Andreas Wiesner

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

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.

Localization of injected apolipophorin III in vivo - new insights into the immune activation process directed by this protein.

A few years ago, it was shown that intrahemocoelic injection of the insect apolipoprotein apolipophorin III (apoLp-III) stimulates an immune response in larvae of the greater wax moth, Galleria mellonella. Since the mode of action of this activation process is unknown, we followed apoLp-IIIs pathway in the early phase of the immune-stimulating process, using biotin as a probe. Biotinylated apoLp-III was injected and localized using avidin-coupled horseradish peroxidase. The labeled protein was fully functional; the added amount of biotin per apoLp-III molecule used in this study only slightly decreased its ability to associate with phospholipase C-treated human low-density lipoprotein, as well as the immune-stimulating capability of apoLp-III.Gel electrophoresis with subsequent staining of biotin moieties and lipids revealed that apoLp-III undergoes lipid association in vivo within the first few minutes after injection. After two hours, no biotinylated apoLp-III was detectable in cell-free hemolymph. At this time, a subpopulation of hemocytes showed a distinct peroxidase staining. Control injections of biotinylated bovine serum albumin did not lead to similar results, giving evidence for the specificity of the phenomena observed. The results indicate that lipid association of apoLp-III occurs prior to endocytosis by immune-competent hemocytes, which is followed by the induction of a humoral immune response.

Characteristics of inert beads provoking humoral immune responses in Galleria mellonella larvae

Abstract Injection of ion exchange beads or sterile latex beads into the haemocoel of wax moth ( Galleria mellonella ) larvae provokes an humoral immune response measurable as antibacterial activity in cell-free haemolymph. The influence of provocator surface properties on the intensity of response was investigated. Comparing the antibacterial activities provoked by injection of anion and cation exchangers it can be concluded, that anion exchangers are better provocators than cation exchangers. The influence of physicochemical parameters was confirmed by the fact, that injection of latex beads pretreated with sulphuric acid lead to enhanced antibacterial activities in larvae as compared to the activities provoked by injection of untreated beads. In order to prove if it is possible to enhance the provocation capacity of beads by binding molecules to their surfaces, the following substances were covalently coupled onto the beads prior to injection: fibronectin, peptides with a cell adhesive signal of fibronectin (GLY-ARG-GLY-ASP-SER-PRO-LYS, GLY-ARG-GLY-ASP-SER, ARG-GLY-ASP-SER), poly-lysine, adjuvant peptide, lysozyme, bovine serum albumin, bovine plasma, cell-free haemolymph and haemolymph lysate supernatant (haemolymph preparations from G. mellonella ) Coupling of adjuvant peptide, fibronectin or haemolymph preparations enhanced the provocation capacity of beads very clearly. Likewise, but to a lower extent, an enhancement was noticed for nearly all the other substances tested in comparison to results obtained by injection of unloaded beads. The results will be discussed in relation to the known facts about the influence of provocator characteristics onto the onset of a cellular reaction by arthropod and vertebrate cells.

Induction of immunity by latex beads and by hemolymph transfer in Galleria mellonella

Injection of sterile latex beads into the hemocoel of last instar larvae of Galleria mellonella provoked a strong defense reaction. Cellular defense by hemocytes was followed by enhanced antibacterial activity in hemolymph. Latex-injected insects showed increased survival rates after a challenge injection with high doses of bacteria. Factors which stimulate the production of antibacterial activity could be demonstrated soon after injection by transfer of hemolymph from preinjected to untreated larvae. A large induction capacity in donor hemolymph was accompanied by a strong decrease in the total hemocyte count of free floating hemocytes, resulting from a decrease in number of plasmatocytes and granular cells, the cell types involved in the cellular defense against the injected latex beads. The results presented support the hypothesis that during cellular defense reactions, factors are released from the hemocytes which stimulate the production of antibacterial substances.

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.

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.

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.

A SMALL PHAGOCYTOSIS STIMULATING FACTOR IS RELEASED BY AND ACTS ON PHAGOCYTOSING GALLERIA MELLONELLA HAEMOCYTES IN VITRO

We established an in vitro transfer system with monolayers of isolated plasmatocytes from Galleria mellonella. The plasmatocytes represent the main phagocytically active haemocyte type in this lepidopteran insect. Plasmatocytes to which hydrophilic silica beads were added as a phagocytosing agent served as ‘donor’ cells. Supernatants from these donor cultures were transferred to freshly prepared ‘recipient’ plasmatocyte monolayers. Subsequently, FITC (fluorescein-isothiocyanate) labelled yeast cells were added to the recipient monolayers and the phagocytic activity was determined using an FITC quenching assay with trypan blue. The phagocytic activity in plasmatocyte monolayers which received supernatants from phagocytically active donor cells was significantly higher than the activity of cells receiving supernatants from non-activated donor cells. Time course studies revealed that the inducing capacity of the donor cell supernatants was highest 2–4h after starting phagocytosis of the silica beads. Isolation of the responsible phagocytosis stimulating factor is still underway. From our investigations we can conclude that it must be a very small (<500 Da), hydrophobic, and heat-sensitive molecule. This allows us to speculate that it could belong to the eicosanoids or to the biogenic amines. In addition, we could show that donor cell supernatants possess opsonic activity and that the injection of donor cell supernatant into intact larvae induces an antibacterial humoral response in vivo.