Apolipophorin III lysine modification: Effect on structure and lipid binding

Apolipophorin III (apoLp-III) from Locusta migratoria was used as a model to investigate apolipoprotein lipid binding interactions. ApoLp-III contains eight lysine residues, of which seven are located on one side of the protein. To investigate the role of positive charges on lipid binding, lysine residues were acetylated by acetic anhydride. The degree of acetylation was analyzed by SDS-PAGE and MALDI-TOF, indicating a maximum of eight acetyl additions. Modified apoLp-III remained α-helical, but displayed a decreased α-helical content (from 78 to 54%). Acetylation resulted in a slight increase in protein stability, as indicated by a change in the midpoint of guanidine-HCl induced denaturation from 0.55 (unmodified) to 0.65 M (acetylated apoLp-III). Lipid bound apoLp-III, either acetylated or unmodified, displayed similar increases in helical content and midpoint of guanidine-HCl-induced denaturation of ∼ 4 M. The ability to solubilize vesicles of dimyristoylphosphatidylcholine remained unchanged. However, the rate to solubilize dimyristoylphosphatidylglycerol vesicles was reduced two-fold. In addition, a decreased ability to stabilize diacylglycerol-enriched low density lipoproteins was observed. This indicated that lysine residues are not critical for the protein's ability to bind to zwitterionic phospholipids. Since binding interactions with ionic phospholipids and lipoproteins were affected by acetylation, lysine side-chains may play a modulating role in the interaction with more complex lipid surfaces encountered in vivo.     

Tyrosine fluorescence analysis of apolipophorin III-lipopolysaccharide interaction

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein that binds to lipopolysaccharides (LPS). Polyacrylamide gel electrophoresis analysis demonstrated that apoLp-III from Galleria mellonella associated with various truncated LPS variants, including lipid A. Subsequent binding studies were performed employing the intrinsic tyrosine fluorescence properties of apoLp-III, which is highly quenched in the unbound state. A marked increase in tyrosine fluorescence intensity was observed upon binding to LPS or detoxified LPS, indicating a new microenvironment for Tyr-142. This also implies that the LPS carbohydrate region is involved in LPS binding. Dissociation constants (Kd) measured by apoLp-III titration were estimated at approximately 1 microM. Increasing the ionic strength did not decrease the Kd, neither did LPS phosphate removal. In addition, truncation apoLp-III mutants, lacking two complete helices, were still able to associate with LPS. This indicates that the association of apoLp-III with LPS may not be governed by charge but by hydrophobic interactions.     

Apolipophorin III: a lipid-triggered molecular switch

Apolipophorin III (apoLp-III) is a low molecular weight exchangeable apolipoprotein that plays an important role in the enhanced neutral lipid transport during insect flight. The protein exists in lipid-free and lipid-bound states. The lipid-bound state is the active form of the protein and occurs when apoLp-III associates with lipid-enriched lipophorins. ApoLp-III is well characterized in two evolutionally divergent species: Locusta migratoria and Manduca sexta. The two apolipoproteins interact in a similar manner with model phospholipid vesicles, and transform them into discoidal particles. Their low intrinsic stability in the lipid-free state likely facilitates interaction with lipid surfaces. Low solution pH also favors lipid binding interaction through increased exposure of hydrophobic surfaces on apoLp-III. While secondary structure is maintained under acidic conditions, apoLp-III tertiary structure is altered, adopting molten globule-like characteristics. In studies of apoLp-III interaction with natural lipoproteins, we found that apoLp-III is readily displaced from the surface of L. migratoria low-density lipophorin by recombinant apoLp-III proteins from either L. migratoria or M. sexta. Thus, despite important differences between these two apoLp-IIIs (amino acid sequence, presence of carbohydrate), their functional similarity is striking. This similarity is also illustrated by the recently published NMR solution structure of M. sexta apoLp-III wherein its molecular architecture closely parallels that of L. migratoria apoLp-III.     

Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae

Larvae of Galleria mellonella exposed to mild heat-shock (38 degrees C) showed an enhanced humoral immune response after microbial infection in comparison to infected animals grown at 28 degrees C. This enhanced response was manifested by increased expression of antimicrobial peptide (AMP) genes leading to enhanced antimicrobial activity in the hemolymph. We found an increased level of Hsp90 and changes in the level of a 55kDa protein recognized by anti-Hsp90 antibodies in fat bodies of infected animals reared at 28 degrees C as well as in uninfected animals exposed to elevated temperature. Pre-treatment of animals with an inhibitor of Hsp90, 17-DMAG, prior to immunization resulted in increased expression of AMP genes encoding gallerimycin and cecropin at 38 degrees C. This observation was correlated with the changes in Hsp90 protein and increased level of 55kDa protein. Also G. mellonella larvae pre-treated with 17-DMAG and exposed to mild heat-shock for 30min showed an increased survival rate after infection with entomopathogenic bacteria Pseudomonas aeruginosa. We also show the effect of 17-DMAG on the phosphorylation state of ERK MAP kinase. We postulate that Hsp90 may play a significant role in converging pathways involved in the insect immune response and heat-shock.     

Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. After the peroral inoculation of Galleria mellonella by the Bt in 5% sublethal concentration (LC₅), a 1.5-fold increase in the phagocytic activity of infected larvae has been registered on the second and third days after the inoculation. With the increase of Bt-inoculum amount to 15% of sublethal concentration (LC₁₅), a further increase of the phagocytic activity and enhanced encapsulation rates in the haemolymph of infected larvae has been observed. The enhanced cellular immunity during the bacteriosis seems to have resulted from the destruction of midgut epithelium cells followed by the subsequent exposure of gut content to lymph factors activating the immune system of haemocoel.     

Immunocompetence of Galleria mellonella: sex- and stage-specific differences and the physiological cost of mounting an immune response during metamorphosis

The antibacterial immune response of the wax moth, Galleria mellonella, was analysed by use of an inhibition zone plate assay. We demonstrated significant stage-specific differences as the immune response was most effective in the pupal, next the larval and then the adult stage. In addition, we demonstrated that an immune challenge at the onset of, or during metamorphosis does not increase nor decrease the strength of the antibacterial immune response in the subsequent developmental stage(s). These findings illustrate that induced immunity is not preserved during metamorphosis but also deny any cost to the immune system itself. However, an immune challenge does induce a significant shortening of the direct development time and affects the mass loss during metamorphosis in a sex-dependent manner: males emerged smaller whereas the mass of females was not significantly affected. These observations indicate that there are sex-specific costs to mounting an immune response during metamorphosis which affect physiological traits, implicating a trade-off between immunity and development.     

The glutathione-related detoxication responses to juvenile and ecdysone hormones in Galleria mellonella

The effect of 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the glutathione pathway of the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) was determined by investigating glutathione peroxidase (GSH-Px), glutathione S-transferases (GST), and glutathione reductase (GR) activities as well as reduced and oxidized glutathione (GSH and GSSG) content with respect to developmental stage. The continuous decreases of GSH-Px and GST activities dependent on the growth period of G. mellonella occurred in JH and 20E groups over and under their controls, respectively. While the GR activities of G. mellonella showed increases in young pupa (YP) for both control and in old larvae (OL) for the 20E groups after the minimum at these periods, they also increased after old pupa (OP) for the JH group with a maximum in OL period. Although GR activity levels in the JH group were significantly higher compared with controls and 20E groups up to OP period, the activity levels for the control and 20E groups were higher than those of the JH group at adult (AD) and old pupa (OP) periods, respectively. In spite of increases in the GR activity of 20E and control groups of G. mellonella, decreased GSH and increased GSSG levels were observed at aging period. GSH levels in the JH group reached a maximum at prepupa (PP) and then decreased with non-significant changes from OL to AD period. According to the results, GSH and GSSG levels, as well as GSH/GSSG ratios, were below and over control levels in 20E and JH groups, respectively, during all of the investigated developmental stages. On the contrary, the LPO levels were higher than the control for 20E and lower for the JH groups during the developmental period. These results show that while ecdysone hormone has a negative effect on the glutathione-related detoxication capacity of G. mellonella, the juvenile hormone has a positive effect on this process.     

Facultative scavenging as a survival strategy of entomopathogenic nematodes

Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode-bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

The influence of chronic eicosanoid biosynthesis inhibition on life history of the greater waxmoth, Galleria mellonella and its ectoparasitoid, Bracon hebetor

Eicosanoids are oxygenated metabolites of three C20 polyunsaturated fatty acids, mainly arachidonic acid (AA; 20:4n-6), but also 20:3n-6 and 20:5n-3. Aside from their importance in biomedicine, eicosanoids act in invertebrate biology. Prostaglandins (PGs) influence salt and water transport physiology in insect rectal epithelia and in Malpighian tubules. PGs also influence a few insect behaviors, including releasing oviposition behavior and behavioral fever. Eicosanoids act in ovarian development and in insect immunity. Because eicosanoids act in several areas of insect biology, we posed the hypothesis that chronic inhibition of eicosanoid biosynthesis, in the absence of microbial challenge, can influence insect life table parameters, including developmental time, survival, adult longevity and parasitoid fecundity. Here we report that inhibiting eicosanoid biosynthesis throughout the larval life exerted minor influences on some life table parameters of the greater wax moth, Galleria mellonella and its ectoparasitoid, Bracon hebetor, however, the inhibitors strongly reduced the production and hatchability of the parasitoids’ eggs. The significance of the work relates to the potentials of understanding and targeting eicosanoid systems as a platform for developing new technologies of insect pest management. As seen here, the impact of targeting eicosanoid systems is seen in crucial moments of insect life histories, such as reproduction or immune challenge rather than in overall larval development.     

Immunosuppressive effect of cyclosporin A on insect humoral immune response

Cyclosporin A suppressed humoral immune response of Galleria mellonella larvae. Insects were immunized with LPS Pseudomonas aeruginosa and then injected with cyclosporin A. Immunosuppressive effects were expressed both, in larvae treated with cyclosporin A at the initial phase of immune response and at the effector phase of antibacterial immunity. Cyclosporin A moderately decreased lysozyme activity and significantly decreased antibacterial activity peptides against Escherichia coli. Immunosuppressive effects of cyclosporin A were observed after immunoblotting with antibodies anti-G. mellonella lysozyme. Tricine SDS/PAGE shown that synthesis of antibacterial peptides of larvae treated with cyclosporin A was considerably inhibited. Insects of impaired immune response by cyclosporin A action lost protective immunity to insect bacterial pathogen P. aeruginosa.     

The role of lipopolysaccharide moieties in macrophage response to Escherichia coli

Lipopolysaccharide (LPS) is the main component of Gram-negative bacteria that - upon infection - activates the host immune system and is crucial in fighting pathogens as well as in the induction of sepsis. In the present study we addressed the question whether the key structural components of LPS equally take part in the activation of different macrophage immune responses. By genomic modifications of Escherichia coli MG1655, we constructed a series of strains harboring complete and truncated forms of LPS in their cell wall. These strains were exposed to RAW 264.7 macrophages, after which phagocytosis, fast release of pre-synthesized TNF and activation of NF-κB signal transduction pathway were quantified. According to our results the core and lipid A moieties are involved in immune recognition. The most ancient part, lipid A is crucial in evoking immediate TNF release and activation of NF-κB. The O-antigen inhibits phagocytosis, leading to immune evasion.     

Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

Is cell surface calreticulin involved in phagocytosis by insect hemocytes?

The innate immune system of insects consists of humoral and cellular components involved in the recognition of and responses to intruding foreign micro- or macroorganisms. Several molecules have been identified so far that recognize molecular patterns present on microorganisms, such as lipopolysaccharides, peptidoglycans and lipoteichonic acid. These molecules, acting as opsonins, trigger immune responses such as phagocytosis, nodule formation, melanization and encapsulation. Here, we investigated the role of calreticulin (CRT) present on the surface of Pieris rapae hemocytes in phagocytosis. Comparative phagocytosis assays using yeast cells showed that hemocytes from different insects exhibit significant variation in their phagocytosing potential and relative CRT involvement.     

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode,Steinernema riobrave (Rhabditida: Steinernematidae)

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.     

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

The role of Pseudomonas aeruginosa elastase B in activation of the humoral immune response in Galleria mellonella larvae was investigated. The results of our study showed that elastase B injected at a sublethal concentration was responsible for eliciting the humoral immune response in G. mellonella larvae. The insects exhibited increased antibacterial activity, namely, we observed appearance of antimicrobial peptides and a higher level of lysozyme in cell-free hemolymph. Elastase B seems to be a more potent elicitor than thermolysin because similar maximal antibacterial activity levels were observed at a 5-fold lower concentration. We also demonstrated that there were differences in the kinetics of induction of antimicrobial activity between thermolysin and elastase B. The maximum level was observed 18 h post challenge of thermolysin and 38 h after injection of elastase B. It was also shown that, 24 h after elastase injection, the relative levels of apoLp-III in the hemolymph significantly increased in comparison with control G. mellonella larvae. The activation of immune responses in metalloproteinase-challenged larvae involved synthesis of metalloproteinase inhibitors which increased the survival rates of insects both against the lethal dose of thermolysin as well as against viable pathogenic bacterial cells of P. aeruginosa.     

Effect of Steinernema glaseri-infected host exudates on movement of conspecific infective juveniles

The entomopathogenic nematode's decision to infect a host is paramount because once the decision is made it is irrevocable; nematodes that invade a host either develop and achieve reproductive success, or they die. Entomopathogenic nematodes that have a cruiser foraging behavior, such as Steinernema glaseri, follow host-associated cues to locate insects to infect. Most of the host finding and infection dynamics research has focused on the infective juvenile nematodes' responses to cues from live insects such as host-associated volatiles and host contact cues. Few studies focus on how previously infected hosts influence infective juvenile infection behaviors. We investigated how exudates from nematode-infected hosts affect the behavior of S. glaseri infective juveniles. We hypothesized that the infective juvenile's behavioral response to cadavers would change as the state of a nematode-infected host changes during pathogenesis. We examined the effect of exudates collected from infected hosts on infective juvenile locomotory behavior. We detected no effects on nematode repulsion or attraction from exudates produced within the first 48h post-infection. We observed repulsion from accumulated exudates during the 3-48, 3-72, 3-120, and 3-144h intervals. Repulsion from exudates was observed during the 48-66, 72-90, and 120-138h intervals in experiments evaluating daily exudate emissions. The repellent effect of infected host exudates may result in an infective juvenile discriminating between suitable and unsuitable hosts.     

Effect of storage temperature on survival and infectivity of Steinernema rarum (OLI strain) (Rhabditida: Steinernematidae)

Nematode strains of the entomopathogenic family Steinernematidae differ in their ability to infect insects at different temperatures. Survival and infectivity of infective juveniles (IJs) of Steinernema rarum (OLI) were studied after their storage at 23 ± 2 °C and at 5 ± 1 °C. Survival at 23 ± 2 °C was always above 95%. At 5 ± 1 °C, survival decreased at week 5, but infectivity did the same after week 2. Unlike other steinernematids, both infectivity and survival of IJs would be higher for S. rarum (OLI) when stored at 23 ± 2 °C.     

Entomopathogenic fungi in cornfields and their potential to manage larval western corn rootworm Diabrotica virgifera virgifera

Entomopathogenic ascomycete fungi are ubiquitous in soil and on phylloplanes, and are important natural enemies of many soil-borne arthropods including larval western corn rootworm, Diabrotica virgifera virgifera, which is a major pest of corn. We measured the prevalence of Beauveria bassiana and Metarhizium anisopliae sensu lato in ten cornfields in Iowa, USA by baiting with larval insects. B. bassiana and M. anisopliae s.l. were present in 60% ± 6.3% and 55% ± 6.4% of soil samples, respectively. Subsequent laboratory bioassays found that some M. anisopliae s.l. strains collected from cornfields killed a greater proportion of D.v. virgifera larvae than a standard commercial strain.