In vitro growth of bacteria in tissue of Galleria mellonella

The in vitro growth patterns of three nonpathogenic and three pathogenic species of bacteria in tissue of Galleria mellonella were compared to growth curves in broth controls. Over the 12-hour observation period, the nonpathogenic species never attained growth in crushed tissue that was comparable to that of the controls. However, the pathogens grew very similarly in crushed tissue and in broth controls. The results suggest that nonpathogenic species of bacteria are unable to grow in crushed tissue of G. mellonella whereas the tissue appears to be analogous to culture media for the pathogenic species. The possible implications of these results are discussed.     

Anti-Listeria activities of Galleria mellonella hemolymph proteins

We report the use of antimicrobial hemolymph proteins from the model host Galleria mellonella as an inhibitor for various Listeria strains, providing a novel source for antilisterial therapeutics. We also have shown that specific virulence-associated genes known to mediate antimicrobial resistance of Listeria in mammalian models indicated a similar function in Galleria.     

Purification and characterization of epidermis-origin hemolymph protein in Galleria mellonella

Epidermis-origin hemolymph protein (EOHP) was identified and purified from the last instar larval hemolymph of Galleria mellonella by anion exchange chromatography, chromatofocusing chromatography, and Sephadex G-100. The EOHP has a native molecular mass of 47 kDa and is composed of one subunit. The isoelectric point of the EOHP was determined to be 5.3. The amino acid composition of the EOHP was rich in aspartic acid, glutamic acid and lysine, but poor in tyrosine, methionine, and tryptophan. EOHP is present in hemolymph over the period from the 4th instar larvae to the adult stage examined. Concentration of EOHP is high during the larval stage but gradually decreased during the developmental stage from pupal to adult stage. EOHP is present in the cuticle, fat bodies and trachea but not in hemocytes, fore gut, mid gut and hind gut.     

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.     

Purification and characterization of eight peptides from Galleria mellonella immune hemolymph

Defense peptides play a crucial role in insect innate immunity against invading pathogens. From the hemolymph of immune-challenged greater wax moth, Galleria mellonella (Gm) larvae, eight peptides were isolated and characterized. Purified Gm peptides differ considerably in amino acid sequences, isoelectric point values and antimicrobial activity spectrum. Five of them, Gm proline-rich peptide 2, Gm defensin-like peptide, Gm anionic peptides 1 and 2 and Gm apolipophoricin, were not described earlier in G. mellonella. Three others, Gm proline-rich peptide 1, Gm cecropin D-like peptide and Galleria defensin, were identical with known G. mellonella peptides. Gm proline-rich peptides 1 and 2 and Gm anionic peptide 2, had unique amino acid sequences and no homologs have been found for these peptides. Antimicrobial activity of purified peptides was tested against gram-negative and gram-positive bacteria, yeast and filamentous fungi. The most effective was Gm defensin-like peptide which inhibited fungal and sensitive bacteria growth in a concentration of 2.9 and 1.9 microM, respectively. This is the first report describing at least a part of defense peptide repertoire of G. mellonella immune hemolymph.     

Hemagglutinating properties of apolipophorin III from the hemolymph of Galleria mellonella larvae

In search for factors that cause encapsulation of foreign bodies in insect hemolymph we discovered that larval hemolymph of Galleria mellonella caused aggregation of mammalian erythrocytes. The hemagglutinating agent was identified as an 18-kDa protein that did not react with lectins. The sequence of 81 amino acids in three protein fragments and the properties of the protein revealed that it was Galleria homologue of apolipophorin III (apoLp-III*). ApoLp-III was found in high amounts in the hemolymph of Galleria larvae, pupae, and adults, as well as in the molting fluid. The hemagglutinating action of the whole hemolymph or the purified apoLp-III was independent of the presence of sugars in the medium. This indicated that it was not mediated by carbohydrates on the erythrocyte surface. The hemagglutination was inhibited at low pH (3.0), in the absence of calcium ions, and in the presence of certain bacterial lipopolysaccharides or their essential component, the 2-keto-3-deoxyoctonate-3-deoxyoctulosonic acid (KDO). It is suggested that interaction of apoLp-III with lipopolysaccharides in bacterial cell walls may play a role in insect immune reactions. Arch. Insect Biochem. Physiol. 38:119–125, 1998. © 1998 Wiley-Liss, Inc.     

Gut flora of Galleria mellonella suppressing ingested bacteria.

Streptococcus faecalis, the only bacterium occurring almost invariably at high populations in guts of Galleria mellonella larvae, suppresses bacteria ingested with food by producing bacteriocin, an antibioticlike substance having a narrow range of bactericidal activity, and by releasing a lysozymelike enzyme, especially in the presence of proteolytic enzymes. The insect intestinal fluid apparently increases the activity of S. faecalis lytic enzyme. Unlike other organisms tested, S. faecalis has shown a strong bactericidal action against various species of unrelated bacteria. Microscopical examination of the sensitive organisms used as indicators has revealed changes resembling formation of protoplasts, gradually leading to destruction of bacterial cells. The insect guts could not be infected, even when the larvae had ingested a high dose of Pseudomonas aeruginosa, Proteus mirabilis, or Bacillus thuringiensis. The mechanism by which S. faecalis could suppress the ingested bacteria is suggested.     

Characterization and immunological analysis of ferritin from the hemolymph of Galleria mellonella.

Ferritin, an iron-binding protein, was purified from the larval hemolymph of the wax moth, Galleria mellonella by KBr density ultracentrifugation and FPLC (Superose 6). The iron content of ferritin was determined by atomic emission spectroscopy and Ferene S stain. Native molecular mass of ferritin was estimated as 630 kDa. SDS-PAGE revealed that the ferritin consists of two major polypeptides of 26 and 32 kDa and one minor polypeptide of 30 kDa. An isoelectric point of ferritin was measured to be approximately 7.3 and only the 32-kDa subunit is glycosylated. The ferritin contains large amounts of lysine, glutamine, glutamic acid and leucine but tryptophan was not detected. Electron microscopic examination of negatively stained preparations showed an 11-nm particle in external diameter and 7-nm iron core. Ferritin is present in both the ovary and testis. Localization of ferritin by immunoelectron microscopy in ovary and testis revealed that the gold particles were located in vitelline membrane and yolk granules but not in follicular epithelium of ovary. In the testis, the gold particles were located in testicular fluid and lumen of vas deferens.     

Isolation of two cDNA clones coding for larval hemolymph proteins of Galleria mellonella

Galleria mellonella a group of four larval hemolymph proteins (LHP) (74, 76, 81 and 82 kDa), which had been earlier shown to be storage proteins, exhibit a stage-specific synthetic pattern. The 82 kDa LHP is synthesized only in day-3 to day-5 last instar larvae, while the other three LHPs are synthesized both in the penultimate (six) and the last instar larvae. None of these LHPs are synthesized in day-0 last instar. With a view to isolate one or more cDNA clones corresponding to these LHPs a cDNA library was prepared in pBR322 starting with poly(A)⁺ RNA from day-5 last instar larval fat body. By differential screening of 714 clones with poly(A)⁺ RNA 39 day-5 larval stage-specific clones were isolated. Two of these clones, designated as 26–38 and 17–36, had 1200–1300 base pair cDNA inserts. Their cDNA inserts did cross hybridize to each other, exhibited different restriction endonuclease digestion patterns and hybridized in northern blots to transcrips of different sizes, thereby suggesting that they represent two separate genes. In addition, the genomic fragments that hybridized in southern blots to the two cDNAs differed in their size. On translation, mRNAs hybrid selected by 26–38 and 17–36 cDNAs produced 76 and 79 kDa polypeptides respectively. Both these genes are expressed in the fat body but not in the midgut, silk glands, Malpighian tubules or carcass. While 26–38 was expressed both in the sixth and seventh (last) instars, 17–36 was expressed only in the last instar. On the basis of tissue and developmental stage specificity of their expression and the sizes of their hybrid selected translation products, these clones are tentatively identified as two LHP-specific cDNA clones. The genes coding for these LHPs appear to be single copy genes.     

Boric acid-induced effects on protein profiles of Galleria mellonella hemolymph and fat body

The dietary effects of boric acid (BA) on the protein profiles of greater wax moth, Galleria mellonella (L.), were investigated in hemolymph and fat body of final instar (VIIth) and pupae. The insects were reared from first-instar larvae on an artificial diets containing 156, 620, 1250 or 2500 ppm of BA. We detected many undetermined protein fractions (6.5-260 kDa) in addition to well-defined protein fractions such as lipophorins and storage proteins in the tissues by using sodium dodecyl-sulphate polyacrylamide gradient gel electrophoresis. A marked quantitative change in the 45 kDa protein fraction of the hemolymph was observed in the VIIth instar larvae reared on 2500 ppm dietary BA.     

Purification and characterization of a flavin-binding storage protein from the hemolymph of Galleria mellonella

The 85K storage protein that accumulates in the hemolymph of Galleria mellonella during the final larval instar was isolated and purified from newly molted pupae. The separation of fresh hemolymph proteins from larvae or pupae by different chromatographic and electrophoretic procedures indicated the native protein had a Mr of 170,000 and consisted of two identical 85K subunits. Crosslinking experiments using fresh hemolymph followed by Western blotting also indicated a dimeric structure for the native protein. Analyses of the dimer purified from pupal hemolymph indicated that 85K was a glycoprotein, containing approximately 6.5% neutral sugar and about 1.9% amino sugar. Like other insect flavin-binding proteins, 85K has a relatively high histidine content but an uncharacteristically high arginine content. The purified 85K dimer did not bind riboflavin, suggesting that the integrity of the molecule had been altered during purification. However, 85K purified in low yield by Affi-Gel Blue chromatography, did bind riboflavin, indicating that under certain, undefined conditions the functional integrity of the protein could be retained during purification. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

In vitro bioassay for the brain allatotropic hormone of Galleria mellonella (Lepidoptera).

An in vitro sensitive bioassay for the Galleria mellonella brain allatotropic hormone (ATTH) was developed. This assay measures the rate of juvenile hormone (JH) synthesis in corpora cardiacacorpora allata complex (CC-CA) stimulated in vitro by ATTH released from the brain during short-term in vitro incubation, or by ATTH extracted from the tissue with methanol. CC-CA of the late VIth instar (VI3) larvae were used for assessment of ATTH. The maximum activation of test CC-CA by ATTH occurred at a concentration of 2 brain equivalents (per 100 ul medium). The highest ATTH activity was exhibited by the brains of chilled VII1 larvae: ATTH extracted from freshly dissected brains, or ATTH released from these brains during 6 h in vitro incubation, activated JH synthesis in the CC-CA nearly five or four times, respectively. The brain of VII1 hydroprenetreated larvae were ATTH inactive.     

Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization

A juvenile-hormone-binding protein (JHBP) has been isolated from Galleria mellonella hemolymph by gel filtration, phosphocellulose chromatography, and by chromatofocusing. The isolated protein is homogeneous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. It has a relative molecular mass of 32,000, Stokes radius 2.4 nm, sedimentation coefficient of 2.3 S, molar absorption coefficient at 280 nm epsilon = 2.34 X 10(4) M-1 cm-1, and is composed of a single polypeptide chain. Chromatofocusing analysis (pI 8.6) and isoelectric focusing (pI 8.1) indicate that the JHBP is an alkaline protein. Its amino acid composition and fluorescence absorption spectra indicate that the protein does not contain tryptophan residues. The protein exhibits one class of binding sites for juvenile hormone (JH), 0.8 per molecule, with the following dissociation constants: JH I, 8.5 X 10(-8) M; JH II, 7.2 X 10(-8) M; JH III, 47 X 10(-8) M. The JHBP binds (10R, 11S)-JH II enantiomer with 2.3-times higher affinity then (10S, 11R)-JH II enantiomer. The pH optimum of binding is 7.0.     

An atomic force microscopy study of Galleria mellonella apolipophorin III effect on bacteria

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. As revealed by LIVE/DEAD staining, incubation of Gram-negative and Gram-positive bacteria in the presence of Galleria mellonella apoLp-III led to growth inhibition of selected bacteria. An atomic force microscopy (AFM) study of bacterial cells after apoLp-III treatment showed considerable alterations in the cell surface of Bacillus circulans, Klebsiella pneumoniae and Salmonella typhimurium. Our results clearly demonstrate that apoLp-III disturbed the proper structure of the bacterial cell surface. The alterations were dissimilar to those caused by cationic antimicrobial peptide, cecropin B, suggesting a different mode of action against bacteria. The present results indicate that AFM provides a powerful tool for studying the interactions of apoLp-III with microbial cells.