Peter Götz

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.

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

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.

Parasitic Fungi and their Interactions with the Insect Immune System

Abstract Recent advances in research on parasitic fungi and their molecular and cellular interactions with the insect immune system have led to new insights into the complex relationships and mechanisms involved in fungal pathogenesis. This review focuses on molecules which mediate virulence of the producing fungi (fungal proteases and toxins) and on molecules contributing to the antifungal humoral immune responses of insects (antifungal proteins, protease inhibitors and detoxification proteins). Among the molecules produced by parasitic fungi during infection, at least proteolytic enzymes and toxins interfere with the host immune system in a sophisticated manner. Some of these compounds support fungal development within the infected host by suppression of its potent immune system. In particular, they impair circulating haemocytes, e.g. by damaging cytoskeletal structures and inducing apoptosis. Resistance of insects to microorganisms relies on cellular and humoral defence reactions. Although research on insect immunity attracted much attention in past decades, studies on the identification, characterization and mode of action of antifungal compounds, inducible protease inhibitors and proteins which detoxify fungal toxins in insects are more recent. Present data suggest a synergistic contribution of these proteins to antifungal defence. Successful development of parasitic fungi in infected host insects obviously requires combined activity of enzymes and immunosuppressive toxins. Current frontiers concerning the research on insect-fungal interactions at the cellular and molecular levels are outlined, with emphasis on applied aspects. Finally, a comparison of parasitic fungi with other groups of entomopathogens reveals parallels in strategies employed for evasion of host immune responses.

Inhibition of phagocytic activity of plasmatocytes isolated from Galleria mellonella by entomogenous fungi and their secondary metabolites

The phagocytic activity of isolated plasmatocytes from Metarhizium anisopliae- or Beauveria bassiana-infected Galleria mellonella larvae was examined and compared to that observed from untreated larvae. Mycosis reduced plasmatocyte phagocytic activity against either yeast cells or blastospores of both entomopathogenic fungi. In contrast, the phagocytic activity of isolated plasmatocytes from diseased larvae against hydrophilic silica beads was only moderately inhibited compared to controls. In addition, the influence of solubilized or surface bound fungal secondary metabolites from M. anisopliae (destruxin A and E, cytochalasin B and D) on plasmatocytes in monolayers was investigated. Fungal cyclic peptides dissolved in sublethal concentrations were found to inhibit phagocytic activity of plasmatocytes in a dose-dependent manner. Fungal blastospores coated with secondary metabolites were not ingested. The phagocytic activity of plasmatocytes obtained from larvae which were preinjected with sublethal concentrations of destruxin A was not significantly reduced. The corresponding inhibition of phagocytic activity found in toxin-treated plasmatocytes in vitro and in plasmatocytes isolated from infected larvae support the assumption that entomopathogenic fungi release secondary metabolites to impair the cellular recognition and defense reactions of their host.

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.

A fluorescence assay demonstrating stimulation of phagocytosis by haemolymph molecules of Gallerta mellonella

Abstract An in vitro microscopic fluorescence assay determining the phagocytic activity of isolated plasmatocytes of Galleria mellonella is described. It was developed to quantify insect cellular immune reactions. The assay, a modification of a method originally established for vertebrate blood cells, is based on the quenching effect of trypan blue on fluorescein-isothiocyanate-labelled yeast cells. Only ingested yeast cells retain their fluorescence after quenching and can be easily distinguished from adhering ones. The technique is highly reproducible and easy to perform. Using this method a phagocytosis-stimulating effect caused by a haemolymph fraction > 100 kDa isolated from G. mellonella is demonstrated.

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.

Silica beads induce cellular and humoral immune responses in Galleria mellonella larvae and in isolated plasmatocytes, obtained by a newly adapted nylon wool separation method

Intrahaemocoelic injection of silica beads into Galleria mellonella (wax moth) larvae provoked strong cellular and humoral reactions similar to those normally occuring during an antibacterial defence. The cellular reactions of the haemocytes—investigated by light and scanning electron microscopy—comprised degranulation of granular cells and phagocytosis by plasmatocytes. The humoral responses—measured in cell free haemolymph as the increase of antibacterial activity against Echerichia coli K12 D31 and of lysozyme activity against Micrococcus luteus cell walls—were significantly enhanced in comparison to controls. Only hydrophylic but not hydrophobic silica beads provoked strong reactions. Plasmatocytes (PLs), the main phagocytic haemocytes of G. mellonella, were isolated by using a newly adapted nylon wool column technique. PL monolayers, prepared from isolated cells, exhibited a high purity by consisting of at least 90% PL. Scanning electron microscopy studies revealed that 64% of the isolated PLs showed phagocytic activity against the sterile silica beads in vitro. Intrahaemocoelic injection of supernatants from monolayers with phagocytosing plasmatocytes into naive larvae led to a clearly higher antibacterial activity against E. coli in haemolymph of recipients than the injection of supernatants from monolayers with non-phagocytosing cells. The earlier supposition that haemocyte-released factors induce the humoral immune response is further supported by these results.

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.