A study of the surface proteins of Entomophthora egressa protoplasts and of larval spruce budworm hemocytes

Entomophthora egressa protoplasts either exposed to or not exposed to trypsin were not attacked by either trypsinized or non-trypsinized larval spruce budworm granulocytes. Granulocytes adhered to protoplasts exposed to papain, and this adhesion could be prevented by papainizing the hemocytes. Differences were observed in the responses of two E. egressa isolates when exposed to papain or to the papain-control solutions. Exposure of hemocytes to trypsin did not reduce either the number of Absidia repens sporangiospores per granulocyte or the percentage of granulocytes with spores, whereas, exposure to papain did. The role of surface proteins, particularly glycoproteins, in hemocyte-fungal cell interactions is briefly discussed.     

Response of eastern hemlock looper hemocytes to selected stages of Entomophthora egressa and other foreign particles

Indirect evidence for the natural existence of the free-protoplast stage of the fungus Entomophthora egressa in the eastern hemlock looper, Lambdina fiscellaria fiscellaria, is presented. The protoplasts were viable after 72 hr postinjection and subsequent development in the host produced conidia characteristic of E. egressa. The hemocytes studied (plasmatocytes, granular cells, and spherule cells) did not adhere to the protoplasts either in vivo or in vitro. Cells of Escherichia coli and sporangiospores of Absidia repens adhered to the granular cells in vitro. The granular cells adhered to the hyphae of Rhizopus nigricans in vitro. The spherule cells strongly adhered to the hyphae and hyphal bodies of E. egressa in vitro. The protoplasts, hyphae, and conidia of E. egressa and the hemocytes of L. fiscellaria fiscellaria adhered to positively charged DEAE-Sephadex beads and not to negatively charged CM-Sephadex beads. Aspects of active and passive strategies for protoplast evasion of host hemocytes are discussed with some emphasis on hemocyte-protoplast electrostatic repulsion and active secretion of hemocyte inhibitors by the protoplasts.     

Strains of protoplasts of Entomophthora egressa in spruce budworm larvae

Protoplasts of strain 458 (S458) and strain 521 (S521) of Entomophthora egressa had LD₅₀s of 620 and 8.8 cells/insect, respectively, for sixth-instar spruce budworm larvae. Both protoplast strains exhibited biphasic growth profiles with comparable growth rates in the larval hemocoel. The growth rates of the fungal strains increased as the total hemocyte counts declined. Hemocytopenia was greatest and most rapid in larvae containing S521 protoplasts. Protoplasts of S521 exposed to α-mannosidase and β-N-acetylglucosaminidase were as virulent as the control cells. β-Galactosidase reduced protoplast virulence.     

Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilusgalloprovincialis

In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated.In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components.     

No evidence for mycoviruses in Entomophthora egressa

Summary Two strains of Entomophthora egressa which differ in their pathogenicity towards the spruce budworm were surveyed for the presence of double-stranded RNA mycoviruses. There was no evidence for the occurrence of any mycovirus in either strain. This indicates that virulence in E. egressa is not associated with a mycovirus.     

Effects of hormones on Entomophthora egressa morphogenesis

The morphological effects of a fungal sex hormone, trisporic acid (TA), and a synthetic insect juvenile hormone (JH) on mycelial cultures derived from protoplasts of the fungus Entomophthora egressa were determined. In comparison with the control treatment (no added hormone), only one treatment (JH and TA both added at 40 μg/50 ml) produced all of the control cell types. Under both experimental conditions, normal hypha (without swollen tips), spherical hyphal bodies, irregularly shaped hyphal bodies, and thick-walled spheres were present. This JH plus TA treatment differed from the control in that normal rod-shaped hyphal bodies were also present and the thick-walled structures tended to be variable in shape. Hyphal tip swelling which did not lead to conidium production was common to all treatments in which only JH was added. The results of the addition of various concentrations and combinations of JH and TA with regard to the production of thick-walled cell types and three forms of hyphal bodies are given and discussed.     

Losing the battle against fungal infection: suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2 × 10³, 2 × 10⁶ or 2 × 10⁸ conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.     

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.     

The Involvement of Hemocyte Prophenoloxidase in the Shell-Hardening Process of the Blue Crab,Callinectes sapidus

Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.     

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 10⁶ cells/ml (n = 15, range 2–7 × 10⁶ cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.     

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d’ être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

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

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

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

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

Ultrastructural aspects of wall regeneration byPythium protoplasts

Electron microscope studies were made of wall regeneration byPythium protoplasts. Wall regeneration began with the formation of a loose network of fibrils on the surface of the protoplast followed by increase in density of the fibrillar mesh and deposition of granular matrix material. The majority of the protoplasts did not develop beyond the loose fibrillar network stage, however a small percentage were able to complete wall formation and to form hyphal tubes. A clear zone of demarcation was visible between the fibrillar surface of the protoplast and the smooth surface at the base of the developing hyphal tube.     

Transformation ofSchizophyllum commune: An analysis of specific properties

Several properties of transformation in the basidiomycete,Schizophyllum commune, were examined. The transformation efficiency of protoplasts made from germinating basidiospores is dependent upon the length of time that the spores are incubated under conditions that promote germination. Protoplasts prepared from ungerminated spores transform at least 10 times more efficiently than protoplasts prepared from germlings (25 μm in length) or from mycelium. Transformation frequencies of 1000 transformants/μg of control plasmid DNA and 10⁷ protoplasts are sufficient for obtaining transformants with 2 × 10⁷ protoplasts and 10 μg of bank DNA from a genomic plasmid library. The probability of cotransforming with two plasmids is dependent on the DNA concentrations of each; concentrations can be adjusted to yield nearly 100% cotrasformants. The presence of a nonselected plasmid in the reaction mix improves the transformation frequency of a selected marker carried on another plasmid; this is not true if linear fragments ofSchizophyllum genomic DNA are used as the nonselected DNA. Transformation of aSchizophyllum protoplast does not require its fusion to another protoplast.     

Regulatory mechanism of silkworm hemocyte adhesion to organs

Circulating hemocytes in the body fluid of the silkworm are increased during the larval-larval molting period. We investigated hemocyte adhesion to organs mediating the selectin-selectin ligands during the feeding period and the larval-larval molting period using the lectin staining method, sugar chain digestion test with glycoside hydrolases, and the hemocyte adhesion inhibition test using monosaccharides. The results of these tests suggested that the selectin ligand involved in hemocyte adhesion was the Sialyl Lewis x-type, and the structure was changed from the feeding period to the larval-larval molting period. Beta-galactosidase appears to be an enzyme that eliminates N-acetylgalactosamine and sialylated N-acetylgalactosamine from the terminal of Sialyl Lewis x. Beta-galactosidase activation in skin basement membranes, muscle, fat bodies, midguts, and hemocytes increased markedly during the larval-larval molting period, and at that time, hemocytes were detached from organs. Adding 20-hydroxyecdysone or its analog, tebufenozide to cultured fat bodies increased β-galactosidase activity in these tissues. Therefore, 20-hydroxyecdysone may induce a structural change in Sialyl Lewis x type sugar chains on the cell surface of silkworm's organs by increasing the β-galactosidase activity to detach hemocytes from organs and increase the number of circulating hemocytes during the larval-larval molting period.     

Morphology ofEntomophthora muscae protoplasts grownin vitro

Summary Entomophthora muscae (C.) Fres. can be grownin vitro as protoplasts. Light and electron microscopical studies of thein vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape ofE. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.Abbreviations Con A concanavalin A - DH Drosophila cell culture medium - FITC fluorescein isothiocyanate - GLEN glucose-lactal-bumin-yeast extract-NaCl culture medium for protoplasts - HBL hyphal body-like protoplasts - MM Mitsuhashi and Maramorosch' insect cell culture medium - PATAg periodic acid-thiocarbohydrazide-silver proteinate technique - PBN phosphate buffer with NaCl - S spherical protoplasts - WGA wheat germ agglutinin     

Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the Mitten Crab, Eriocheir sinensis

Hemocytes are the main immune defense cells in crustacean, and its in vitro culture can be a useful tool for the study of host and pathogen interaction. In the present study, the primary hemocyte culture of Chinese mitten crab (Eriocheir sinensis), including mixed and single hemocyte, was set up for the first time. In this study, different pH (6.4, 6.8, 7.2, 7.6, and 8.0), temperature (26, 28, and 30°C), and osmolarity (500, 700, 900, 1,100, and 1,300 mOsm kg^?1) values were tested. Moreover, the effects of two types of medium (1× L-15 and 3× L-15) with the same osmolarity on hemocyte culture were evaluated. After incubation at different culture conditions, the morphological changes (degranulation, lysis, shrinkage, and detachment) and survival rate of hemocytes were taken into account in order to evaluate the culture condition effect. Our results showed that the total hemocyte counts of Chinese mitten crab were about 2.5?×?10⁷ cells ml^?1, and three subpopulations of hemocytes were distinguished as granulocytes (43.46?±?4.98%), semigranulocytes (31.04?±1.95%), and hyalinocytes (25.50?±4.89%). The optimal culture condition for primary hemocytes of Chinese mitten crab was 3× L-15 medium, 1,100 mOsm kg^?1, pH 6.8 at 28°C. Hemocytes at optimal culture condition could retain a better morphology and higher survival rate: hemocytes retained a survival rate >60% after 5 d and >40% after 7 d. Furthermore, the hemocyte subpopulations were isolated by Percoll step gradient centrifugation and cultured in optimized hemocyte culture conditions. The results showed that hyalinocytes and semigranulocytes could maintain a survival rate of >50% after 15 d, while granulocytes only retained a survival rate of 26% after 5 d.