Juvenilizing effect of cooling on Galleria mellonella

Cooling of larvae of G. mellonella in the final instar results in a serious disturbance in their later development. Such disturbance concerns the transition to additional larval stages, loss of the ability to spin cocoons, and the formation of individuals incapable of continuing normal development.The kind of disturbances and their degree depends on the age of cooled larvae and the cooling time applied. Cooling of 1- and 2-day-old larvae results in their transition to additional larval stages, while cooling larvae aged from 3 to 7 days of the duration of instar VII causes the formation of a large number of individuals incapable of further development. The loss of the capacity to spin cocoons depends solely on the cooling time applied.     

Immunological activation of phagocytic cells in Galleria mellonella

A phagocytosis-stimulating activity against the normally nonphagocytosable cells of Bacillus thuringiensis subtoxicus develops in the hemolymph of larvae of Galleria mellonella at different periods after injection with readily phagocytosable latex beads. The phagocytosis-stimulating factor can be transferred into new animals with the cell-free hemolymph of treated larvae. It is not detectable in the hemolymph of normal larva but is present in the supernatant of homogenates of the skin. The fractionation of activated hemolymph on Sephadex shows that the active substance has a low molecular weight. It appears to have a biological effect in cellular defense reactions as in the case of lymphokine in vertebrates.     

Disturbances in the development of the egg chambers of Galleria mellonella L. (Lepidoptera) after reserpine administration

Reserpine causes some changes in the morphology of developing egg chambers when administered to the last (7th)-instar larvae of Galleria mellonella. Among the alterations, there is a numerical change in the number of egg chambers produced in one ovariole. The number of previtellogenic and postvitellogenic egg chambers was decreased significantly. Histological study revealed a series of pathological alterations in the follicular epithelium, oöcytes and trophocytes. Atrophy or proliferation of follicular cells was often observed. Strong vacuolation of the cytoplasm of cells forming the egg chambers appeared in all developmental stages, mainly at the vitellogenic stage. Changes also occurred in the surface of the ovariole tube.The observed disturbances in the developing ovarioles of the wax moth are probably the consequence of reserpine action on the neuroendocrine system.     

The fate of exotoxin of Bacillus thuringiensis in Galleria mellonella caterpillars

Three hours after parenteral administration of ³²P-labeled exotoxin of Bacillus thuringiensis to caterpillars of Galleria mellonella, 80% of the radioactivity was localized in the hemolymph in the form of the original exotoxin. The remaining radioactivity occurred in the organs of the caterpillar, especially in the spinning glands and the intestine. After peroral administration, the exotoxin does not pass the intestinal wall into the hemolymph to a measurable degree. In this case, the exotoxin is split in the intestinal wall and the products of ³²P reutilization have been found in the hemolymph. The mechanism of action of the exotoxin in the insect organism is discussed; presumably it depends on different ways of administration of the substance.     

Control of prothoracic gland activity in larvae of Galleria mellonella

Prothoracic glands of last instar wax moth larvae maintain spontaneous secretory activity both in decapitated larvae and in isolated abdomens into which they have been transplanted, as judged by their ability to induce secretion of a new cuticle. Their activity is hormonally stimulated by the brain and inhibited by the prothoracic and mesothoracic ganglia. The subesophageal ganglion seems to suppress the inhibitory influence of the thoracic ganglia. The prothoracic glands of larvae decapitated at different times during the last instar all respond to brain implantation, and this response does not change when brains are implanted at increasing intervals after decapitation. The prothoracotropic activity of the isolated brain is highest in brains of pupae and adults but is relatively and consistently low in brains of last instar larvae. The results demonstrate that the control of prothoracic glands is a complex process governed by the nervous integration of various stimuli.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Rate of synthesis of nucleic acids and their precursors in developing ovaries of Galleria mellonella

Three groups of ovarioles were selected for study: previtellogenetic (I), those in the course of vitellogenesis (II), and postvitellogenetic (III). Previtellogenetic ovarioles showed the highest content of DNA and acid-soluble nucleotide fraction. The mean rate of incorporation of [³H]-thymine into the mononucleotide fraction within 2·5 min mainly depends on the rate of transport of the pyrimidine base into the ovarioles, and was found to be highest (35 × 10⁴ dis/min per μmole of nucleotide per min) in vitellogenetic ovarioles. The rate for [³H]-uracil is highest in previtellogenetic ovarioles.Incorporation of pyrimidine bases into the polynucleotide fraction was corrected for the mononucleotide pool available for synthesis. [³H]-Thymine is incorporated into the polynucleotide fraction most actively in group III of the ovarioles, being probably restricted to numerous follicular cells, whereas [³H]-uracil is mainly incorporated during vitellogenesis.     

Sex differences in the lipolytic activity of Galleria mellonella fat body

The sexual differences in the intensity of production and release of free fatty acids (FFA) as well as the differences in the absolute amount of FFA produced and released from the fat body are most conspicuous at the sixth day after the larval-pupal ecdysis. Furthermore, it is demonstrated that a significant difference exists in the lipolytic activity of the fat body between the two sexes.     

Lactate dehydrogenase isoenzymes in the larvae of Barathra brassicae and Galleria mellonella during microsporidan infection

Changes in the activities of lactate dehydrogenase isoenzymes in the gut and fat body of Galleria mellonella and Barathra brassicae larvac infected by the microsporidans Nosema plodiae and Pleistophora schubergi were studied by means of dise electrophoresis. In the normal last instar G. mellonella gut and fat body three isoenzymes, LDH-1, LDH-2-3, and LDH-4, and in B. brassicae two isoenzymes, LDH-1 and LDH-2-3, were present. In the fat body of both the animals infected by N. plodiae, the isoenzyme LDH-2-3 increased in activity substantially by the fifth day of infection. The gut LDH isoenzymes were not affected by the microsporidan. The same LDH-2-3 effect could be provoked by some enzymes toxic for G. mellonella larvae such as phospholipase-C and protease preparations.     

Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae

The high molecular fraction of the extract from Metarhizium anisopliae grown on wheat bran contains proteolytic enzymes which are toxic for Galleria mellonella larvae. The complex of proteases was fractionated using precipitation with ammonium sulfate, gel filtration, and electrofocusing. Two components have been found: one with the optimum of activity on hemoglobin at pH 6.5, and the second with the optimum around pH 9. The prevailing protease acting at pH 6.5 was inhibited by phenylmethylsulfonyl fluoride and the inhibition was followed by decrease of toxicity. The molecular weights of the enzymes are 35 × 10³ and 71 × 10³.     

Investigations on cellular defense reactions with Galleria mellonella against Bacillus thuringiensis

In contrast to a great number of foreign particles (bacteria and inorganic materials), cells of some strains of Bacillus thuringiensis are not phagocytosed in the first hours after injection into the hemocoel of Galleria mellonella. This phenomenon is not caused by the production of β-exotoxin or exoenzymes, because heat-killed cells are not phagocytosed and the phagocytosis of latex particles is not prevented by the presence of living B. thuringiensis. The phagocytosis of heat-killed B. thuringiensis subtoxicus can be encouraged by treatment of the cells and by simultaneous injection of latex particles. A factor stimulating the phagocytosis is discussed. It is induced by the injection of phagocytosable latex particles into the hemocoel but not by injection of living or killed B. thuringiensis subtoxicus.     

Moulting hormone level during the last instar of Galleria mellonella larva

Using radioimmunoassay the moulting hormone titres of the greater wax moth were determined during the last larval instar. Two peaks were observed, one when the larvae start to spin and another just before the pupation. The second peak exhibits the higher MH level, equivalent to 3600 ng/g ecdysterone. By TLC-RIA analysis three compounds were detected: ecdysone, ecdysterone and a very polar metabolite (VPM). The pattern of MHs during the last larval instar is described and the possible changes in the activity of enzymes of MH metabolism and ecdysone-ecdysterone conversion is discussed.     

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.     

The granular cells of Galleria mellonella during clotting and phagocytic reactions in vitro

Light and electron-microscopic observations of the blood-cells (hemocytes) of the wax-moth Galleria mellonella showed that hemolymph coagulation was initiated by the rapid release of material from the granular cells. During incubation for short terms in vitro these cells showed progressive degranulation as material derived from the granules was discharged into the hemolymph. Attempts to determine the nature of this material by staining with ruthenium red proved mainly unsuccessful. When challenged with bacteria in vitro the granular cells failed to phagocytose these particles and instead the bacteria became embedded in the granular material surrounding these cells. The mode of coagulation reported here is compared with previous reports of the role of invertebrate hemocytes in hemolymph clotting.     

Selective depletion of the plasmatocytes in Galleria mellonella following injection of bacteria

Injection of a suspension of a bacterial pathogen, Bacillus cereus, into larvae of the wax moth, Galleria mellonella, resulted in the disappearance of plasmatocytes from the haemolymph. This depletion effect was dose dependent, and occurred within 5 min of injection of the bacteria. Similar effects, though of lesser intensity, followed injection of a number of other species of both pathogenic and nonpathogenic bacteria. This rapid and specific reaction may play a part in the natural response of insects to the injection of foreign bodies.     

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.     

Persistence of Bacillus thuringiensis in foundation beeswax and beecomb in beehives for the control of Galleria mellonella

Beecomb was protected against wax moth attack by impregnating foundation beeswax with Bacillus thuringiensis. Bakthane, a serotype I product, gave good protection of broodcomb for 2 yr at 0.5% in wax and partial protection for 6 yr at 2%. The active component was exotoxin present at about 0.9% in Bakthane in a very insoluble state. No harm to the bees was detected, even when a partially purified preparation of exotoxin was used, but further tests on the leaching of pure exotoxin from comb into honey are required before the latter can be regarded as a practical method of wax moth control. The action of exotoxin on Galleria mellonella larvae was slower than that of the spore-crystal complex and less than that of exotoxin on some Diptera. Spores and crystals of serotype V were × 500 as potent in G. mellonella as those of serotype I and exotoxin on honey-rich artificial food in laboratory assays, but their activity deteriorated in the first hive trials with treated foundation wax. Prehive deterioration was due both to Teepol and soap in the liquid lubricating the wax mill rollers and to moist storage of the foundation. This deterioration was prevented by the use of Triton X-100 as lubricant, by drying the newly impregnated foundation wax and by storing it in dry conditions. This resulted in good protection of comb for one hive season by 1% of serotype V Thuricide in foundation wax. However, protection after two seasons varied, making the use of serotype V spores and crystals uneconomical for commercial practice, although safe for bee and man.     

Some aspects of regulation of fat body lipolytic activity in Galleria mellonella

The degradation of ¹⁴C-trioleate by the homogenate of fat body of normally developing specimens of both sexes of Galleria mellonella at different days after the larval-pupal ecdysis, and of specimens subjected to different experiments was studied. It has been confirmed that the lipolytic activity of female fat body being low after the larval-pupal ecdysis, rises distinctly by about 6 days later. In contrast to this, the lipolytic activity of this tissue in males is high and does not appear to undergo changes.Ovariotectomy causes a significant fall of lipolytic activity.Preincubation of fat body of ovariotectomized females in the medium containing ovaries of pharate adults 6 days after the larval-pupal ecdysis brings about a rise of lipolytic activity of this tissue.     

A re-investigation on the ecdysteroids during embryogenesis in the desert locust, Schistocerca gregaria

Comparison of gas chromatography and high-pressure liquid chromatography methods for determination of ecdysteroids in insect eggs revealed that published values for Schistocerca gregaria were very low. The error has been traced to incomplete hydrolysis of conjugates. Revised values for the levels of ecdysone, 20-hydroxy-ecdysone and 2-deoxy-ecdysone are given here. The same general pattern of falling ecdysteroid titre during early stages of embryo development followed by an increase and a final decrease is observed. Experiments have been made on the effect of phosphate, ionic strength and a specific β-glucuronidase competitive inhibitor on the hydrolysis of the polar ecdysteroid conjugates, in an attempt to provide more evidence on the chemical nature of these compounds.