Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host's fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A(2), B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.     

Immuno-gold localisation of arabinogalactan protein in the developing style of Nicotiana alata

Arabinogalactan-protein was localised ultrastructurally in the transmitting tissue of the Nicotinana alata Link & Otto style from the bud stage to anthesis using monoclonal antibody directed to terminal α-L-arabinosyl residues followed by goat anti-mouse IgG linked to 20 nm gold particles. Gold particles were localised on the Golgi apparatus, multivesicular bodies, cell wall and intercellular matrix with the highest concentration over multivesicular bodies. During flower development there was an increase in the total number of gold particles with a relative decrease on the multivesicular bodies and an increase on the intercellular matrix. There was also an increase during development in the proportion of intercellular matrix relative to all other components of the transmitting tissue. Vesiculation of the endoplasmic reticulum and the production of cup-shaped vesicles by the Golgi bodies were observed during development. It is concluded that secretion of arabinogalactan-protein from the intracellular to the extracellular location occurs via multivesicular bodies produced by the Golgi apparatus and possibly the endoplasmic reticulum.     

Role of the parasporal body in causing toxicity of Bacillus thuringiensis toward Aedes aegypti larvae

A unique type of microorganism has been found causing an unusual disease in larvae of the clover cutworm, Scotogramma trifolii (Lepidoptera; Noctuidae). The organism contains DNA and reproduces exclusively by self-assembly forming enveloped reniform/bacilliform particles which measure 170 × 450 nm in negatively stained preparations. During initial stages of development, the organism apparently reproduces primarily within vesicles in the cytoplasm of a variety of cell types including hemocytes, and epidermal, fat body, and tracheal matrix cells. Most reproduction, however, occurs in vesicles that circulate in the hemolymph. These vesicles, most of which are derived from host cells, measure 2–10 μm in diameter, are highly refractile, reach populations as high as 10⁸/ml of hemolymph, and are diagnostic for the disease. The pathology caused by this organism, its shape and ultrastructure, and reproduction within vesicles indicate it is either a peculiar type of rickettsia, possibly related to those of the genus Rickettsiella, or a new type of invertebrate virus. Among its unusual features are its ability to induce the formation of reproductive vesicles from host cell components, and its apparent control of de novo ribosome and membrane synthesis within these vesicles as it develops. The possible relationship of this organism to baculoviruses and rickettsia is discussed.     

Comparative analysis of the binding of antibodies prepared against the insect Spodoptera exigua and against the mycopathogen Nomuraea rileyi

Polyclonal antibodies were produced in mice against Spodoptera exigua (beet armyworm) larval hemolymph and hemocytes and against cell wall surfaces of hyphal bodies and hyphae of the entomopathogenic hyphomycete Nomuraea rileyi. In addition to exhibiting strong activity against their original antigenic substrates, all of the antibodies cross-react extensively with other substrates. The hemolymph antibody binds to hemocytes and vice versa, and both antibodies cross-react to the insect fat body basement membrane (extracellular matrix (ECM) and to N. rileyi and Beauveria bassiana (another entomopathogenic fungus) cell wall surfaces (ECM). Likewise, the anti-fungal antibodies cross-react with S. exigua hemolymph and hemocytes, especially the granules that may contain ECM components, and with fat body basement membrane. These cross-reactivities are specific as indicated by negative controls in the microscopy and Western blotting assays. Parallel labeling experiments using Con A suggest that the reactive epitopes contain mannose; however, none of the antibodies bind to mannose residues of nonentomopathogenic Candida albicans or Saccharomyces cerevisiae yeast cells. Thus, these cross-reactivities suggest that the host mimicry expressed by surface components of entomopathogenic fungi represents an important pathogenic determinant.     

Integument and hemocyte peptides

There are four routing classes of integument peptide in the caterpillar of Calpodes ethlius. The epidermis secretes peptides apically into the cuticle (C), basally into the hemolymph (H) and in both directions (BD). Peptides in a 4th class (T), are presumed to be transported across the epidermis, because the epidermis does not synthesize them although they occur in both cuticle and hemolymph. In a search for the origin of the presumed transepidermal peptides we found that hemocytes contain some peptides from all four routing classes. Peptides prepared from washed hemocytes reacted in immunoblots to antibodies against integument peptides prepared from hemolymph and cuticle. These peptides are probably synthesized by hemocytes because they matched those from medium containing [³⁵S]methionine in which hemocytes had been incubated. Calpodes hemolymph contains four hemocyte types. Immunogold labelling localized integument peptides in the secretory pathway of granulocytes and spherulocytes and in the cytosol of oenocytoids but not in plasmatocytes. Each peptide was localized in a particular kind or kinds of hemocyte. Granulocyte secretory vesicles reacted with antibodies to C180, C55 and BD82 kDa peptides. Spherulocytes secretory vesicles reacted with antibodies to C180, C55, BD89, BD82 and a 78 kDa peptide presumed to be the precursor of T66. Oenocyotoids reacted with antibodies to H45, 38, 32, 23 and BD89 kDa peptides. Spherulocytes were the only tissue to react with antibodies to the T66 kDa peptide that is found abundantly in cuticle and hemolymph. Spherulocytes are therefore presumed to secrete the 66 kDa peptide into the hemolymph from where it is transported to the cuticle. The C180 and C55 kDa peptides do not occur in hemolymph. Their presence in granulocytes and spherulocytes may be associated with hemocyte functions such as basal lamina formation, since immunogold localized them in that part of the basal lamina next to the hemolymph, as would be expected if hemocytes deposited components onto the exposed hemolymph surface. The presence of hemolymph peptides in oenocytoids is more difficult to interpret, since the antigenic reactions are localized in the cytosol rather than in the secretory pathway expected for exported proteins. We conclude that integument peptides are not secreted only by the epidermis, nor is the cuticle their only destination.     

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.     

Evasion of host defense by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana.

In vivo cells (hyphal bodies) of the hyphomycetous insect pathogen Beauveria bassiana collected from host Spodoptera exigua larval hemolymph were osmotically sensitive and lacked a well-defined cell wall. In light and electron microscope studies, a galactose-specific lectin purified from S. exigua hemolymph, concanavalin A (specific for alpha-mannose), and a polyclonal antibody to B. bassiana cell walls all bound to surfaces of in vitro-produced B. bassiana blastospores; however, none of these probes labelled the thin layer of extracellular material covering the plasma membranes of hyphal bodies. These cells were observed freely circulating in S. exigua hemolymph at 36 h postinfection, although immunocompetent hemocytes were known to be present. Additionally, association of hyphal bodies with hemocytes in monolayers was significantly less than for opsonized in vitro blastospores or submerged conidia. The absence of antigenically important galactomannan components on in vivo cells may therefore allow these cells to escape recognition and phagocytosis. Lack of structural components (e.g., chitin, as evidenced by the absence of binding of wheat germ agglutinin) may also be important with respect to evasion of host cellular defense mechanisms. Production of wall material resumed 48 to 60 h postinfection and therefore may coincide with loss of phagocytic capabilities of the hemocytes due to immunosuppressive effects of fungal metabolites. The protoplast-like cells may be formed by the action of hydrolytic enzymes in the hemocytes or by inhibition of fungal cell wall synthetases.     

Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions

Initiation of atherosclerosis is characterized by accumulation of aggregates of small lipid droplets and vesicles in the extracellular matrix of the arterial intima. The droplets and vesicles have features that suggest that they are formed from modified plasma-derived low density lipoprotein (LDL) particles. A variety of hydrolytic enzymes and prooxidative agents that could lead to extracellular assembly of LDL-derived droplets and vesicles are present in the arterial intima. In fact, in vitro studies have demonstrated that extensive oxidation of LDL and treatment of LDL with either proteolytic or lipolytic enzymes will induce LDL aggregation and fusion and treatment of LDL with cholesterol esterase will cause formation of vesicles. Fusion of LDL particles proceeds faster in vitro when they are bound to components of the extracellular matrix derived from the arterial intima, such as proteoglycans, and, depending on the type of modification, the strength of binding of modified LDL to the matrix components may either increase or decrease. In the present article, we discuss molecular mechanisms that provide clues as to how aggregated lipid droplets and vesicles may be derived from modified LDL particles. We also describe how these modified forms of LDL, by means of their trapping to the extracellular matrix, may lead to extracellular lipid accumulation in the arterial intima.     

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.     

Helix pomatia lectin and annexin V,two molecular probes for insect microparticles: possible involvement in hemolymph coagulation

Insect hemolymph coagulation involves a complex reaction with contributions from hemocytes and soluble factors. Here we present evidence for the presence of microparticles in the coagulation reaction. These particles are formed by hemocytes in a calcium-dependent process. Both the particles and the remaining cells are labelled by annexin V indicating the presence of phosphatidylserine on the outer membrane. Microparticles are enriched in hemomucin, a surface protein of Drosophila hemocytes that is specifically recognised by a snail (Helix pomatia) lectin. Hemomucin is shown to bind to lipophorin, a multifunctional hemolymph molecule previously implied in coagulation. Our findings suggest similarities at a biochemical and cellular level between vertebrate blood and insect hemolymph coagulation.     

Production of reactive oxygen species by hemocytes from the cattle tick Boophilus microplus.

We have investigated the phagocytic activity and the production of reactive oxygen species (ROS) by hemocytes from the cattle tick Boophilus microplus. Two main types of hemocytes were detected in tick hemolymph: plasmatocytes and granulocytes. The plasmocytes were the most abundant cells, being responsible for the in vivo phagocytosis of yeast. ROS production was evaluated by luminol-amplified luminescence and phenol red oxidation. The luminescence increased when hemocytes were incubated with bacteria, zymosan, or phorbol 12-miristate 13-acetate (PMA). The luminescence was inhibited by superoxide dismutase and catalase, which are antioxidant enzymes that remove superoxide and hydrogen peroxide, respectively. The phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with bacteria and PMA. Taken all together, our data indicate that tick hemocytes are able to produce ROS during the phagocytic process similarly to vertebrate phagocytes.     

Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.     

Observations on the phagocytosis and elimination of carmine particles injected into the abdominal musculature of the white shrimp, Penaeus setiferus

White shrimp, Penaeus setiferus, were injected in the abdominal musculature with 0.03 ml of a 1.4% carmine-saline solution and were kept at a temperature of 26°–28°C and a salinity of 23–26‰. Tissue samples were taken at hourly intervals of 1, 6, 12, 18, 24, 30, 36, 42, and 72 hr and at 4, 6, 8, 10, 12, 16, 19, and 33 days post-injection and were examined histologically to determine the sites of phagocytosis and elimination of foreign particulate matter. Within 1 hr post-injection, extracellular clumps of the carmine particles were formed in the hemolymph. These clumps had been invaded by hemocytes at 18 hr, but they persisted throughout the study. Phagocytosis of the particles was accomplished by hemocytes circulating in the hemolymph and by fixed phagocytes in the gill, heart, loose connective tissue, and blood sinusoids in the abdomen. The fate of some phagocytized carmine was climination by the migration of hemocytes through the epithelium of the gills, gut, hepatopancreas, and through the extremities of the pereiopods and the pleopods. Encapsulations or brown nodules were formed in the musculature of the pereiopods around necrotic hemocytes that had phagocytized carmine. A large blister or cyst filled with carmine was formed in the gill cover of one specimen. The obsevation of carmine particles at 33 days post-injection indicates a slow clearance rate of large amounts of abiotic particulate matter in penaeid shrimp.     

In vitro bacteridical capacity of Blaberus craniifer hemocytes

Blaberus craniifer hemocytes, maintained in short-term culture, are capable of phagocytosing and destroying Staphylococcus aureus, Staphylococcus albus, Streptococcus faecalis, Serratia marcescens, and Proteus mirabilis. The observed bactericidal activity of the hemocyte suspensions was entirely a function of the phagocytes; the medium, the hemolymph, and cell products elaborated during incubations were not bactericidal. No humoral opsonic factors were required for, or facilitated, bacterial phagocytosis in vitro. Washed hemocyte monolayers bathed by hemolymph-free medium were capable of phagocytosing bacteria. The addition of hemolymph concentrated by ultrafiltration did not increase the bactericidal capacity of the hemocytes. Bacteria opsonized with concentrated hemolymph were not killed more efficiently than were untreated bacteria.A partial blockage of bactericidal capacity was induced by prior exposure of the hemocytes to bacteria or to latex particles. The functional blockade was more complete with bacteria than with latex particles.Pseudomonas aeruginosa, Escherichia coli, Salmonella typhosa, and Diplococcus pneumoniae were phagocytosed but not killed by the hemocytes. This lack of bactericidal activity suggests that roaches may encounter difficulty in eliminating these organisms from the hemocoel. However, deficient bactericidal capacity probably does not entirely correlate with pathogenicity since the known insect pathogens, Staphylococcus albus, Serratia marcescens, and Proteus mirabilis, are killed by the hemocytes. Pathogenicity seems to depend on a complex of factors including bacterial strain, dose received, and intracellular survival of ingested bacteria. A possible connection between the lack of hemocytic bactericidal capacity and the role of roaches as potential disease vectors warrants further investigation.     

Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.     

Enzymatic activities in European flat oyster, Ostrea edulis, and pacific oyster, Crassostrea gigas, hemolymph

Enzymatic activities in the hemolymph of healthy and Bonamia-infected Ostrea edulis and Crassostrea gigas were studied with a commercial kit for the detection of 19 enzymes: 15 and 16 enzymes, respectively, were detected in the hemolymph of O. edulis and C. gigas and 10 of them showed relatively high activity levels. Most of them existed in both the cell-free fraction of the hemolymph and in the hemocytes. The cell-free hemolymph fraction of Bonamia ostreae-infected European flat oysters showed an elevated enzymatic activity level compared with that of healthy individuals. C. gigas hemocytes possessed higher enzymatic activity levels than O. edulis hemocytes. Differences in enzymatic activities existed in granulocytes and hyalinocytes in both oyster species. The enzyme release from oyster hemocytes seemed to be selective. The infection by B. ostreae induced enzymatic activity variations in European flat oysters. Higher enzyme levels within hemocytes may contribute partly to the natural resistance of C. gigas to the infection by B. ostreae.     

Phagocytosis in invertebrates: Studies on the hemocytes of the clam Tridacna maxima

The two types of cells found in the hemolymph of the clam Tridacna maxima have been examined in vitro by light microscopy, and their morphological charcteristics described and illustrated. It was shown that the hemocytes, which exhibited rapidly spreading cytoplasm and extensive ruffled membrane activity, were able to phagocytose carbon particles experimentally introduced into the animals. Attention is drawn to the part that the phagocytic hemocytes of invertebrates may have to play in putative host defense mechanisms and physiological waste removal processes.     

A contribution to the pathobiology of Biomphalaria glabrata hemocytes

This study attempts to investigate the relationship between the hemocytes in the two compartments: circulating peripheral lymph and the connective tissues. The hemocytes are compared with the vertebrate macrophages and constitute the principal line of defense against external aggression. The hemocytes were counted in circulating hemolymph and their phagocytic capability was evaluated in Schistosoma mansoni-infected Biomphalaria glabrata and the results were compared with those obtained from normal intact control snails. Although the number of circulating hemocytes revealed a mild increase in snails at the 6th week of infection, the overall findings were similar and pointed out that the cells in the two compartments are not functionally connected. However, the hemocytes found within the connective tissues of infected snails showed definite ultrastructural differences in the number and disposition of cytoplasmic prolongations and organelles in comparison with the hemocytes from non-infected snails. Histochemically, the staining for acid phosphatase activity served as a marker to hemocytes, sometimes being found in extracellular material at the foci of parasite-hemocyte interactions.