Ultrastructure of the hemocytes of Cetonischema aeruginosa larvae (Coleoptera,Scarabaeidae): involvement of both granulocytes and oenocytoids in in vivo phagocytosis

In the context of comparative studies on immunity defence mechanisms of adults and larvae of the coleopteran Cetonischema aeruginosa (Drury, 1770) the ultrastructure of the circulating hemocytes of the third instar larval stage has been investigated by means of light and transmission electron microscopy (TEM). Six types of hemocytes were found in the hemolymph of C. aeruginosa and they were identified as prohemocytes, granulocytes, plasmatocytes, coagulocytes, oenocytoids and spherule cells. In order to identify the "professional" phagocyte cell, phagocytosis assays were performed in vivo by injection of 0.9 microm carboxylate-modified polystyrene latex beads. It was demonstrated that the granulocytes and the oenocytoids of C. aeruginosa were the only hemocyte types involved in this cellular response.     

Passive evasion of encapsulation in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae), a polyembryonic parasitoid of Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

The hymenopteran Macrocentrus cingulum usually deposits one egg into the larval body cavity of lepidopteran Ostrinia furnacalis, and the egg subsequently splits into several dozens of embryos during its development. How the parasitoid eggs and embryos avoid encapsulation by the host's immune response remains unknown. We compared hemocyte counts, morphologies and behaviors between unparasitized O. furnacalis larvae, and larvae parasitized by M. cingulum. No distinct differences were observed. Sephadex A-25 beads elicited a strong encapsulation response when injected into the parasitized host larvae, which indicates that parasitism by M. cingulum does not affect host's cellular immunity. However, there were significant differences in the host's encapsulation reactions towards injected eggs from different sources. Injected M. cingulum mature eggs excised from the lateral oviducts of the female wasps were not encapsulated, while immature eggs or driselase treated mature ones provoked an encapsulation response within 2 h after injection. Inspection of eggs by transmission electron microscopy revealed that the driselase collapsed the surface fibrous layer of the eggs, indicating that surface fibrous layer may play a role in protecting eggs from host's immune attack.     

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.     

Cytotoxic activity of blowfly Calliphora vicina hemocytes

The cytotoxic activity of hemocytes isolated from larvae of the blowfly Calliphora vicina was tested using human myelogenous leukemia K562 cells as target. Both single cell and cytotoxicity assays demonstrated that the hemocytes recognize the K562 cells as nonself, firmly attach to their surface and induce target destruction in a manner resembling the effect of mammalian cytotoxic lymphocytes. The cytotoxic activity increased dramatically in the course of larval metamorphosis and was considerably higher shortly before the onset of pupariation, compared to the activity of human peripheral blood or mouse spleen lymphocytes. In insects, the cytotoxic hemocytes may take part in defense against eukaryotic parasites and in the elimination of aberrant self cells, as well as in developmental processes such as metamorphosis.     

Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation

Insect hemocytes play a major role in developmental processes where they disassociate and rebuild metamorphosing tissues while undergoing physiological changes themselves. We identified hemocyte changes from the last larval to the beginning of the pupal stage of the tobacco hornworm, Manduca sexta. Larval and pupal hemocytes behaved differently in a 40% Percoll density gradient. Larval granular cells were found in almost all density layers, pupal granular cells were abundant in high density layers; larval plasmatocytes occurred in dense layers, pupal plasmatocytes became enriched in less dense layers of the gradient. Using a panel of monoclonal antibodies generated against purified hemocytes, several different antibody binding patterns were identified. Quantitative differences in staining intensities were observed more often than qualitative changes, e.g. a loss or a gain of staining. Both phenomena were related to both plasmatocytes and granular cells. The distribution of the corresponding antigens in tissues was tested on cross sections of larvae and pupae as well as in Western blot analyses using organ homogenates. Several antibodies were specific for hemocytes only, among which two antibodies bound to molecules of the hematopoietic organ. Other antibodies had an additional reactivity to other tissues, mainly to the basal lamina.     

四种对虾血细胞组成及超微结构

本文应用电镜超薄切片技术,对中国对虾,日本对虾,长毛对虾和草虾的血细胞进行超微结构的研究,根据血细胞形态,超微结构特点,特别是胞质中特征性颗粒的大小和内部结构,区分为四类：１．透明细胞,其胞质不含特征性颗粒,具有较强的吞噬能力;２．小颗粒细胞,其胞质含有高电子密度的小圆形颗粒和条纹状大颗粒,在对虾防御反应中起着关键性作用;３．大颗粒细胞,其胞质中含有高电子密度的均质大颗粒,在宿主防御反应中起着细胞     

Morphology of hemocyte lysis and clotting in the ridgeback prawn,Sicyonia ingentis

Summary Coagulation of hemolymph in the shrimp Sicyonia ingentis was studied using light and electron microscopy. Differential counts of unclotted hemolymph show that 54% of the hemocytes are deposit cells characterized by a high nucleocytoplasmic ratio, a few granules, and cytoplasm filled with distinctive deposits. The remaining hemocytes have numerous large or small granules filling the cytoplasm. Examination of clotted hemolymph to which trypan blue had been added shows that deposit cells lyse, whereas the granulocytes exclude the dye, attach to slides, and extend filopodia. This suggests that deposit cells, not granulocytes, initiate coagulation. Ultrastructural changes in deposit cells were studied at specific times after mixing hemolymph and seawater. Deposit cells fixed immediately after removal from shrimp were shaped like elliptical discs and contained abundant, 50 nm diameter cytoplasmic deposits. After 30 s in seawater, deposit cells displayed several cytoplasmic blebs, and had aggregated the deposits. Cytolysis occurred by 45 s. Linear arrays of deposit appeared to extend through breaks in the plasma membrane, forming filamentous strands that hydrated to produce the clot. At 1 min after withdrawal, spheres of clotted hemolymph were seen, each surrounding a lysed deposit cell. Granulocytes remained relatively unchanged and trapped between adjacent expanding clots. Coagulation via hemocyte lysis is compared with other clotting mechanisms observed in various crustaceans and arthropods.     

华北大黑鳃金龟幼虫血细胞的超微结构观察

应用光学和电子显微镜技术检查了华北大黑鳃金龟3龄幼虫血淋巴内的血细胞,识别出5种类型的血细胞(原血胞、浆血胞、颗粒血胞、珠血胞和凝血胞)并对每一种血细胞的超微结构特点进行描述。     

Novel analogs of alloferon: Synthesis,conformational studies,pro-apoptotic and antiviral activity

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1_MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala⁹]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys⁹]-, [Phg⁹]-, [Lys¹²]-, and [Phe¹²]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.