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.     

The role of the haematopoietic tissue in haemocyte production and maturation in the black tiger shrimp (Penaeus monodon)

The haematopoietic tissue (HPT) of the black tiger shrimp (Penaeus monodon) is located in different areas in the cephalothorax, mainly at the dorsal side of the stomach and in the onset of the maxillipeds and, to a lesser extent, towards the antennal gland. In young and in experimentally stimulated animals, the HPT is expanded in relatively larger and more numerous lobules throughout the cephalothorax. Four cell types could be identified in the HPT by electron microscopy. The type 1 cells are the presumed precursor cells that give rise to a large- and a small-granular young haemocyte, denominated as the type 2 and type 3 cells, respectively. A gradient of maturation from the type 1 towards the type 2 or 3 cells could frequently be observed. The presumed precursor cells are located towards the exterior of the lobules and maturing young haemocytes towards the inner part, where they can be released into the haemal lacunae. The type 4 cells show typical features of interstitial cells. Different stimulation experiments were carried out and various techniques were used to study the HPT in relation to the (circulating) haemocytes. The majority of the cells in the HPT are able to proliferate and proliferation can be increased significantly after the injection of saline and, to a much higher extent, after LPS injection. The circulating haemocytes of crustaceans are generally divided into hyaline (H), semigranular (SG) or granular (G) cells, of which large- and small-granular variants of each of these were suggested in the present study. Even after stimulation in this study, the circulating haemocytes scarcely divide. The high variations that were found in the total haemocyte count in the stimulation experiments were not accompanied by significant differences in differential haemocyte count and, therefore, appeared to be a less useful indicator of stress or health in P. monodon. Light and electron microscopical observations support the regulation of the populations of the different haemocyte types in the circulation by (stored) haemocytes from the connective tissue. In conclusion, according to morphological and immuno-chemical criteria, it is proposed in the present study to divide the haemocytes into a large-and a small-granular developmental series. After extensive morphological observations, it is suggested that the hyaline cells are the young and immature haemocytes of both the large- and small-granular cell line that are produced in the HPT, and can be released into the haemolymph. Indications were found that the granular cells, of at least the large-granular cell line, mature and accumulate in the connective tissue and are easily released into the haemolymph. Combining the results of the present study with literature, this proposed model for haemocyte proliferation, maturation and reaction will be discussed.     

红螯光壳螯虾白斑综合症血液病理学研究

采用Wright-Geimsa染色法和电镜技术对人工感染的红螯光壳螯虾(Cherax quadricarinatus)白斑综合症(White spot syndrome,WSS)血液病理学进行了研究。结果显示:患病螯虾血细胞总数、透明细胞(AH)数量极显著减少(P<0.01),大颗粒细胞(LGH)极显著增加(P<0.01);病毒感染后3种血细胞大小均有增加趋势,透明细胞和大颗粒细胞的核质比(NP)较感染病毒前极显著下降(P<0.01)。显微病理学变化主要表现为血涂片中血细胞明显减少,病变、破损或解体的细胞增多,至濒死期螯虾血液呈典型的溶血状态。超微病理学变化表现为血细胞受到了损伤。高尔基体变形、线粒体结构模糊破损;核膜变形核固缩、细胞核高度异染色质化;濒临死亡的螯虾血细胞细胞器和染色质溶解,胞浆水肿,细胞溶解坏死。在患病螯虾的血细胞核中清晰可见WSSV粒子。     

中华稻蝗血细胞染色方法的比较

通过Giemsa、Wright和Giemsa-Wright混合染色3种方法,对中华稻蝗Oxya chinensis(Thunberg)的血细胞进行了染色和血细胞的形态学观察,其结果是Giemsa染液染色时间较长,对细胞核染色效果较好,细胞质界限清晰,但对细胞质中的颗粒染色较差;Wright染液染色时间较短,对细胞核和细...     

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...     

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.     

Induced inflammatory process in Peripatus acacioi Marcus et Marcus (Onychophora)

The inflammatory response induced by the implant of a suture thread in Peripatus acacioi muscle was characterized under light and transmission electron microscopy (TEM). After 24 and 48 h granulocytes were observed migrating through the connective tissue toward the suture thread. These cells contain cytoplasmic eosinophilic granules as well as free granules near to the thread. There were few spherule cells with eccentric smooth kidney-shaped acidophilic nuclei and basophilic granules. Cells with intermediary characteristics as well as cells with a central basophilic nucleus with scarce acidophilic cytoplasm devoid of granules were also found. Under TEM, the granulocytic coelomocytes show small and homogeneous electron dense granules, while the spherule cells possess spherules that can be heterogeneous, granular, or with myelin figures. An acute induced inflammatory process is described for the first time in Onychophora and contributes to the scarce available literature on the function of the coelomocytes within this group.     

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.     

Immunohistologic and immunoelectron microscopic characterization of the mucosal lymphocytes of human small intestine by the use of monoclonal antibodies

Monoclonal antibodies reactive with T cells, T cell subsets, B cells, monocytes, and natural killer cells were used to characterize the nature of mucosal lymphocytes in the human small intestine by application of the immunoperoxidase technique to tissue sections for light and electron microscopic examination. In addition, for comparison, peripheral blood mononuclear cells (PBL) were studied by immunoelectron microscopy. Most of the intraepithelial lymphocytes (IEL) were T cells (Leu-1+, T3+) and expressed the phenotype associated with cytotoxic/suppressor T cells (Leu-2a+, T8+). In contrast, a majority of T lymphocytes in the lamina propria expressed the phenotype associated with helper/inducer T cells (Leu-3a+, T4+). These observations confirm and extend the findings previously reported. In addition, a small number of cells in the lamina propria with the ultrastructural features of macrophages were found to react with anti-Leu-3a and anti-T4 antibodies. Although many IEL contained cytoplasmic granules and had ultrastructural features similar to those of circulating granular lymphocytes, none of these cells reacted with anti-Leu-7 (HNK-1), anti-T10, or anti-M1 antibodies. This suggests that IEL may not be related to circulating large granular lymphocytes, which are Leu-7+, T10+, M1+ and are associated with natural killer activity. Not only Leu-7+ PBL, but T8+, T4+, or T3+ mucosal lymphocytes or PBL also may contain cytoplasmic granules. Therefore, the cytoplasmic granules are not restricted to one cell type, in particular, to Leu-7+ cells.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D₁, B, D₂, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.     

Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses

For the first time, morpho-functional characterisation of haemocytes from the cockle Cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. Haemocyte mean number was 5.5 (x 10(5)) cells/mL haemolymph. Two main haemocyte types were found in haemolymph: granulocytes (85%), about 10 microm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 microm in diameter, with a few or no granules. Most of the cytoplasmic granules stained in vivo with Neutral Red, indicating that they were lysosomes. On the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. Acidophil hyalinocytes were the largest haemocyte type (about 14 microm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. Both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2%). It increased significantly (up to 26%) after pre-incubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. Haemocytes also produced superoxide anion. Moreover, both granulocytes and hyalinocytes (except acidophils) were positive for some important hydrolytic and oxidative enzymes. Lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. Results indicate that haemocytes from C. glaucum are effective cells in immune responses.     

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.     

Insight on cellular and humoral components of innate immunity in Squilla mantis (Crustacea, Stomatopoda)

For deeper insights into the function of crustacean haemocytes in immune responses, we studied the morphology and enzyme content of circulating cells of the mantis shrimp Squilla mantis from the North Adriatic Sea, together with their ability to phagocytose foreign cells. We also assayed the enzyme content and the agglutinating and haemolytic activities of cell-free haemolymph. Three haemocyte types, i.e., hyalinocytes, semigranulocytes and granulocytes, can be distinguished, according to cell and nuclear morphology and the presence of cytoplasmic granules. All of them share the same patterns of enzyme activities and are recognised by the same lectins. Spreading cells (hyalinocytes and semigranulocytes) can ingest foreign cells; granules of semigranular and granular cells have similar cytochemical properties. Injection of Micrococcus luteus into the heart sinus results in an increase in the frequency of hyaline cells and a decrease in the frequency of granulocytes. After 24 h from the injection, a decrease in the number of phagocytosing hyalinocytes, and a general decrease in the frequency of acid phosphatase-positive cells was reported. Our data match previous results and suggest the existence of a single differentiation pathway for Squilla haemocytes with the three haemocyte morphs as different stages of cell differentiation. Results also indicate that Squilla haemolymph performs immunosurveillance, through rapid changes in haemocyte distribution, increase of antimicrobial and antioxidant enzymes and secretion of lectins stimulating agglutination, phagocytosis and encapsulation.     

Ultrastructure of the haemocytes of Tetrodontophora bielanensis Waga (Collembola)

Five types of haemocytes: prohaemocytes, plasmatocytes, granular haemocytes, spherule cells and phagocytes, have been distinguished on the basis of ultrastructural studies. Prohaemocytes are ovoid cells with a simple structural organization. Plasmatocytes are larger; their cytoplasm contains well-developed rough endoplasmic reticulum, numerous mitochondria and free ribosomes. Granular haemocytes are the most numerous of the blood cells, characterized by the presence of electron-dense granules. The cytoplasm of spherule cells contains many spherules made up of filamentous material of medium electron density. Rough endoplasmic reticulum, free ribosomes and mitochondria are also found in the cytoplasm. Phagocytes are the largest haemocytes. Their cytoplasm contains an abundance of lysosomes and myelin structures. In addition to haemocytes, cells intermediate between plasmatocytes and granular haemocytes have been observed, which indicates that the granular haemocytes are derived from plasmatocytes.     

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.     

Mast cells in the developing avian eye

Mast cells are present in the eye of Gallus domesticus, appearing in the anterior uvea in embryos at stage 39 HH (13th day). In hatching and adult birds, they are present in the sclera, uvea, pectinate ligament, and conjunctiva. Mast cells are absent in the cornea, retina, and pecten oculi. Maturing mast cells in the anterior eye segment appear as round cells having eccentric nuclei and a few cytoplasmic metachromatic granules, whose fluorescence increases during development. Mature cells are more numerous in late development, and their cytoplasm is rich in metachromatic and intensely fluorescent granules. Ultrastructurally, maturing mast cells display progranules and a few electron dense and homogeneous granules on one side of the cell. Mast cells of adult birds possess homogeneous cytoplasmic granules, some of which display protuberances that penetrate hollows of adjoining granules. Heterogeneous granules exhibiting latticed and mottled patterns are also present. The existence of mast cells in the anterior eye segment indicates that these cells might perform a physiological role during development and in aqueous humor outflow. They might modulate exchanges between blood and aqueous humor through chemical mediators present in their granules. © 1996 Wiley-Liss, Inc.     

Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca: Pelecypoda)

Summary The fine structure of oyster leucocytes resembles to a great extent, that of typical eucaryotic cells. Organelles which have been described for the first time in this report are light granules, dense granules, protocentriole and X structure. Light microscopy reveals two morphological types of oyster leucocytes: agranular and granular. Based upon nuclear morphology and cytoplasmic compositions revealed in electron microscopy, at least three types of agranular and one type of granular cells are recognized.In the Giemsa-stained preparations, granular leucocytes exhibit three distinct types of cytoplasmic granules: refractile, dark blue, and pink, which presumably correspond to light granules Type A, B, and C seen in the electron micrographs. A granular leucocyte may contain one or more types of granules. Cytochemical investigations show that oyster leucocytes contain at least three hydrolytic enzymes: non-specific esterases, acid, and alkaline phosphatase. The latter two enzymes constitute 63% of the enzyme activity detected. These intracellular enzymes may be associated with the light granules and/or lysosome-like bodies.It is also demonstrated that the granular leucocyte population is significantly higher (P<0.001) in the oysters experimentally infected with Bacillus mycoides (72.19±4.71%) as contrasted with that of the controls (37.18±4.48%).Leucocytes in progressive stages of degeneration are also described.Contribution No. 71 from Marine Research Laboratory, University of Connecticut.The initial phase of this investigation was carried out at the Department of Zoology, Rutgers, The State University, New Brunswick, New Jersey, and supported by Public Health Service Research Grant AI-00781 from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, awarded to Dr. L. A. Stauber. Supported by a grant from the University of Connecticut Research Foundation and Faculty Summer Fellowship to S. Y. Feng.     

Ultrastructure of the epithelial cells of the ventral prostate from the hopping mouse Notomys alexis

The large ventral prostate of the hopping mouse has abundant secretory units whose epithelial cells vary in height and which often have nuclei in the apical region of the cell. TEM observations indicated two epithelial cell types in which some unusual features occurred. Type A cells had granular endoplasmetic reticulum (GER) whose membranes often formed intracytoplasmic confronting cisternae. Type B cells had more fragmented and vesiculated GER with sparse ribosomes and less frequently also intracytoplasmic confronting confronting cisternae. In the latter cells, two types of granules were found, one of which was derived from the Golgi and the other possibly directly from the GER. Type A cells only had one type of granule present. A highly convoluted membrane was also found at the basal region in many of the cells. The significance of these unusual ultrastructural features has yet to be ascertained.     

A comparative ultrastructural study of the trigeminal ganglion in the rat and chicken

The neurons of the trigeminal ganglia of the rat and chicken were characterized by means of light microscopic, electron microscopic, and histochemical methods. Light microscopy disclosed four types of neurons, based on the characteristics of Nissl granules: (1) large neurons with diffusely distributed and very fine granules, (2) neurons containing coarse and sparsely distributed Nissl granules, (3) neurons containing dense Nissl granules of varying size, and (4) small neurons with granules concentrated peripherally. Electron microscopy allowed further definition of these four types of neurons by the length and arrangement of flattened cisterns of granular endoplasmic reticulum (gER) and the number of neurofilaments. Type 1 cells were largest, with a mean nuclear area of 139.8 ± 28.3 μm². Type 4 cells were smallest, with a mean nuclear area of 74.6 ± 20.9 μm². The mean nuclear areas of type 2 and 3 cells were intermediate to those of the type 1 and 4 cells. Type 3 and 4 neurons lacked neurofilaments. Four forms of Golgi apparatus were found: (1) large bent grains forming a network throughout the soma, (2) dispersed fine granular deposits, (3) fine or small granules, and (4) coarse bent deposits arranged confluently in the perinuclear zone. In some rat neurons, the concentration of acid phosphatase reaction products suggested a high enzymatic activity, whereas the chicken ganglion cells showed no such concentration. These findings are discussed and compared with the classifications of previous studies.     

中国鲎变形细胞超微结构的进一步观察

中国鲎的变形细胞可在血液中发育成熟,根据其超微结构,可分为原始变形细胞、幼变形细胞和变形细胞三个阶段。 变形细胞受到外环境或内毒素的刺激后,第一型颗粒出现一系列变化,最终把内容物释放到细胞外。与此同时,细胞表面形成若干胞质性突起,边缘带分散断裂,质膜下和胞质性突起中出现微丝。第二型颗粒无变化,提示它们和血液凝胶化过程无直接关系。