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Injection of heat-killed bacteria into larvae of the greater wax moth Galleria mellonella is followed by changes in lipoprotein composition in the hemolymph. Density gradient centrifugation experiments revealed that within the first four hours after injection, a part of larval lipoprotein, high-density lipophorin (HDLp), was converted into a lipoprotein of lower density. SDS-polyacrylamide gel electrophoresis analysis of the gradient fractions and sequencing of protein fragments, established that the exchangeable apolipoprotein apolipophorin III (apoLp-III), a potent immune-activator, was associated with this newly formed lipophorin. To investigate further the influence of lipophorin-associated apoLp-III on immune-related reactions, we performed in vitro studies with isolated hemocytes from G. mellonella and lipophorins from the sphinx moth Manduca sexta, as a natural source of high amounts of low-density lipophorin (LDLp) and HDLp. The hemocytes were activated to form superoxide radicals upon incubation with LDLp, but not with HDLp. Fluorescence-labeled LDLp was specifically taken up by granular cells. This process was inhibited by adding an excess of unlabeled LDLp, but not by HDLp. We hypothesize that larval lipophorin formed in vivo is an endogenous signal for immune activation, specifically mediated by the binding of lipid-associated apoLp-III to hemocyte membrane receptors.

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.

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3'-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pET-vector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae.

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

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 (

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.

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.

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.

Apolipophorin III (apoLp-III) is a prototype exchangeable apolipoprotein that is amenable to structure-function studies. The protein folds as a bundle of five amphipathic alpha-helices and undergoes a dramatic conformational change upon lipid binding. Recently, we have shown that a truncation mutant of Galleria mellonella apoLp-III comprising helices 1-3 is stable in solution and able to bind to lipid surfaces [Dettloff, M., Weers, P. M. M., Niere, M., Kay, C. M., Ryan, R. O., and Wiesner, A. (2001) Biochemistry 40, 3150-3157]. To investigate the role of the C-terminal helices in apoLp-III structure and function, two additional 3-helix mutants were designed: a core fragment comprising helix (H) 2-4, and a C-terminal fragment (H3-5). Each truncation mutant retained the ability to associate spontaneously with dimyristoylphosphatidylcholine (DMPC) vesicles, transforming them into discoidal complexes. The rate of apolipoprotein-dependent DMPC vesicle transformation decreased in the order H1-3H2-4H3-5. Truncation of two helices led to a significant decrease in alpha-helical content in buffer in each case, from 86% (wild-type) to 50% (H1-3), 28% (H2-4), and 24% alpha-helical content (H3-5). On the other hand, trifluoroethanol or complexation with DMPC induced the truncation mutants to adopt a high alpha-helical structure similar to that of wild-type protein (84-100% alpha-helical structure). ApoLp-III(H1-3) and apoLp-III(H2-4), but not apoLp-III(H3-5), were able to prevent phospholipase-C-induced low density lipoprotein aggregation, indicating that interaction of the C-terminal fragment with spherical lipoprotein surfaces was impaired. As lipoprotein binding is significantly affected and DMPC transformation rates are relatively slow upon removal of N-terminal helices, the data indicate that structural elements necessary for lipid interaction reside in the N-terminal part of the protein.

In this study the authors give immunocytochemical evidence for the presence of interleukin (IL)-1alpha- and tumour necrosis factor (TNF) alpha-like molecules in the haemocytes of last instar larvae from the greater wax moth Galleria mellonella. Similar results are demonstrated in a continuous haemocyte line (BTI-EA-1174-A) from the salt marsh caterpillar Estigmene acraea. In Galleria mellonella larvae granular cells show a strong positive reaction with both primary antibodies, whereas plasmatocytes are stained to a lesser extent. Cell line haemocytes also react positively with both antibodies. After activating the cells with lipopolysaccharide (LPS) staining of Estigmene acraea cells is decreased, whereas Galleria mellonella haemocytes show no visible reaction in comparison to non-activated cells.

The entomopathogenic nematodes Heterorhabditis megidis and Steinernema feltiae turned out to be successful antagonists of the orthopteran insects Locusta migratoria and Schistocerca gregaria. The death rate of locusts maintained on nematode-inoculated sand was remarkably high. Even dosages as low as one nematode per cubic centimeter of sand killed approximately 50% of the locusts within 10 days. The impact of parasitation on locusts' immune defense was closely investigated for L. migratoria parasitized by H. megidis. Adult locusts died within 30-35 h after being fed with 50 infective H. megidis juveniles. Within the first 30 h after ingestion of the nematodes, locust hemolymph was assayed for alterations in the humoral and cellular defense components and for the presence of the nematode-associated Photorhabdus luminescens bacteria. Humoral defense was generally low without any correlation to the state of parasitation. There was no detectable activity against Escherichia coli and only little lysozyme-like activity against Micrococcus luteus. In contrast, cellular defense components were strongly influenced by parasitation. Most interestingly, the phagocytic capacity of the hemocytes was already hampered 12 h after oral application of the nematodes, whereas considerable hemocyte death occurred not earlier than 24 h after feeding. The nematode-associated bacteria could be detected in hemolymph of some of the nematode-fed locusts as early as 3 h after feeding and in all hemolymph samples after 24 h. Supernatants from isolated P. luminescens cultures were able to inhibit the L. migratoria phagocytes in vitro; thus the successful parasitation appears to be dependent on an inhibition by bacteria-released compounds rather than on overloading or simply killing of the phagocytic active hemocytes.

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.

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.

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.