Fine structure of the hemocytes and nephrocytes of Argas (Persicargas) arboreus (Ixodoidea: Argasidae)

The fine structure of the hemocytes and nephrocytes in Argas (Persicargas) arboreus is described and compared with that of similar cells in other tick species and insects. The hemocytes are of three types: prohemocytes, with a relatively undifferentiated cytoplasm lacking granular inclusions and probably serving as progenitors of the other hemolymph cell types; plasmatocytes, containing abundant mitochondria, cisternae of rough endoplasmic reticulum (RER), and free ribosomes, as well as some small granular inclusions; granulocytes, the predominant cell type in the hemolymph, containing numerous granules of variable electron density and maturity, and pseudopodia-like processes on the cell surface. Plasmatocytes and granulocytes are phagocytic and possibly also have other functions in the tick body. Cells with intermediate features appear to be in a stage of transition from plasmatocyte to granulocyte. Nephrocytes contain vacuoles enclosing fibrillar material, some electrondense granules, and moderate amounts of the active organelles—mitochondria, RER, and ribosomes. The nephrocyte is surrounded by a basal lamina and its plasma membrane infolds to form many deep invaginations coated by a fine fibrillar material. Openings to these invaginations are closed by membranous diaphragms. Coated tubular elements connect the surface invaginations with large coated vesicles, which appear to be specialized for internalization of proteins from the hemolymph. The dense granules may represent an advanced stage of condensation of ingested protein and thus may be lysosomal residual bodies, or they may develop by accumulation of secretory products.     

Fine structure of the midgut epithelium in the developing brown shrimp,Penaeus aztecus

The midgut epithelium of larval and early postlarval brown shrimp has been studied with light and electron microscopy. Ultrastructurally the features of the midgut do not change during these stages of development. On the basis of electron density, two epithelial cell types can be distinguished, and these are referred to as light and dark cells. The dark cells contain more rough endoplasmic reticulum and more free ribosomes than the light cells. Mitochondria in the dark cells have a matrix which is less electron dense than the mitochondrial matrix of the light cells. Both cell types have a microvillous border with a surface coat. The microvilli lack microfilaments within their core, and a terminal web is not differentiated in the stages examined. Tubular smooth endoplasmic reticulum is abundant in the basal portions of the cells. Electron dense, membrane bound vesicles are consistently seen in association with the Golgi apparatus, apical cell surface, and gut lumen and therefore are believed to be secretory granules. Cells in the anterior portion of the midgut often contain very large lipid droplets in the cytoplasm.     

Reexamination of hemocytes in brine shrimp (Crustacea, branchiopoda)

In 1941, a single type of hemocyte was described in the blood of the brine shrimp Artemia salina using light microscopy. This condition is unusual because most crustaceans examined using morphological, cytochemical, and functional methods have at least two types of hemoctyes. Upon examining A. franciscana, we found a single type of disk-shaped hemocyte, with a centrally located nucleus and about 15 large (6 microm diameter) granules. The granules stain for the presence of acid phosphatase and react with L-DOPA suggesting, respectively, that they are involved in degrading ingested material and possess the phenoloxidase system. Hemocytes require calcium for adhesion, bind together to mend small wounds in the body wall, and are able to phagocytose bacteria. Blood cells of A. franciscana are morphologically and functionally similar to those of the primitive chelicerate, Limulus polyphemus, and both forms have apparently given rise to more advanced taxa with multiple types of hemocytes. The major difference between the two species is the presence of the phenoloxidase system in the Crustacea and its apparent absence in the chelicerates.     

Antimicrobial activity of histones from hemocytes of the Pacific white shrimp.

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 microm while a mixture of histones H2B and H4 was active at 3 microm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.     

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。     

Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.     

Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon)

An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 x 10(5). Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.     

Aspects of classification of Hemiptera hemocytes from six triatomine species.

The objective of this work was to characterize, and compare different morphological types of hemocytes of Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, Triatoma infestans, Panstrongylus megistus, and Dipetalogaster maximus. This information provides the basis for studying the cellular immune systems of these insects. Seven morphological hemocyte types were identified by phase-contrast microscopy: prohemocytes, plasmatocytes, granular cells, cystocytes, oenocytoids, adipohemocytes and giant cells. All seven types of hemocytes are not present in every species. For example, adipohemocytes and oenocytoids were not observed in P. megistus and P. infestans, and giant cells were rarely found in any of the species studied. The hemocytes of Rhodnius and Dipetalogaster are more similar to each other than those from Triatoma and Panstronglus which in turn closely resemble each other. Emphasis is placed on methodological problems arising in this work which are discussed in detail.     

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.     

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.     

Fine structure of interphase nuclei

The initiation and replication sites of DNA synthesis in the plasmodial nuclei of Physarum polycephalum were studied with electron microscopic autoradiography. By using both thin sectioning and whole mount techniques, it was shown that the dense chromatin masses in the nucleus consisted of predominantly elementary chromatin-like fibrils, approximately 300 Å in diameter while the electron transparent region in the nucleus consisted of predominantly finer fibrils, less than 100 Å in diameter. With electron microscopic autoradiography it was found that (1) the initiation sites of DNA synthesis were definitely in the boundary regions between the dense chromatin masses and the electron transparent region, (2) the initiation and replication sites of DNA synthesis were definitely not on the nuclear membrane, (3) within a few minutes, replication sites migrated from the initiation sites to the electron transparent region and (4) in this electron transparent region, almost all of the nuclear DNA was synthesized.