Studies on the role of protein kinase A in humoral immune response of Galleria mellonella larvae

Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate-kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1-4muM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36kDa in G. mellonella fat body.     

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     

Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses

Proteolytic activity released within an organism by wounded tissues or invading pathogens can strongly impair the physiological homeostasis when it remains non-regulated. Thus, an efficient mechanism that enables recognition and inactivation of non-regulated proteolytic activity is essential to limit toxic effects. In larvae of the Greater wax moth Galleria mellonella we discovered that injection of bacterial thermolysin at a sublethal concentration mediates both acquired resistance against a subsequently injected lethal concentration of this metalloproteinase and stimulation of humoral immune response accompanied by the synthesis of an inducible metalloproteinase inhibitor (IMPI) which is released within the haemolymph. In search of a putative mechanism mediating recognition and regulation of released microbial metalloproteinases we determined that thermolysin-mediated hydrolysis of G. mellonella haemolymph proteins in vitro yields small (<3 kDa), heat-stable molecules which were discovered to represent potent elicitors of humoral immune responses when injected into untreated larvae. Obtained results allowed to design a model explaining for the first time regulation of released metalloproteinases within the haemolymph of insects. The determined coherence between regulation of released metalloproteinases by IMPI and the simultaneous induction of antimicrobial proteins provides a new insight into the mechanisms leading to expression of genes in course of humoral immune responses.     

JNK MAP kinase is involved in the humoral immune response of the greater wax moth larvae Galleria mellonella

We investigated the participation of MAP kinases in the response of Galleria mellonella larvae to immune challenge. JNK MAP kinase was activated in fat body 10-15 min after LPS injection in vivo. The level of JNK activation was time- and LPS dosage-dependent. JNK MAP kinase isolated from cell-free extract of fat bodies dissected from immune stimulated larvae phosphorylated c-Jun protein in vitro. The activity of Gm JNK kinase was abolished in the presence of the JNK specific inhibitor SP600125. Our data indicate a correlation between JNK phosphorylation and induction of antimicrobial activity in the insect hemolymph after immune stimulation. Hemolymph from larvae pre-treated with JNK specific inhibitor SP600125 showed a reduced level of antibacterial activity after LPS injection. JNK inhibition by SP600125 abolished antibacterial activity of the in vitro culture of G. mellonella fat body. Finally, we also show a correlation between JNK-dependent immune response of G. mellonella larvae and elevated temperature.     

Effect of Pseudomonas aeruginosa crude proteolytic fraction on antibacterial activity of Galleria mellonella haemolymph

The antibacterial activity of immune haemolymph Galleria mellonella directed against Escherichia coli D31 was destroyed by Pseudomonas aeruginosa crude proteolytic fraction. This was demonstrated by diffusion well assay and acid gel electrophoresis and subsequent bioautography. On the contrary, lysozyme activity appeared to be insensitive to extracellular proteases of P. aeruginosa when activity was determined using the bioautography method. In addition, no change in lysozyme protein level was observed by immunoblotting with specific antibodies directed against G. mellonella lysozyme, which confirmed that lysozyme was not degraded by the crude proteolytic fraction of P. aeruginosa. However, a significant decrease of lysozyme activity in naive and immune haemolymph exposed to the action of P. aeruginosa proteins determined by using diffusion well assay was observed. Mechanisms of the observed inhibition require further studies.     

Inhibition of Beauveria bassiana Proteases and Fungal Development by Inducible Protease Inhibitors in the Haemolymph of Galleria mellonella Larvae

Injection of zymosan or dead yeast cells enhanced the inhibitory activity against exocellular Beauveria bassiana proteases in the cell - free haemolymph of Galleria mellonella larvae . Pre - injected larvae exhibited no decreased mortality after subsequent injection with living B. bassiana blastospores but survived for a prolonged time before death . Increased levels of protease inhibitors in the haemolymph were also observed after injection of B. bassiana proteases . In contrast , no enhanced inhibitory activity against B. bassiana proteases was detected in infected larvae when mycosis was initiated with conidia which enabled the fungus to invade host larvae through the integument in a natural manner . B. bassiana proteases were not completely inhibited by the addition of cell - free haemolymph . Protease inhibitors obtained after heat and trichloroacetic acid precipitation of cell - free haemolymph were added to the protein medium of B. bassiana to study the effect on its growth in vitro. Enriched fractions from pre - injected larvae delayed fungal growth in comparison with fractions from untreated larvae , suggesting that delayed mortality of immunized G. mellonella larvae infected with B. bassiana is due to enhanced levels of protease inhibitors . A non - virulent form of the same strain exhibited reduced capacity to release proteases in vitro. The results strongly suggest that the capacity of insects to release inhibitors against fungal proteases influences their susceptibility against entomopathogenic fungi .     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

Influence of fungal infection on the DOPA-semiquinone and DOPA-quinone production in haemolymph of Galleriamellonella larvae

The formation of reactive oxygen metabolites in haemolymph of Galleria mellonella larvae was studied by ESR spectroscopy. The inhibition of the production of the reactive oxygen metabolites of DOPA in haemolymph under the action of fungal infection was shown using spin trap 1-hydroxy-3-carboxy-pyrrolidine. This inhibition correlated with decrease of phenoloxidase activity in haemolymph of infected insects. Simultaneously, the decrease of production of DOPA-semiquinone was detected using method of spin stabilization by diamagnetic metal ions. Moreover, it was shown that the formation of DOPA-quinone was slowed down in haemolymph of infected insects. Our results suggest that the DOPA-derived quinones/semiquinones may be involved in immune response of insects as part of its defense mechanism.     

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.     

Antibacterial activity in vivo and in vitro in the hemolymph of Galleria mellonella infected with Pseudomonas aeruginosa

The antibacterial activity of hemolymph from Galleria mellonella infected with entomopathogenic strain of Pseudomonas aeruginosa and non-pathogenic bacterium Escherichia coli was studied. In vivo, the antimicrobial activity appeared shortly after P. aeruginosa infection, reached the maximum level 18 h postinjection, while 30 h later only trace activity was noted. The activity induced by E. coli sustained on the high level until 48 h after infection. We also noted that the antimicrobial activity level induced by the non-pathogenic bacterium was higher in comparison to that measured in insects infected with the pathogenic strain of P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides of G. mellonella larvae were digested by P. aeruginosa elastase B. After 1 h incubation of cell-free hemolymph of immune-challenged larvae with elastase B, no antibacterial activity was observed. It was also shown that elastase B degraded synthetic cecropin B while in the presence of 6 mM EDTA antibacterial activity of cell-free hemolymph as well as cecropin B, was not changed which confirmed that the activity was abolished by the metalloprotease.     

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.     

Pre-exposure to yeast protects larvae of Galleria mellonella from a subsequent lethal infection by Candida albicans and is mediated by the increased expression of antimicrobial peptides

Pre-exposure of the larvae of Galleria mellonella to Candida albicans or Saccharomyces cerevisiae protects against a subsequent infection with 10(6) C. albicans cells. This protection can also be induced by exposing larvae to glucan or laminarin prior to the administration of the potentially lethal inoculum. Analysis of the genes coding for galiomicin, a defensin in G. mellonella, a cysteine-rich antifungal peptide gallerimycin, an iron-binding protein transferrin and an inducible metalloproteinase inhibitor (IMPI) from G. mellonella demonstrated increased expression, which is at its highest after 24 h of the initial inoculum. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increased expression of a number of proteins associated with the insect immune response to infection 24 h after the initial exposure. This study demonstrates that the larvae of G. mellonella can withstand a lethal inoculum of C. albicans if pre-exposed to a non-lethal dose of yeast or polysaccharide 24 h previously which is mediated by increased expression of a number of antimicrobial peptides and the appearance of a number of peptides in the challenged larvae.     

Apolipophorin III is a substrate for protease IV from Pseudomonas aeruginosa

Our results demonstrated that Pseudomonas aeruginosa serine protease IV degraded apolipophorin III from the haemolymph of Galleria mellonella larvae. ApoLp-III protein was degraded in a stepwise manner. Four intermediate forms of 15, 13.3, 11.9 and 9.5 kDa were detected after 30 min digestion while only one of 5.6 kDa was released after 1-h incubation time. N-terminal amino acid sequence analysis of 5.6 kDa peptide revealed that it was released from apoLp-III after cleavage between lysine 70 and 71. ApoLp-III degradation by protease IV was inhibited by 1 mM TLCK but not 1 mM EDTA, additionally demonstrating that digestion was catalysed by a serine protease. Our data also indicated apoLp-III degradation in vivo during P. aeruginosa infection of G. mellonella larvae.     

Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

Hydrolytic degradation of juvenile hormones in haemolymph and corpora allata of Galleria mellonella (L).

Hydrolytic rates of juvenile hormones (JHs) I, II and III by the corpora cardiaca-corpora allata complex (CC-CA) and by the haemolymph of Galleria mellonella remain in the same order (III greater than I greater than II in CC-CA and I greater than III greater than II in haemolymph) throughout the last larval instar. Haemolymph hydrolytic activity shows peak at the end of feeding when 80 pmol JH I versus 15 pmol JH II is degraded per 1 microliter and minute; hydrolysis rapidly declines in the apolysing insects. Hydrolytic rates in CC-CA reach a maximum of 240 fmol/pair per min for JH III and 85 fmol/pair per min for JH II in pharate pupae. Brain implantations or chilling of freshly ecdysed last instar larvae, which are known to elevate JH titer and induce supernumerary larval molt, do not affect JH hydrolysis. The results indicate that the dominance of JH II in Galleria may be at least partly controlled by preferential hydrolysis of homologs I and III.     

Changes in <Emphasis Type="Italic">Galleria mellonella</Emphasis> lysozyme level and activity during <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> infection

The level of lysozyme in fat body, hemocytes and cell-free hemolymph from Galleria mellonella larvae infected with Pseudomonas aeruginosa was determined and evaluated. In the samples of fat body and hemocytes, an increase in lysozyme content was detected 1 d after infection and then a significant decrease was observed after a prolonged infection time. In the case of cell-free hemolymph, an increase in the lysozyme level was noticeable during the first 30 h post injection and stayed at a similar level for 42 h. The smaller decrease of the lysozyme level after 42 h might be associated with the development of bacteremia of P. aeruginosa in insects. In addition, the gradual increase in the content of lysozyme correlated with the increase of its activity in the hemolymph of the infected larvae as a response to injection with P. aeruginosa. The G. mellonella lysozyme appeared to be insensitive to extracellular proteinases produced in vivo by P. aeruginosa.     

Immune reactions of Chironomus larvae (Insecta: Diptera) against bacteria

Humoral encapsulation (“melanization”) represents the predominant defence reaction of Chironomus larvae against injected bacteria. Only low levels of phagocytic activity were observed; cellular encapsulation and nodule formation were completely missing due to low numbers of haemocytes. No other humoral antibacterial activity was detected in normal Chironomus haemolymph and even haemolymph of preinjected (“immunized”) Chironomus larvae showed little inhibition of bacterial growth on agar test plates.Low cellular and lytic activity of Chironomus haemolymph against bacteria is well compensated for by its fast and efficient capacity of humoral encapsulation. Within 5–10 min, even high numbers of injected bacteria (up to 10⁵ per larva) were surrounded by capsular material. Within this range of injection dose, the fates of pathogenic and non-pathogenic strains were identical, and bacteria which are highly pathogenic for many other insects, e.g. larvae of Galleria mellonella, proved to be harmless to Chironomus larvae. The rapidity of humoral encapsulation may prevent the release of toxins or enzymes by which pathogenic bacteria normally damage its host and weaken its immune system.     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

Development of an insect model for the in vivo pathogenicity testing of yeasts

Conventional in vivo assays to determine the relative pathogenicity of yeast isolates rely upon the use of a range of mammalian species. The purpose of the work presented here was to investigate the possibility of using an insect (Galleria mellonella) as a model system for in vivo pathogenicity testing. The haemolymph of G. mellonella larvae was inoculated with PBS containing different concentrations of stationary phase yeasts of the genus Candida by injection at the last pro-leg. Larvae were incubated at 30 degrees C and monitored over 72 hours. Results indicate that G. mellonella can be killed by the pathogenic yeast Candida albicans and by a range of other Candida species but not to a significant extent by the yeast Saccharomyces cerevisiae. The kill kinetics for larvae inoculated with clinical and laboratory isolates of C. albicans indicate the former class of isolates to be more pathogenic. Differences in the relative pathogenicity of a range of Candida species may be distinguished using G. mellonella as a model. This work indicates that G. mellonella may be employed to give results consistent with data previously obtained using mammals in conventional in vivo pathogenicity testing. Larvae of G. mellonella are inexpensive to culture, easy to manipulate and their use may reduce the need to employ mammals for routine in vivo pathogenicity testing with a concomitant reduction in mammalian suffering.     

Presence of IL-1- and TNF-like molecules in Galleria mellonella (Lepidoptera) haemocytes and in an insect cell line Fromestigmene acraea (Lepidoptera).

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.