis exustus and Pila globosa. It revealed two basic types of hemocytes. They are agranulocytes and granulocytes. Agranulocytes are hyalinocytes which are round, unspread hemocytes and have a large nucleo-cytoplasmic ratio. Granulocytes are spreading hemocytes, forming numerous pseudopodia. For the purpose of differential counting, we present a categorization of the granulocytes into three sub-categories based on cell dimensions, nucleo-cytoplasmic ratio, distribution of granules in the cytoplasm and position of the nucleus. The smaller granulocytes are younger cells, and are termed Granulocytes I (Progranulocytes). The larger ones are fully developed cells that have been differentiated into Granulocyte II (basophilic) and Granulocyte III (eosinophilic).     

Salivary gland ultrastructure of the unfed adult and feeding female of Haemaphysalis (Rhipistoma) leachi (Ixodoidea: Ixodidae)

The paired salivary glands of unfed adult Haemaphysalis (Rhipistoma) leachi contain one type of agranular and three types of granular alveoli connected to a salivary duct system. Type I agranular alveoli consist of one large, central cell surrounded by peripheral cells with numerous basal membrane infoldings indicative of epithelia involved in fluid transport. Glycogen particles, lipid-like droplets, and the parallel pattern of infolded membranes disappeared from the peripheral cells during feeding. Types II, III, and IV granular alveoli contain some agranular interstitial epithelial cells, cap cells, and fundus cells, but are predominantly composed of structurally different granular cell types a, b, c, d, e, and f. Agranular cells develop during the early stages of feeding. Granular a, c, e, and f cells release their granules directly after attachment to the host and possibly are involved in cement secretion required for firm attachment to it. The b cell granules are replaced by b₁ filamentous granules during feeding. Golgi bodies and rough endoplasmic reticulum (RER) participate in the formation of most types of granules. The d cells contain lamella-like structures and condensing vacuoles, probably responsible for lysosome formation. The main salivary duct and all types of alveoli are innervated by neurosecretory axons.     

Interaction of molluscs and foreign substances: The morphology and behavior of hemolymph cells of the American oyster, Crassostrea virginica, in vitro

Hemolymph cells of Crassostrea virginica have been studied in the living state with apochromatic and brightfield-phase contrast microscopy and in the fixed and stained state with brightfield and Nomarski optics. Cells of two size populations have been recognized. The large cells are of two classes: granulocytes and fibrocytes. Granulocytes commonly include mixtures of acidophilic, basophilic, and refractile granules and are believed to be of one general type. Fibrocytes are subcategorized as being of the primary and secondary types, with the first enclosing lobate nuclei and the second enclosing spherical or ovoid nuclei. The small cells, designated as hyalinocytes, are either agranular or slightly granular.     

Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.     

Comparative studies on the statoblasts of higher phylactolaemate bryozoans

Statoblasts of five higher phylactolaemates were compared morphologically. As a result, they were divided into two groups: Group I comprising Lophopus crystallinus, Lophopodella carteri, and Pectinatella gelatinosa, and Group II comprising Pectinatella magnifica and Cristatella mucedo. These two groups are thought to represent independent evolutionary series. In Group I and in P. magnifica, the statoblasts are curved to varying degrees after the manner of a saddle. When the dorsal and ventral valves are flattened, therefore, the contour is different between the two. In Group I, the outermost layer of a mature statoblast is hard-gelatinous and basophilic; it remains intact after the statoblast is set free. The statoblast does not float until it is dry, and the float is similar in size on both valves. In Group II, a mature statoblast is covered by a softgelatinous basophilic layer, which decays after the statoblast is released. The statoblast floats without drying, and the float is better developed on the dorsal valve than on the ventral. Moreover, in the members of Group II, large yolk granules are first formed, followed by much smaller yolk granules. When their statoblasts are treated with KOH, the shell is separated completely into two valves. These characters are common to many lower phylactolaemates. By contrast, in L. carteri and P. gelatinosa, the yolk granules are uniformly small and the capsule proper resists KOH treatment. On these points, L. crystallinus is somewhat different from these two species, suggesting its primitive nature.     

The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles

Hemocytes of Mytilus edulis were examined cytologically and cytochemically. On the basis of structure, staining reactions, and phagocytic behavior, they were divided into two main groups: basophilic hemocytes and eosinophilic granular hemocytes (granulocytes). The basophilic cells were further divided into small lymphocytes and larger phagocytic macrophages reactive for lysosomal hydrolases. Mitosis was observed in granulocytes and in small lymphoid cells, believed to be the stem cells for the basophilic cell line. A few cells appeared to be intermediate between lymphocytes and small granulocytes. Macrophages were the main cell type involved in the clearance of injected carbon particles. However, granulocytes did show some phagocytic activity. Brown cells displaying apparent amoebocytic behavior were found to contain Fe³⁺ and Pb²⁺ in cytoplasmic inclusions, some of which were also reactive for β-glucuronidase and glucosaminidase. These cells appear to have a separate origin from the hemocytes.     

Cell types and hydrolytic enzymes of soft shell clam (Mya arenaria) hemocytes

Hemocytes of the soft shell clam, Mya arenaria, based on appearance after Romanowsky-type staining, can be shown to be either granulocytes or agranulocytes comprising 76.5 and 23.5% of the total cell population, respectively. Cytoplasmic granules are basophilic, eosinophilic, or refractile. Cytochemical studies indicate that these cells are markedly heterogeneous with respect to certain hydrolytic lysosomal enzymes and in cytoplasmic glycogen and lipofuscin. The overall activities of these enzymes in clam hemocytes, as estimated by the number of reactive sites, were unrelated to one another. Using consecutive double-staining techniques, individual cells were also found to vary in their enzyme content. These findings emphasize the biochemical individuality of circulating hemocytes and the variations noted probably reflect differences in number and composition of lysosomes.     

Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

The time course of clearance of an injected dose of 10⁶ CFU ml^?1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.