Apoptosis and adhesion of hemocytes during molting stage of silkworm, Bombyx mori

To clarify the regulatory mechanism of the rapid changes in the hemocyte density in the silkworm, Bombyx mori, during ecdysis, we evaluated the relationship between the hemocyte density and the incidence of apoptosis during this stage. We also evaluated the role of the sugar chains on the adhesion of hemocytes by analyzing the effects on the hemocyte density of the injection of enzymes that cut sugar chains and monosaccharides into the body cavity. The hemocyte density was increased in the molting stage and spinning, and then decreased after the ecdysis. During spinning, the diameter of the granulocytes markedly increased, in which fatty granules in the cytoplasm increased, becoming foamy. They were identified to be apoptotic hemocytes using the Hoechst staining and the Comet assay. The decrease in the hemocyte density during spinning was mainly caused by the apoptosis of granulocytes. Next, we focused on the fluctuation of hemocyte density during the molting stage. Examination of the changes in the hemocyte density induced by injecting glycoside hydrolases, neuraminidase, sialic acid, or monosaccharides into the body cavity during the fourth molt stage and the third day in fifth instar larva demonstrated that the alteration of hemocyte density was regulated by the attachment and detachment of hemocytes via a selectin ligand, sugar chains. As with the injection of glycoside hydrolase, neuraminidase, sialic acid and fucose raised the hemocyte detachment, and it was assumed that the selectin ligands include the sialyl Lewis x like sugar chains, the same as mammalian lymphocytes.     

Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda

Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs. In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda. For both species, hemocyte densities per microl of blood increased with instar. Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar. Hematopoietic organs were located in the meso- and metathorax of S. Frugiperda and P. Includens. These organs contained large numbers of hemocytes in S. Frugiperda, but contained few hemocytes in P. Includens. The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes. Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA. BrdU labeling indices for both species also fluctuated with the molting cycle. Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S. Frugiperda but not in P. Includens. Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation. Arch.     

Effects of high temperature on the hemocyte cell cycle in silkworm larvae

To understand the inhibitory effects of high temperature on insect growth at the cellular level, we investigated the influence of high temperature on the proliferation and division of larval hemocytes in the silkworm, Bombyx mori. Although the total number of hemocytes in the larval body increased enormously over time at 26 degrees C, no increase was observed at 38 degrees C. The number of mitotic hemocytes in circulation increased between days 1 and 2 of the fourth larval stage at 26 degrees C, whereas fewer hemocytes were observed at 38 degrees C. Laser scanning cytometry revealed that the DNA content of hemocytes collected from the fourth-stadium larvae was predominantly 2C, 4C, and 8C, and the proportion of each type of hemocyte changed dynamically with development during the fourth instar. Specifically, the proportion of hemocytes with a higher DNA content increased gradually during the feeding phase then decreased during the molting phase at 26 degrees C; in contrast, no decrease was observed at 38 degrees C. The heat-induced accumulation of 8C hemocytes was mainly detected in granulocytes and plasmatocytes. These data suggest that high temperatures induce a G(2) arrest in larval hemocytes.     

Cell interactions in the differentiation of a melanotic tumor in Drosophila.

The cellular events in the formation of melanotic tumors in the tu-W mutant larva of Drosophila melanogaster are described. The first step is the differentiation of spherical hemocytes to flattened cells, the lamellocyte variants. Subsequently, the surface of the caudal fat body undergoes changes to which the hemocytes respond by forming cellular capsules. The hemocytes utilize two mechanisms in this process: (1) phagocytosis of small particulate materials escaping from the adipose cells, (2) adhesion to form a multilayered wall of lamellocytes. Differentiating hemocytes in the vicinity of the tumor-forming site extrude membrane-bound vesicles that tend to adhere to the hemocyte surfaces. These vesicles are trapped between the lamellocytes as they pile in layers to form the capsule wall. It is suggested that the vesicles play a role in lamellocyte-to-lamellocyte adhesion during the initial stages of hemocyte aggregation at the tumor-forming site.     

Cell Interactions in the Differentiation of a Melanotic Tumor in Drosophila

The cellular events in the formation of melanotic tumors in the tu-W mutant larva of Drosophila melanogaster are described. The first step is the differentiation of spherical hemocytes to flattened cells, the lamellocyte variants. Subsequently, the surface of the caudal fat body undergoes changes to which the hemocytes respond by forming cellular capsules. The hemocytes utilize two mechanisms in this process: (1) phagocytosis of small participate materials escaping from the adipose cells, (2) adhesion to form a multilayered wall of lamellocytes.
Differentiating hemocytes in the vicinity of the tumor-forming site extrude membrane-bound vesicles that tend to adhere to the hemocyte surfaces. These vesicles are trapped between the lamellocytes as they pile in layers to form the capsule wall. It is suggested that the vesicles play a role in lamellocyte-to-lamellocyte adhesion during the initial stages of hemocyte aggregation at the tumorforming site.     

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes

Larval Galleria melonella(L.)hemocytes form microaggregates in response to stimulation by Gram-positive bacteria Hemocyte adhesion to foreign materials is mediated by the CAMP/protein kinase A pathway and the B-subunit of cholera toxin using a cAMP-independent mechanism.Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide,implying integrins may be part of the mechanism.Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses ars,ory,Ai,and B3 subunits occred on both granular cell and plasmatocyte hemocyte subtypes.A fluorescent band representing the binding of rabbit as-integrin subunit antibodies occurred between adhering heterotypic hemocytes.The frequency of the bands was increased by cholera toxin.The as andβrabbit integrin subunit antibodies inhibited removal of Bacillus subtilis(Cohn)from the hemolymph in vivo,A as ir-specific synthetic peptide blocker similarly diminished hemocyte function whereas the 0v Bs-specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities.Western blots revealed several proteins reacting with a given integrin-antibody subtype.Thus integrin-antibody reactive proteins(which may include integrins)with possible as and B epitopes modulate immediate hemocyte function.Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followved by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.     

Isolation of cDNA clones coding for humoral lectin of silkworm, Bombyx mori, larvae.

In the hemolymph of the silkworm, Bombyx mori, lectin with hemagglutinating activity against sheep red blood cells increases at larval-larval ecdysis and at spinning stage (Suzuki and Natori, 1983) and is induced by infection with cytoplasmic polyhedrosis virus. A Bombyx lectin polypeptide with molecular weight approx 280K is responsible for hemagglutinating activity, since antiserum raised against this polypeptide inhibited hemagglutinating activity. The site of synthesis of Bombyx lectin was determined by primary tissue cultures of fat body and hemocytes. A hemagglutinating activity assay demonstrated that hemocyte is responsible for the release of hemagglutinin into the culture medium. Isolation of cDNA clones coding for Bombyx lectin was carried out on the cDNA library prepared in an expression vector lambda gt11 starting with poly(A)+ RNA from spinning larval hemocytes. As a result of immunoscreening, several positive clones were obtained, and the cDNA clones were characterized.     

Tickcidal effect of monoclonal antibodies against hemocytes, Om21, in an adult female tick, Ornithodoros moubata (Acari: Argasidae)

In the present study, monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established. Afterward, artificial feeding was performed to assess the tickcidal effect of fetal bovine serum meal containing each mAb. As a result, Om21 showed the strongest tickcidal effect on adult female O. moubata. The reactivity of various tick cells and organs, including the hemocyte, midgut, trachea, ovary, fat body, and muscle, to Om21 was then examined by an indirect immunofluorescent antibody test and by immunoelectron microscopy. Om21 reacted with not only hemocytes but also with fat body cells, epidermis, cuticle of the trachea, connective tissue of the muscle, and the basement membrane of the midgut, trachea, fat body, oocyte, and epidermis. These results suggest that Om21 passing through the midgut epithelium induced a tickcidal effect on hemocytes or various organs. However, the target of Om21 could not be identified in the present study. The antihemocyte mAb produced in this study, Om21, may be useful for the immunological control of ticks.     

Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti

Hemocytes are an essential component of the mosquito immune system but current knowledge of the types of hemocytes mosquitoes produce, their relative abundance, and their functions is limited. Addressing these issues requires improved methods for collecting and maintaining mosquito hemocytes in vitro, and comparative data that address whether important vector species produce similar or different hemocyte types. Toward this end, we conducted a comparative study with Anopheles gambiae and Aedes aegypti. Collection method greatly affected the number of hemocytes and contaminants obtained from adult females of each species. Using a collection method called high injection/recovery, we concluded that hemolymph from An. gambiae and Ae. aegypti adult females contains three hemocyte types (granulocytes, oenocytoids and prohemocytes) that were distinguished from one another by a combination of morphological and functional markers. Significantly more hemocytes were recovered from An. gambiae females than Ae. aegypti. However, granulocytes were the most abundant cell type in both species while oenocytoids and prohemocytes comprised less than 10% of the total hemocyte population. The same hemocyte types were collected from larvae, pupae and adult males albeit the absolute number and proportion of each hemocyte type differed from adult females. The number of hemocytes recovered from sugar fed females declined with age but blood feeding transiently increased hemocyte abundance. Two antibodies tested as potential hemocyte markers (anti-PP06 and anti-Dox-A2) also exhibited alterations in staining patterns following immune challenge with the bacterium Escherichia coli.     

Hemocytes and hemocytopoiesis in Silkworms.

A brief review is presented of the current state of ultrastructure, cytochemistry, and physiology of the hemocytes and meso- and metathoracic peri-imaginal-wing organs in silkworms. According to the accepted morphological classification, five circulating types of hemocytes are recognized in Bombyx mori as well as in Antheraea pernyi. They are prophemocytes or stem cells, plasmatocytes or pre-differentiated cells and three specialized cells, granulocytes, spherule cells and oenocytoids. During post-embryonic development the last four types are the most common in the circulating hemolymph. Plasmatocytes are considered to be pluripotent cells from which granulocytes, spherule cells and oenocytoids are derived. Contrary to the situation in most insects the plasmatocytes are not phagocytic in Antheraea. The granulocytes are efficient phagocytes. Both plasmatocytes and granulocytes are involved in pinocytosis. Another possible function of the granulocytes is hemolymph coagulation. The function of the spherule cells which contain a paracrystalline material (muco- or glycoproteins) is by no means clear. The phenoloxidase activity found within the cytosol of oenocytoids appears effective against the natural monophenol and diphenol substrates. The involvement of oenocytoids in the complex metabolism of phenols and particularly in the production of plasma phenolases has been reported. The mitotic division of five circulating hemocyte types is well known and was long regarded as the only mechanism of postembryonic hemocyte production. We present for silkworms, experimental evidence of the hemocytopoietic function of the meso- and metathoracic organs surrounding the imaginal wing discs. Ablation experiments demonstrate that the mitotic activity of free hemocytes is unable to maintain the normal hemocytogram in the absence of the two paris of organs. These organs are typically divided into cell islets ensheathed by a connective tissue membrane. Two types of islets may be classified by the disposition of the cells : the compact islets or aggregations of stem cells and the reticulate islets which are mainly composed of hemocytes at different steps of differentiation. The relative number of prohemocytes in the total hemocyte population ranges from 84 to 97 p. cent in organs of Antheraea pernyi. This well-defined cell type appears to be the major hemocyte type in hemocytopoietic organs. In Antheraea, the mitotic index (the relative number of mitotic hemocytes in the total cell population) varies from 0.5 to about 3 p. cent. Finally, our data direct attention to cyclic functional changes such as mitotic divisions and hemocyte differentiation which run parallel to the molting cycle.     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

Insulin signaling is involved in hematopoietic regulation in an insect hematopoietic organ

Only a few extracellular hematopoietic factors have been identified in insects. We previously developed an in vitro culture system for the larval hematopoietic organ (HPO) of the silkworm Bombyx mori, and found that cell proliferation is linked to hemocyte discharge from the HPO. In this study, we tested hematopoietic activity of bombyxin, a peptide in the insulin family. When silkworm HPO was cultured with synthetic bombyxin-II, the number of discharged hemocytes increased in a dose-dependent manner, indicating that bombyxin promoted cell proliferation in the HPO. However, a neutralization experiment using anti-bombyxin-II antibody revealed that bombyxin is not the primary effector in larval plasma. Similarly, bovine insulin showed hematopoietic activity. Addition of molting hormone, 20-hydroxyecdysone, circumstantially enhanced the hematopoietic activity of bombyxin and insulin. Bombyxin and insulin induced phosphorylation of different sets of proteins in the HPO, suggesting that their signaling pathways are different.     

Developmental changes of storage proteins and biliverdin-binding proteins in the haemolymph and fat body of the common cutworm,Spodoptera litura

The concentrations of three storage proteins (SL-1,SL-2 and SL-3, hexamers of 70-80kDa subunits) and two biliverdin-binding proteins (BP-A and BP-B, dimers of 165kDa) in the haemolymph and fat body during larval and pupal development of Spodoptera litura were determined by immunodiffusion tests using polyclonal antisera. SL-1 and SL-2 (methionine-rich) first appeared in the haemolymph of one-day-old sixth (final) instar larvae, prominently increased in the haemolymph during the later feeding period and were almost totally sequestered by the fat body after gut purge. SL-3 (arylphorin) was first detected in the haemolymph during the molting period to the final larval ecdysis, increased in concentration throughout the entire feeding period of the final larval instar and was partly sequestered by the fat body several hours later than the other storage proteins. BP-A showed nearly the same pattern in the haemolymph as SL-3: BP-B increased during feeding period and decreased during molting period and attained a maximum level during the penultimate larval instar, however its concentration decreased considerably and remained low in the final larval instar. BP-A was partly and BP-B was almost totally sequestered by the fat body 8 h after sequestration of SL-1 and SL-2, rendering the fat body blue in colour. These facts suggest an additional function of biliverdin-binding proteins as amino acid storage proteins and the results show a differential uptake mechanism for these proteins by the fat body.     

Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP(3) signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP(3) receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP(3) receptor by increased levels of 20-hydroxyecdysone.     

An integrin-tetraspanin interaction required for cellular innate immune responses of an insect, Manduca sexta

In their encounters with foreign intruders, the cells of the insect innate immune system, like those of the mammalian immune system, exhibit both humoral and cell-mediated responses. Some intruders can be dispatched by the humoral immune system alone, but many must be phagocytosed by individual hemocytes or encapsulated by interacting hemocytes. Surface proteins of hemocytes control the abrupt transition of hemocytes from resting, nonadherent cells to activated, adherent cells during these cell-mediated responses. Two of these surface proteins, an integrin and a tetraspanin, interact during this adhesive transition. As demonstrated with a hemocyte adhesion assay and a surface plasmon resonance assay, the large extracellular loop of tetraspanin D76 binds to a hemocyte-specific integrin of Manduca sexta. The interaction between the large extracellular loop domain and hemocyte-specific integrin is interrupted not only by a monoclonal antibody (MS13) that binds to a domain of beta-integrin known to be a ligand-binding site for cell adhesion but also by double-stranded beta-integrin RNA. Transfected S2 cells expressing tetraspanin mediate adhesion of hemocytes. A monoclonal antibody to tetraspanin D76 perturbs the cell-mediated immune response of encapsulation. These studies involving antibody blocking, RNA interference, and binding assays imply a trans interaction of integrin and tetraspanin on hemocyte surfaces.     

Aedes aegypti: characterization of hemocyte polypeptide synthesis during wound healing and immune response to inoculated microfilariae

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80-90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.     

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae)

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.