Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.

Gender differences and individual variation in the immune system of the scorpionfly Panorpa vulgaris (Insecta: Mecoptera).

From investigations of the vertebrate immune system gender specific differences in individual immunocompetence are well known. In general, females seem to possess more powerful immune systems than males. In invertebrates, the situation is much less clear. Therefore, we investigated the immune system of an invertebrate species, the scorpionfly Panorpa vulgaris. We found a high degree of individual variation in both traits studied, the lysozyme-like antibacterial activity of hemolymph and the capacity for in vitro phagocytosis of artificial particles. These two immune traits were positively correlated. As expected, hemolymph derived from females had higher lysozyme-like activity and hemocytes from females phagocytosed more particles. The difference in phagocytosis was mainly based on higher total hemocyte counts and higher proportions of phagocytically active cells in females, while the average number of ingested particles per active phagocyte was not significantly different. The observed gender differences are discussed in the context of reproductive strategies and parasite-mediated sexual selection.

Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth, Galleria mellonella.

The effects of beauverolide L and cyclosporin A, cyclic peptidic metabolites, produced by several genera of entomopathogenic fungi on immune responses of last instar larvae of the greater wax moth Galleria mellonella have been examined. Intrahemocoelic injection of either metabolite-coated silica particles or dissolved metabolites in a concentrations ranging between 10 and 30 micrograms per larvae caused no mortality but activated humoral responses in G. mellonella larvae. The challenge induced a significant release of lysozyme and cecropin-like activity into the hemolymph, suggesting stimulatory activity on humoral immune responses. Injected metabolite-coated particles were rapidly surrounded by hemocytes which subsequently accomplished formation of melanized nodules, which increased in size and number compared with controls. In vitro assays with dissolved metabolites indicated no adverse effects of beauverolide L or cyclosporin A on attachment or spreading of isolated plasmatocytes but dose-dependent inhibition of their phagocytic activity. Isolated plasmatocytes incubated with cyclosporin A or beauverolide L exhibited cytoskeleton alterations that differed from those observed in plasmatocytes from infected G. mellonella larvae or reported from other fungal secondary metabolites. The experiments provided further data to elucidate the role of fungal secondary metabolites in development of mycoses in insects.

LPS (lipopolysaccharide)-activated immune responses in a hemocyte cell line from Estigmene acraea (Lepidoptera).

The suitability of the hemocyte cell line BTI-EA-1174-A from Estigmene acraea (Lepidoptera) to serve as a tool for studying insect immune reactions in vitro was investigated. Addition of bacterial lipopolysaccharides to the cultures caused enhanced phagocytosis of silica beads, as well as increased lysozyme activity in the cell culture supernatants. Addition of fungal beta 1,3-glucans did not result in any activation. The LPS-influenced (1 mg/mL) increase of phagocytic reactions against the silica beads was at its highest within 24 h after LPS-addition. Activated cells exhibited drastic changes in their morphology in connection with reduced cell numbers in the cultures but without increased mortality rates. LPS-dosages higher than 10 micrograms/mL LPS provoked significantly enhanced lysozyme activities. A maximal induction took place with 1 mg/mL LPS. The lysozyme activity started to rise 2 days after LPS-addition, further increase was observed up to the seventh day. The responsible protein was isolated from cell culture supernatants and N-terminally sequenced. The exact molecular mass was determined by mass spectrometry as 14.080 kDa. The amino acid sequence of the analysed portion revealed high sequence-similarity to the lysozymes of other lepidopteran insects as well as to hen egg lysozyme. Further results presented in this paper give indications for the existence of soluble molecules which are released by the cells and which enhance the LPS-triggered activation.

Isolated Apolipophorin III from Galleria mellonella Stimulates the Immune Reactions of This Insect.

Apolipophorin III (apoLp-III) was isolated from the haemolymph of last instar larvae of Galleria mellonella. The ultraviolet (u.v.) spectrum and the N-terminal amino acid sequence reveal high similarities with the apoLp-III from Manduca sexta. The protein is heat-stable. The molecular mass of apoLp-III was determined to be 18 077 Da using mass spectrometry. The heat treatment (90 degrees C, 30 min) resulted in a pI shift from 6.6 for the non-heated to 6.1 for the heat-treated apoLp-III without change in the molecular mass, indicating that a conformational change might have been caused by the heat treatment, rather than covalent alterations. Intrahaemocoelic injection of pure apoLp-III into last instar G. mellonella larvae is followed by a dose-dependent increase of antibacterial activity in cell-free haemolymph of treated larvae 24 h after injection. Furthermore, pure apoLp-III enhances the phagocytic activity of isolated haemocytes in vitro. The newly discovered role of apoLp-III in inducing immune-related functions in insects is discussed in regard to the known features of this molecule in lipid metabolism. Arylphorin, another heat-stable protein in G. mellonella haemolymph, was likewise isolated in this study. The protein was identified by N-terminal protein sequencing, the sequence obtained exactly matches the known sequence data for this protein. Copyright 1997 Elsevier Science Ltd. All rights reserved

Effects of the entomopathogenic fungus Metarhizium anisopliae and its secondary metabolites on morphology and cytoskeleton of plasmatocytes isolated from the greater wax moth, Galleria mellonella.

The effects of Metarhizium anisopliae infection and three different secondary metabolites released by the fungus, destruxin A and E and cytochalasin D, on the morphology and cytoskeleton of plasmatocytes of the greater wax moth Galleria mellonella were studied. Plasmatocytes isolated from M. anisopliae infected larvae exhibited impairment of attachment, spreading and cytoskeleton formation accompanied with the occurrence of blebbing and pycnotic nuclei. Plasmatocytes treated with destruxin in vitro exhibited similar morphological and cytoskeleton alterations. The corresponding effects were characterized by inhibition of attachment, spreading and filopodia formation as well as by impaired formation of actin filaments and microtubules. Cytochalasin was shown to affect plasmatocytes in vitro in a different manner than destruxin A and E. The results of our comparative study strongly suggested that the morphology and cytoskeleton alterations of plasmatocytes observed in M. anisopliae infected larvae were predominantly caused by destruxins released by the fungus during mycosis. Its mode of action is discussed with regard to present knowledge about its effects on target cells.

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme.

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.

Investigation of oxygen transfer through the membrane of polymer hollowspheres by oxygen micro-electrodes.

Estimation of oxygen transfer from an aqueous solution into polymer hollow spheres, as used for encapsulation of microorganisms, requires detection of the dissolved oxygen concentration inside the hollow sphere. Using microcoaxial needle electrodes, oxygen kinetics in different penetration depths within single cellulose-sulfate hollow spheres loaded with and without living yeast cells could be measured. Based on the reaction kinetics the diffusion coefficient for oxygen and the oxygen-uptake rate have been calculated. The diffusion coefficient DO2 within the thin membrane of cell-free spheres was in the order of 10(-10)m2s-1. Due to the liquid core of the hollow sphere the corresponding diffusion coefficient DO2 is in the range of the value for water. The oxygen-uptake rate QO2 in cell containing spheres could be estimated to 90 mmol l-1 h-1, which corresponds to a specific oxygen-uptake rate qO2 of 10 mmol g-1 h-1. It is to conclude that oxygen supply of the microorganisms inside the hollow spheres was, in this case, not critically influenced by the thin polymeric wall of the capsules.