A Sericesthis iridescent virus infection of the hemocytes of the waxmoth,Galleria mellonella (Lepidoptera)

The cytopathology of the Seriscethis iridescent virus (SIV) infection of Galleria hemocytes was studied by phase-contrast microscopy, [³H]thymidine autoradiography, and fluorescent antibody and acridine orange staining. Of the five hemocyte classes only the oenocytoid was not infected. Infected plasmatocytes and adipohemocytes undergo nuclear and cytoplasmic hypertrophy accompanied by nuclear DNA synthesis. The timing and identity of nuclear and cytoplasmic DNA synthesis are discussed.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Biomphalaria glabrata (Gastropoda): effect of urethane on the morphology and function of hemocytes, and on susceptibility to Schistosoma mansoni (Trematoda)

Urethane (ethyl carbamate) anesthesia of Biomphalaria glabrata resulted in several rapid changes to the snail's blood picture. At 2 hr postexposure (PE) to the drug, there was an elevation in the prevalence of acid phosphatase (APase)-positive hemocytes, a significant increase in the number of APase granules per cell, a three fold increase in circulating blood cell number, and a decrease in the percentage of hemocytes expressing a cell subpopulation-specific surface membrane epitope (BGH₁). However, urethane had little effect on erythrophagocytosis by host hemocytes. All of the observed changes returned to control levels by 12 hr PE to the drug. Blood cells cultured with various concentrations of the anesthetic in vitro did not exhibit any alterations in the parameters described above. Since circulating hemocytes represent the primary effector component involved in internal defense reactions, the effect of urethane-induced changes in the composition of circulating blood cells on susceptibility to a larval trematode, Schistosoma mansoni, was examined. Such changes had no apparent effect on host susceptibility since the rate of infection for urethane-exposed snails (88.2%) was the same as for nonurethane-treated B. glabrata (82.4%). However, the effects of urethane-induced changes in hemocyte number and composition on other invading organisms is not known. Therefore, it is suggested that such alterations should be considered when internal defense reactions are studied in snails exposed to this drug and other commonly used anesthetics.     

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.     

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.     

Induction of lysozymelike activity in the hemolymph and hemocytes of an insect, Spodoptera eridania

Lysozymelike activity is present in the hemocytes and cell-free hemolymph of Spodoptera eridania. Its level remains essentially constant during larval development and can be induced by injection of various foreign materials. Serum bacteriolytic activity rises 24 hr after injection of saline, BSA, bacteria, bacterial endotoxin (LPS), latex particles, or sham injection. However, the magnitude and subsequent duration of the response depends on the nature of the injected material. The response is transient following sham injection or injection of soluble substances, such as saline and BSA, as compared to treatment with latex or bacteria. Both soluble and insoluble fractions of bacterial LPS preparations stimulated the lysozyme response. The response to a single injection of E. coli LPS was dose dependent and persisted for at least 5 days; however, additional injections had no effect on serum lysozyme level. The basal intracellular lysozyme level was significantly increased by E. coli LPS injection. Lysozyme release by hemocytes was proportional to intracellular concentration and did not increase after phagocytic stimulation of hemocytes.     

Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

The time course of clearance of an injected dose of 10⁶ CFU ml^?1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.     

Hemocytes of Biomphalaria glabrata: Factors affecting variability

Variability in the hemocyte number of two geographic strains of Biomphalaria glabrata was studied. In each strain a logarithmic increase in hemocyte number associated with increasing shell size was observed. A two fold increase in circulating hemocytes occurred 2 hr following the exposure of a susceptible strain of B. glabrata to miracidia of Schistosoma mansoni. The hemocyte number was dependent on the temperatures at which the snails were maintained.     

Role of a small G protein Ras in cellular immune response of the beet armyworm, Spodoptera exigua

Insect cellular immune responses accompany cytoskeletal rearrangement of hemocytes to exhibit filopodial and pseudopodial extension of their cytoplasm. Small G proteins are postulated to be implicated in the hemocyte cellular processes to perform phagocytosis, nodulation, and encapsulation behaviors. A small G protein ras gene (Se-Ras) was cloned from cDNAs prepared from hemocytes of the beet armyworm, Spodoptera exigua. The open reading frame of Se-Ras encoded 179 amino acids with a predicted molecular weight of 20.0 kDa, in which 114 residues at amino terminus were predicted to be a GTP binding domain. It showed high sequence similarities (86.1-92.8%) with known ras genes in other insects. Se-Ras was constitutively expressed in all developmental stages from egg to adult without any significant change in expression levels in response to bacterial challenge. A specific double strand RNA (dsRNA) could knockdown its expression in the hemocytes after 48 h post-injection. While the RNA interference (RNAi) did not show any change in total or differential hemocyte counts, it impaired hemocyte behaviors. The RNAi of Se-Ras significantly suppressed hemocyte spreading, cytoskeleton extension, and nodulation behaviors in response to bacterial challenge. Release of prophenoloxidase from oenocytoids was significantly inhibited by the RNAi, which resulted in significant suppression in PO activation in response to an inducer, PGE₂. These results suggest that Se-Ras is implicated in mediating cellular processes of S. exigua hemocytes. This is the first report of Ras role in insect cellular immune response.     

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.     

中华绒螯蟹血细胞原代培养条件的优化

比较了几种常见血细胞培养基(L-15、2×L-15、3×L-15、M199和RMPI-1640)对中华绒螯蟹(Eriocheir sinensis)血细胞原代培养中细胞形态以及存活率的影响,以及在筛选获得的最佳培养基中添加不同比例胎牛血清(FBS)(0%、5%、10%和15%),进一步观察了血清对中华绒螯蟹血细胞培养效果的比较。结果表明,3×L-15培养基培养效果较好,所培养的细胞形态相对完整,数量较多,培养至96 h时血细胞存活率仍大于60%;而其他4种培养基效果较差,培养12 h存活率均低于50%,且细胞形态结构变化明显。以3×L-15培养基为基础,添加不同比例胎牛血清后发现,对细胞存活有显著影响,存活率明显降低。因此,不添加血清的3×L-15培养基对中华绒螯蟹血细胞的生长较为适宜。     

Infection of the boll weevil by Chilo iridescent virus

When infections with Chilo iridescent virus (CIV) were induced in larvae of boll weevils, Anthonomus grandis, by intrahemocoelic injection or by feeding, and in adults by feeding, the typical blue coloration of adipose tissue developed at 3–7 days postinfection, and mortality occurred after 3 days. The symptomatology and the pathological expressions depended on the initial infectious titer. The virus remained viable when it was added to the feeding stimulant bait used to infect weevils with protozoan pathogens in the field, and weevils feeding on the formulation became infected when it had been exposed 1–3 days in nature.     

Response of the insect immune system to three different immune challenges

Insects rely on an innate immune system to effectively respond to pathogenic challenges. Most studies on the insect immune system describe changes in only one or two immune parameters following a single immune challenge. In addition, a variety of insect models, often at different developmental stages, have been used, making it difficult to compare results across studies. In this study, we used adult male Acheta domesticus crickets to characterize the response of the insect innate immune system to three different immune challenges: injection of bacterial lipopolysaccharides (LPS); injection of live Serratia marcescens bacteria; or insertion of a nylon filament into the abdomen. For each challenge, we measured and compared hemolymph phenoloxidase (PO) and lysozyme-like enzyme activities; the number of circulating hemocytes; and the nodulation responses of challenged and un-challenged crickets. We found that injection of an LD₅₀ dose of LPS from Escherichia coli elicited a more rapid response than an LD₅₀ dose of LPS from S. marcescens. LPS injection could cause a rapid decrease 2 hpi, followed by an increase by 7 dpi, in the number of circulating hemocytes. In contrast, injection of live S. marcescens produced a rapid increase and then decrease in hemocyte number. This was followed by an increase in the number of hemocytes at 7 dpi, similar to that observed following LPS injection. Both LPS and live bacteria decreased hemolymph PO activity, but the timing of this effect was dependent on the challenge. Live bacteria, but not LPS, induced an increase in lysozyme-like activity in the hemolymph. Insertion of a nylon filament induced a decrease in hemolymph PO activity 2 h after insertion of the filament, but had no effect on hemocyte number or lytic activity. Our results indicate that the innate immune system’s response to each type of challenge can vary greatly in both magnitude and timing, so it is important to assess multiple parameters at multiple time points in order to obtain a comprehensive view of such responses.     

Multiplication of Chilo iridescent virus in noninsect arthropods

Attempts to infect noninsect arthropods with Chilo iridescent virus (CIV) originally isolated from Lepidoptera were made by using eight species belonging to four classes. Multiplication of CIV was demonstrated in two species of terrestrial Crustacea (the pill bug, Armadillidium vulgare, and the slater, Porcello scaber) and one species of Chilopoda, the house centipede, Thereuonema higendorfi. The lethality experiment of CIV for A. vulgare suggested that chronic infection is a characteristic of the CIV infection in both classes, Crustacea and Insecta. Neither iridescence nor recovery of virus infectivity was demonstrated in the following arthropod species: the sea slater, Ligia exotica (Crustacea: Isopoda), the grapsid crab, Sesarma haematocheir (Crustacea: Decapoda), the millipede, Oxidus gracillis (Diplopoda: Polydesmoidea), Rhysodesmus semicirculatus (Diplopoda: Polydesmoidea), and the giant crab spider, Heteropoda venatoria (Arachnida: Araneae).     

Modifications of the hemogram and of the hemocytopoietic tissue of male adults of Locusta migratoria (Orthoptera) after injection of Bacillus thuringiensis

The injection of live Bacillus thuringiensis (with the culture medium) into the hemocoel of male adults of Locusta migratoria results in a significant fall of the number of circulating hemocytes followed 2 days later by a sharp increase of the hemocyte figure. Identical doses of washed live bacteria have the same effect on the hemogram, whereas neither culture medium deprived of the bacteria by filtration nor heatkilled bacteria modify the hemocyte number. Injection of isolated β-exotoxin of B. thuringiensis in nonlethal concentrations remain without effect on the hemogram.Morphological studies show that the injected bacteria are essentially taken up by the reticular (phagocytic) cells of the hemocytopoietic tissue, leading to a necrotic evolution of many of these cells. Necrotic zones are rapidly encapsulated by granular hemocytes.One to two days after the injection, the hemocytopoietic tissue shows signs of considerable hypertrophy: both the polymorphous reticular cells and the maturing blood clusters become notably more numerous.The modifications observed in the hemocytopoietic tissue partly explain the alterations of the hemogram after injection of B. thuringiensis.     

Up-regulation of circulating hemocyte population in response to bacterial challenge is mediated by octopamine and 5-hydroxytryptamine via Rac1 signal in Spodoptera exigua

Bacterial challenge induced a significant increase in the total hemocyte population within 4 h in the beet armyworm, Spodoptera exigua. Octopamine and 5-hydroxytryptamine (5-HT) are known to play critical roles in mediating insect immune responses. This study analyzed the effects of both biogenic monoamines on mediating up-regulation of circulating hemocyte population in response to bacterial challenge. Injection of either octopamine or 5-HT induced a significant increase in the total hemocyte count in the hemolymph without any bacterial challenge. On the other hand, the monoamine antagonists, phentolamine (an octopamine antagonist) and ketanserin (a 5-HT antagonist) each suppressed the increase of the circulating hemocyte counts in response to bacterial challenge. This rapid change of circulating hemocyte population did not appear to be the result of de novo hemocyte production from the hematopoietic organ because a physical block (“ligation”) of hemolymph circulation between thorax and abdomen did not inhibit the increase of hemocyte counts in the isolated abdomen in response to bacterial challenge. The effects of the two monoamines on hemocyte numbers were not dependent on the mediatory effects of eicosanoids, because dexamethasone, an eicosanoid biosynthesis inhibitor, had no effect on the hemocyte recruitment induced by the monoamines. On the other hand, an adenylate cyclase inhibitor, NKY80, significantly impaired hemocyte mobilization in response to bacterial challenge, implying involvement of cyclic AMP in the control of hemocyte numbers. Also, a Rac1 inhibitor, NSC23766, significantly antagonized the effects of monoamines in increasing circulating hemocyte numbers. Rac1 activity was necessary to form F-actins in the hemocytes of S. exigua, where its activity showed a quantitative correlation with hemocyte-spreading behavior. This study suggests that octopamine and 5-HT mediate a rapid increase of circulating hemocyte population in response to bacterial challenge via Rac1 signal in S. exigua.     

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.     

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.     

The Capsid Proteins of a Large, Icosahedral dsDNA Virus

Chilo iridescent virus (CIV) is a large (∼ 1850 Å diameter) insect virus with an icosahedral, T = 147 capsid, a double-stranded DNA (dsDNA) genome, and an internal lipid membrane. The structure of CIV was determined to 13 Å resolution by means of cryoelectron microscopy (cryoEM) and three-dimensional image reconstruction. A homology model of P50, the CIV major capsid protein (MCP), was built based on its amino acid sequence and the structure of the homologous Paramecium bursaria chlorella virus 1 Vp54 MCP. This model was fitted into the cryoEM density for each of the 25 trimeric CIV capsomers per icosahedral asymmetric unit. A difference map, in which the fitted CIV MCP capsomers were subtracted from the CIV cryoEM reconstruction, showed that there are at least three different types of minor capsid proteins associated with the capsomers outside the lipid membrane. “Finger” proteins are situated at many, but not all, of the spaces between three adjacent capsomers within each trisymmetron, and “zip” proteins are situated between sets of three adjacent capsomers at the boundary between neighboring trisymmetrons and pentasymmetrons. Based on the results of segmentation and density correlations, there are at least eight finger proteins and three dimeric and two monomeric zip proteins in one asymmetric unit of the CIV capsid. These minor proteins appear to stabilize the virus by acting as intercapsomer cross-links. One transmembrane “anchor” protein per icosahedral asymmetric unit, which extends from beneath one of the capsomers in the pentasymmetron to the internal leaflet of the lipid membrane, may provide additional stabilization for the capsid. These results are consistent with the observations for other large, icosahedral dsDNA viruses that also utilize minor capsid proteins for stabilization and for determining their assembly.