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.     

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).     

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.     

Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis

BACKGROUND: The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD: This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS: It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS: With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.     

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.     

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis.     

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.     

Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrongylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, especially rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER). They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrodensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrodense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasm is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/mm3 of hemolymph in the 4th instar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.     

Fine structure and classification of shrimp hemocytes

The structure of hemocytes from two species of penaeid shrimp was examined by light and electron (TEM) microscopy. Hemocytes from the two species are indistinguishable and are classified as either agranular, small-granule, or large-granule hemocytes. Agranular hemocytes are the smallest of the hemocytes, lack granules, compose only 5–10% of the circulating hemocytes, and are nonrefractile when examined by light microscopy. Small-granule hemocytes are the most abundant type of hemocyte (75% of all hemocytes), appear nonrefractile, and contain a variable number (1–40) of granules (0.4 μm diameter). Large-granule hemocytes compose 10–20% of the hemocytes. They are filled with granules (0.8 μm in diameter) that are highly refractile when examined by light microscopy and are electron-dense when examined by TEM. Our classification scheme is based solely on the absence or presence and relative size of granules. Features used by other researchers, such as cell size, shape, and staining properties, were not used because these features are subtle and/or subjective. The proposed classification is compared with schemes developed for other decapods, and its usefulness and limitations are discussed. This scheme will serve as a basis for further studies on the maturation and physiological function(s) or crustacean hemocytes.     

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.     

Schistosoma haematobium in Bulinus guernei: Electron microscopy of hemocyte-sporocyst interactions

Electron microscopy has revealed that in Bulinus guernei (Gambian strain) snails infected with Schistosoma haematobium (Egyptian strain) daughter sporocysts and cercariae, two kinds of hemocytes called granulocytes and hyalinocytes are found associated with the sporocysts. Granulocytes are small, numerous, plumbophilic, and amoeboid. They contain lysosome-like granules. Hyalinocytes are large, sparse, less plumbophilic than granulocytes, and have intracellular microfilaments (about 9 nm wide), and few or no pseudopods. They are devoid of lysosome-like granules. Granulocytes and hyalinocytes infiltrate near sporocysts, but only granulocytes interact with sporocyst microvilli by contact. Granulocytes induce a restricted multilamellated encapsulation reaction. Extracellular microfilaments (about 12.5 nm wide), with a regular transverse structure pattern of about 50-nm periodicity, frequently are found along the outer surface of granulocytes located adjacent to sporocysts. Intracellular filamentous structures and a prominent glycocalyx also are features of the seemingly more active granulocytes contiguous with sporocysts. Cell adhesions may occur between surfaces of (1) granulocytes and sporocysts, (2) interdigitating pseudopodial processes of capsular granulocytes, and (3) granulocytes and hyalinocytes.     

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.     

贝类血细胞硫代黄素T荧光染色方法

硫代黄素T( thioflavin T,TFT)是一种用于组织学的苯并噻唑荧光染料,因其对淀粉样蛋白有高亲和性而主要被用于淀粉样病变的荧光显微检测.本研究分别以软体动物门双壳纲的栉孔扇贝(Chlamys farreri)和中国蛤蜊(Mactra chinensis)、腹足纲的拟紫口玉螺(Natica janthosto...     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Flow cytometric and light microscopic identification of hemocyte subpopulations in <Emphasis Type="Italic">Modiolus kurilensis</Emphasis> (Bernard, 1983) (Bivalvia: Mytilidae)

Flow cytometry using forward and side light scattering identified three cell subpopulations in the hemolymph of Modiolus kurilensis (Mytilidae). Light microscopic data indicated that they correspond to three hemocyte morphotypes: R1, hemoblasts and agranulocytes (hyalinocytes); R2, semigranulocytes; and R3, granulocytes. The hemoblasts, agranulocytes, and semigranulocytes had a basophilic endoplasm, while the granulocytes were mainly eosinophilic. The proportion of the different cell morphotypes and the level of subpopulation distinction substantially varied among individuals. This seems to be connected with the different functional activities of the hemocytes, depending on the immunological status of the bivalves.     

圆背角无齿蚌血细胞培养

用新设计的培养基培养了圆背角无齿蚌的血细胞,在倒置相差镜下进行了活体观察及扫描电镜摄影,发现培养的血细胞无颗粒细胞、颗粒细胞、透明细胞和类淋巴细胞,前三者均能伸出长的伪足和突起,与瓶壁紧密贴附,呈体外培养的成纤维细胞型;后者呈圆形,不与瓶壁贴附;四者的比例约为4：2：3：1。在活体内注射或在培养基上加入PHA和ConA,培养2－7d中,每天取部分供加入秋水仙素,用空气干燥法制片,作染色体观察,但未观察到转化细胞和有丝分裂相。研究结果表明,圆背角无齿蚌的颗粒细胞、无细胞和透明细胞在体外贴附玻璃表面的特征与高等动物的巨噬细胞类似,而不贴瓶的圆形细胞与高等动物的淋巴细胞类似,但在体外培养均不能繁殖,它们可能是高度分化的细胞。     

Cytological response of hemocytes in the European flat oyster, Ostrea edulis, experimentally exposed to mercury

Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.     

Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 μm in diameter, with small granules (0.05-0.5 μm in diameter), large (5.00-9.00 μm in diameter, with large granules (0.50-2.50 μm in diameter) and combination (5.00-9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae): Effects of parasitism on differential hemocyte counts,micro‐ and ultra‐structures of host hemocytes

Abstract Parasitism by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) by using only its associated venom, can suppress the immunal responses of Pieris rapae (Lepidoptera: Pieridae). However, up to now, current knowledge of the mechanisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy (TEM). Five hemocyte types, prohemocytes (PRs), granulocytes (GRs), plasmatocytes (PLs), oenocytoids (OEs) and coagulocytes (COs), were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.