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.     

The effect of Habrobracon hebetor venom on the activity of the prophenoloxidase system, the generation of reactive oxygen species and encapsulation in the haemolymph of Galleria mellonella larvae

The cellular and humoral immune reactions in haemolymph of the wax moth Galleria mellonella larvae naturally injected by venom of ectoparasitic wasp Habrobracon hebetor were analyzed. A strong decline of phenoloxidase (PO) activity in the haemolymph and the number of haemocytes with PO activity of envenomated wax moth was observed. In addition, it has been shown that the rate of capsule melanization in the envenomated larvae was half that of the control. Also production of reactive oxygen species (ROS) in the haemolymph of envenomated larvae decreased. The obtained data casts light on the suppression of the main immune reactions in G. mellonella larvae during natural envenomation by H. hebetor.     

Pathology of Spiroplasma floricola in Galleria mellonella larvae

Spiroplasma floricola strain 23-6, originally isolated from tulip tree flowers, was injected into larvae of the greater wax moth. Histopathology and cytopathology of disease larvae were studied by histochemical, fluorescent antibody, and electron microscopical methods. The gut was empty, polysaccharides in fat and muscle tissue were reduced, the fat body was broken down, and phospholipids were depleted in larvae 4 days after injection. Fluorescein conjugated S. floricola antibody was adsorbed onto hemocytes, sarcolemma, gut epithelial membrane, and the cortex of the ventral ganglia. By electron microscopy, spiroplasmas were found in hemocoel, hemocytes, pericardial cells, connective tissues, basement membranes, epidermal cells of the cuticle, the neural lamella, and the peripheral glial cells of the ventral nerve cord, and on midgut and epidermal membranes. It is postulated that the cytopathological effects induced in the body of the insect released nutritional elements that allow extensive reproduction of S. floricola.     

Iron contributes to the antibacterial functions of the haemolymph of Galleria mellonella

Studies with Galleria mellonella larvae and the iron chelating agent EDDA showed that iron was essential for the removal of dead Xenorhabdas nematophila and Bacillus subtilis from the haemolymph. The delay in removal of the bacteria from the iron-restricted haemolymph was attributed to reduced adhesiveness of the haemocytes and prophenoloxidase activity. Iron augmentation returned these activities to control levels. Whereas dead B. subtilis had no effect on the concentration of ferrozine-detectable iron (henceforth iron) in the haemolymph, dead X. nematophila was associated with substantially lower levels of iron as the number of damaged haemocytes increased. Haemocyte lysate lowered the concentrations of iron in both FeCl₃ solutions and deproteinized larval serum independent of serum lipids. Haemocyte lysate added to tryptic soybroth lowered the level of iron and limited the growth of X. nematophila. X. nematophila limited iron availability in the plasma by releasing lipopolysaccharides; such a mechanism may be a means of impairing the antimicrobial defences of the insects.     

Thermal and physical stresses induce a short-term immune priming effect in Galleria mellonella larvae

Exposure of larvae of Galleria mellonella larvae to mild physical (i.e. shaking) or thermal stress for 24 h increased their ability to survive infection with Aspergillus fumigatus conidia however larvae stressed in a similar manner but incubated for 72 h prior to infection showed no elevation in their resistance to infection with A. fumigatus. Stressed larvae demonstrated an elevated haemocyte density 24 h after initiation of the stress event but this declined at 48 and 72 h. Larval proteins such as apolipophorin, arylophorin and prophenoloxidase demonstrated elevated expression at 24 h but not at 72 h. Larvae maintained at 37 °C showed increased expression of a range of antimicrobial and immune-related proteins at 24 h but these decreased in expression thereafter. The results presented here indicate that G. mellonella larvae are capable of altering their immune response following exposure to mild thermal or physical stress to mount a response capable of counteracting microbial infection which reaches a peak 24 h after the initiation of the priming event and then declines by 72 h. A short-term immune priming effect may serve to prevent infection but maintaining an immune priming effect for longer periods may be metabolically costly and unnecessary while living within the colony of another insect.     

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live gram-positive bacteria Micrococcus lysodeikticus and gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae.     

Detoxification of destruxins in Galleria mellonella L. larvae

1. Destruxins applied by intrahaemocoelic injection into Galleria mellonella larvae quickly accumulate in hindgut and are extensively metabolized by opening their cyclic structure.2. The process of detoxification corresponds to physiological state of larvae from early symptoms of paralysis to complete recovery within several hours.     

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.     

Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans

Larvae of the greater wax moth (Galleria mellonella) that had been subjected to physical stress by shaking in cupped hands for 2 min showed reduced susceptibility to infection by Candida albicans when infected 24 h after the stress event. Physically stressed larvae demonstrated an increase in haemocyte density and elevated mRNA levels of galiomicin and an inducible metalloproteinase inhibitor (IMPI) but not transferrin or gallerimycin. In contrast, previous work has demonstrated that microbial priming of larvae resulted in the induction of all four genes. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increase in the intensity of a number of peptides showing some similarities with proteins associated with the insect immune response to infection. This study demonstrates that non-lethal physical stress primes the immune response of G. mellonella and this is mediated by elevated haemocyte numbers, increased mRNA levels of genes coding for two antimicrobial peptides and the appearance of novel peptides in the haemolymph. This work demonstrates that physical priming increases the insect immune response but the mechanism of this priming is different to that induced by low level exposure to microbial pathogens.     

大蜡螟幼虫的体色遗传规律

对大蜡螟Galleria mellonella幼虫不同颜色品系的普通遗传学分析表明,大蜡螟幼虫的体色遗传是常染色体遗传且符合复等位基因遗传规律。深黄色基因（AA）对灰黑色基因(BB)和灰色基因(CC)为显性,深黄色基因(AA)对白黄色基因(DD)、灰黑色基因(BB)对白黄色基因(DD)和灰色基因(CC)、灰色基因(CC)对白黄色基因(DD)为不完全显性。基因型为AD、BD、CD的个体,其表现型均为黄色;基因型为AA、BC的个体,其表现型均为深黄色。     

Conformational change of the haemolymph juvenile-hormone-binding protein from Galleria mellonella (L)

A juvenile-hormone-binding protein (juvenile-hormone carrier), isolated from Galleria mellonella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile-hormone-binding activity of this protein. With SDS/PAGE it was shown that juvenile-hormone-binding protein, a 32-kDa peptide, is first slowly converted into a 30-kDa molecule, then into two or three smaller-molecular-mass species (20-25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30-kDa peptide has a 2.4-times-higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20-25-kDa peptides. The rate of proteolysis of juvenile-hormone-binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non-denaturing conditions, free binding-protein molecules could be separated from juvenile-hormone-binding-protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross-linking experiments, binding of the hormone to its haemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71S. It is concluded that juvenile-hormone binding induces a conformational transition of its carrier protein. This hormone-induced change might have a physiological significance for signal transmission.     

Chilled Galleria mellonella larvae: mechanism of supernumerary moulting

ABSTRACT. Supernumerary larval instars were produced when Galleria mellonella L. (Lepidoptera) larvae were chilled at 0°C. Although sensitivity to cooling stress of the last instar and younger larvae were generally the same, only penultimate and the last instar larvae showed a significant correlation between their age and the number of additional larval moults. Chilling stress induced a rapid and persistent increase in the JH titre of the last instar larvae. Severing the ventral nerve cord resulted in a predictable loss of the ability to produce supernumerary moults in chilled last instar larvae. The data suggest that sensory input stimulates allatotropic hormone secretion by the brain of chilled larvae. The possible mechanism controlling supernumerary moulting is discussed.     

Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae

Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides. The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria. The total number of bacteria adhering to all the haemocytes on the slides declined. Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides. Purified apoLp-III bound to B. subtilis. ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min. ApoLp-III bound to B. subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.     

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.