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.     

红褐斑腿蝗血细胞的形态与分类

利用光学显微镜和显微数码拍照系统,对红褐斑腿蝗Catantops pinguis(Stal)血细胞的形态进行观察和分类。结果在红褐斑腿蝗血淋巴中观察到5种血细胞,分别是原血胞、浆血胞、粒血胞、珠血胞和囊血胞。原血胞为小型圆形细胞,边缘圆滑、清晰,核质比例很大。粒血胞多为中型,形状不规则,边缘凹突不平,内含较大的异质性溶酶体颗粒。浆血胞多为中型,刚离体时形状较规则,常呈圆形、卵圆形。浆血胞内缺乏大的颗粒,细胞核大而圆形,细胞质内具许多小型颗粒状物质。浆血胞离体后形状变化较多,常发展出伪足,呈丝状、短芒状、钩状或片状伪足。珠血胞多为大、中型,外形大体呈圆形,但边缘由于大小不等的珠形内含物突出,呈花瓣状。囊血胞多为中型,圆形或椭圆形,细胞质内具有大小不一的带有折光性的颗粒或块状物,细胞边缘比较光滑。     

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.     

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.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

Classification of larval circulating hemocytes of the silkworm,<Emphasis Type="Italic"> Bombyx mori</Emphasis>, by acridine orange and propidium iodide staining

Circulating hemocytes of the silkworm can be classified by fluorescence microscopy following staining with acridine orange and propidium iodide. Based on their fluorescence characteristics, three groups of circulating hemocytes can be distinguished. The first group, granulocytes and spherulocytes, is positive for acridine orange and contain bright green fluorescent granules when observed by fluorescence microscopy. In granulocytes, these green granules are heterogeneous and relatively small. In contrast, in spherulocytes, the green granules appear more homogenous and larger. The second group of hemocytes consists of prohemocytes and plasmatocytes. These cells appear faint green following staining with acridine orange and do not contain any green fluorescent granules in the cytoplasm. Prohemocytes are round, and their nuclei are dark and clear within a background of faint green fluorescence. Inside the nucleus there are one or two small bright green fluorescent bodies. Plasmatocytes are irregularly shaped and their nuclei are invisible. Oenocytoids belong to the third group, and their nuclei are positive for propidium iodide. Therefore, all five types of circulating hemocytes of the silkworm, including many peculiar ones that are difficult to identify by light microscopy, can now be easily classified by fluorescence microscopy following staining with acridine orange and propidium iodide. In addition, we show that hemocytes positive for acridine orange and propidium iodide are in fact living cells based on assays for hemocyte composition, phagocytosis, and mitochondrial enzyme activity.     

A comparative ultrastructural study of blood cells from nine insect orders

Summary An ultrastructual study of hemocytes from 9 different insect orders has led to the identification of 8 cell types: (1) Plasmatocytes, whose cytoplasm is filled with small dense lysosomes and large heterogeneous structures, are phagocytic cells. (2) Granulocytes, filled with uniformly electron dense granules, are involved in capsule formation. (3) Coagulocytes, which contain granules and structured globules and which possess a well developed RER, are involved in phagocytosis. (4) Spherule cells are filled with large spherical inclusions. (5) Oenocytoids are large cells with few cytoplasmic organelles. These 5 hemocyte types represent the majority of insect blood cells. (6) Prohemocytes, blastic cells which are one of the stem cells of hemocytes, are very few in number in each species investigated. (7) Thrombocytoids and (8) Prodocytes are restricted to a small number of insect species.The ultrastructural characteristics of these hemocyte types are discussed.     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

An Ultrastructural Study of the Hemocytes of the Pericardial Body in the Ascidian Ciona intestinalis (L.)

Abstract The hemocytes of the pericardial body of Ciona intestinalis were studied by electron microscopy. Our findings showed that stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes and globular granulocytes are present at the periphery of the smaller-sized pericardial bodies. The stem cells are small round cells with a large nucleus, with or without nucleolus, and homogeneous cytoplasm containing numerous ribosomes. The clear vesicular granulocytes are characterized by an ameboid shape and cytoplasm containing several large electron-lucent vacuoles and small electron-dense granules. The microgranulocytes are variable in shape and contain numerous large electron-dense granules. The unilocular granulocytes show a single large vacuole with an electron-dense or electron-lucent content and a thin layer of peripheral cytoplasm that contains the flattened nucleus. The globular granulocytes are characterized by the presence of large vacuoles containing either fibrogranular material or electron-dense aggregates.     

Hemocytes and hemocytopoiesis in Silkworms.

A brief review is presented of the current state of ultrastructure, cytochemistry, and physiology of the hemocytes and meso- and metathoracic peri-imaginal-wing organs in silkworms. According to the accepted morphological classification, five circulating types of hemocytes are recognized in Bombyx mori as well as in Antheraea pernyi. They are prophemocytes or stem cells, plasmatocytes or pre-differentiated cells and three specialized cells, granulocytes, spherule cells and oenocytoids. During post-embryonic development the last four types are the most common in the circulating hemolymph. Plasmatocytes are considered to be pluripotent cells from which granulocytes, spherule cells and oenocytoids are derived. Contrary to the situation in most insects the plasmatocytes are not phagocytic in Antheraea. The granulocytes are efficient phagocytes. Both plasmatocytes and granulocytes are involved in pinocytosis. Another possible function of the granulocytes is hemolymph coagulation. The function of the spherule cells which contain a paracrystalline material (muco- or glycoproteins) is by no means clear. The phenoloxidase activity found within the cytosol of oenocytoids appears effective against the natural monophenol and diphenol substrates. The involvement of oenocytoids in the complex metabolism of phenols and particularly in the production of plasma phenolases has been reported. The mitotic division of five circulating hemocyte types is well known and was long regarded as the only mechanism of postembryonic hemocyte production. We present for silkworms, experimental evidence of the hemocytopoietic function of the meso- and metathoracic organs surrounding the imaginal wing discs. Ablation experiments demonstrate that the mitotic activity of free hemocytes is unable to maintain the normal hemocytogram in the absence of the two paris of organs. These organs are typically divided into cell islets ensheathed by a connective tissue membrane. Two types of islets may be classified by the disposition of the cells : the compact islets or aggregations of stem cells and the reticulate islets which are mainly composed of hemocytes at different steps of differentiation. The relative number of prohemocytes in the total hemocyte population ranges from 84 to 97 p. cent in organs of Antheraea pernyi. This well-defined cell type appears to be the major hemocyte type in hemocytopoietic organs. In Antheraea, the mitotic index (the relative number of mitotic hemocytes in the total cell population) varies from 0.5 to about 3 p. cent. Finally, our data direct attention to cyclic functional changes such as mitotic divisions and hemocyte differentiation which run parallel to the molting cycle.     

Characterization of the Hemocytes in Larvae of Protaetia brevitarsis seulensis: Involvement of Granulocyte-Mediated Phagocytosis

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.     

华北大黑鳃金龟幼虫血细胞的超微结构观察

应用光学和电子显微镜技术检查了华北大黑鳃金龟3龄幼虫血淋巴内的血细胞,识别出5种类型的血细胞(原血胞、浆血胞、颗粒血胞、珠血胞和凝血胞)并对每一种血细胞的超微结构特点进行描述。     

Morula-like cells in photo-symbiotic clams harboring zooxanthellae

Symbiosis is observed between zooxanthellae, symbiotic dinoflagellates, and giant clams and related clams which belong to the families Tridacnidae and Cardiidae. We have previously shown that a photo-symbiotic clam Tridacna crocea has three types of hemocytes, the eosinophilic granular hemocyte with phagocytic activity, the agranular cell with electron lucent granules, and the morula-like cell with large (ca. 2 mum in diameter) colorless granules. The function of the morula-like cell is not clear, but it has not been reported in any other bivalves except photo-symbiotic clams T. crocea and Tridacna maxima. In order to clarify whether it is specific to photo-symbiotic clams or not, we studied hemocytes in the photo-symbiotic clams Tridacna derasa (Tridacnidae), Hippopus hippopus (Tridacnidae) and Corculum cardissa (Cardiidae), and a closely related non-symbiotic clam Fulvia mutica (Cardiidae). The eosinophilic granular hemocytes and the agranular cells were found in all of the clams examined. However, the morula-like cells which were packed with many large electron dense granules (ca. 2 mum in diameter), were observed only in the photo-symbiotic clams. In F. mutica, a closely related non-symbiotic clam, this type of hemocyte was not found. Instead a hemocyte with vacuoles and a few large granules containing peroxidase activity was observed. The large granules of F. mutica varied in size from ca. 1-9 mum in diameter. Present data suggests that the presence of morula-like cells is restricted to photo-symbiotic clams and that the hemocytes associated with the morula-like cells may have some functional relationship to symbiosis with zooxanthellae.     

Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda

Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs. In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda. For both species, hemocyte densities per microl of blood increased with instar. Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar. Hematopoietic organs were located in the meso- and metathorax of S. Frugiperda and P. Includens. These organs contained large numbers of hemocytes in S. Frugiperda, but contained few hemocytes in P. Includens. The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes. Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA. BrdU labeling indices for both species also fluctuated with the molting cycle. Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S. Frugiperda but not in P. Includens. Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation. Arch.     

Cytochemical aspects of Mercenaria mercenaria hemocytes.

The hemocytes of the hard clam M. mercenaria were of three types: an agranulocyte, a small, and a large granulocyte. The agranulocyte, with only a thin periphery of cytoplasm surrounding the nucleus, had no visible cytoplasmic granules in living preparations but did exhibit a few centers of nonspecific esterase activity. This cell type represented 2% of the hemocyte population. The small granulocyte possessed four distinct granule types and comprised 61% of the total cell population. Large granulocytes accounted fro 37% of all hemocytes. While they contained the same four granule types identified in the small granulocyte, only one-third the total number were present. The nucleus of all three hemocyte types appeared morphologically similar. The four types of granules observed were a blunt, dot-like, a refractile and a filamentous granule. Blunt granules were identified as mitochondria, based on their ability to reduce Janus Green B to diethyl safranin, the presence of NADH dehydrogenase activity and boundary staining with Sudan black B. Dot-like granules were identified as lysosomes on the basis of neutral red staining, localization of acid phosphatase and nonspecific esterase activity and staining with Sudan black B. Refractile granules were demonstrated to be membrane-bound, lipid-filled structures that reacted positively with Sudan black B and Oil red O, respectively; these granules act as lipid storage centers. Nuclear similarity of the three cell types suggest that these cells might represent different stages of maturity, rather than three distinct cell lines. This was also indicated by the similar yet graded cytochemical reactions and the varying degree of motility and phagocytic activity demonstrated by hemocyte types.     

Unaltered hematocrit values and differential hemocyte counts in acanthocephalan-infected Armadillidium vulgare

Three cell types were present in Armadillidium vulgare hemolymph. Hyaline cells, 8.2 to 12.0 μm in size with a few fine granules in the cytoplasm, comprised 42% of the cells. Twelve percent of the cells were nonexplosive granulocytes, 8.8 to 15.0 μm, with many cytoplasmic granules of medium size. A third cell type, an explosive 7.2- to 12.0-μm granulocyte with coarse cytoplasmic granules, comprised 46% of the cells. Within 48 hr after adult specimens of A. vulgare were fed eggs of the acanthocephalan, Plagiorhynchus cylindraceus, isopod hemocytes aggregated in the intestinal epithelium surrounding penetrating acanthors. Encapsulation and death of the parasite routinely followed. No significant difference in hematocrit values or in differential hemocyte counts occurred between infected and uninfected control isopods.     

Lobster (Homarus americanus) hemocytes: Classification,differential counts,and associated agglutinin activity

Four hemocyte types were recognized in the lobster based on size and refractile nature of the granules, the ratios of cytoplasm to nucleus, and Giemsa stain characteristics. Two hyaline types were designated as prohyalocytes (1.8%) and hyalocytes (64.2%), and two granular types were termed eosinophilic granulocytes (12.2%) and chromophobic granulocytes (21.9%). There was no significant difference in the percentages of the different hemocyte types (differential hemocyte counts) between sexes, but hyalocyte and eosinophilic granulocyte percentages varied significantly between populations of lobsters. The data suggested that the difference in agglutinin activity (HA) between lobsters with the same total hemocyte numbers was due to activity associated with fixed hemocytes or quantitative differences in HA activity associated with one or more hemocyte types, rather than an increase in the percentage of any one particular type in circulation.     

Internal defenses of the snail Biomphalaria glabrata.

We describe three distinct types of cells among Biomphalaria glabrata hemocytes: large cells with a tubulo-vesicular compartment, a component of the endocytic system, and with numerous mitochondria and large aggregates of glycogen particles; medium-size cells poor in organelles and glycogen; and small cells with organelles and few secretory granules. Other small hemocytes can be interpreted as juvenile cells. B. glabrata hemocytes contain few enzymes and do not show specific secretory granules, except for a subpopulation of large cells richer in acid phosphatase vesicles. Hemocytes have different aspects corresponding to different physiological states and their transitions: in quiescent hemocytes, the cell cortex is narrow and organelles are scattered in the cytoplasm, both in circulating cells characterized by thin-folded filopods and large macropinocytic vacuoles and in sedentary cells in which extended filopods connect to the extracellular matrix. In stress-activated hemocytes, the cortical region is thickened by polymerization of actin, and organelles are gathered around the nucleus. Fixed phagocytes are components of the connective tissue; the presence of numerous lysosomes and residual bodies and of acid phosphatase and peroxidase activities suggests a high phagocytic activity.     

Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera,Carabidae)

The present study focuses on the ability of Pterostichus melas italicus Dejean to mount cellular and humoral immune responses against invading pathogens. Ultrastructural analyses revealed the presence of five morphologically distinct types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. Differential hemocyte counts showed that plasmatocytes and granulocytes were the most abundant circulating cell types and plasmatocytes exhibited phagocytic activity following the latex bead immune challenge. Macrophage-like cells were recruited after the immune challenge to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules formed to immobilize the latex beads. Total hemocyte counts showed a significant reduction of hemocytes after latex bead treatment. Phenoloxidase (PO) assays revealed an increase of total PO in hemolymph after immune system activation with lipopolysaccharide (LPS). Moreover, the LPS-stimulated hemocytes showed increased protein expression of inducible nitric oxide synthase, indicating that the cytotoxic action of nitric oxide was engaged in this antimicrobial collaborative response. These results provide a knowledge base for further studies on the sensitivity of the P. melas italicus immune system to the environmental perturbation in order to evaluate the effect of chemicals on non-target species in agroecosystems.