Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 10⁴ times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.     

Aspects of the cross transmission to Galleria mellonella of a Baculovirus from the alfalfa looper, Autographa californica

The nuclear polyhedrosis virus originally isolated from the alfalfa looper, Autographa californica, was successfully transmitted to the greater wax moth, Galleria mellonella. Both the many polyhedra per nucleus (MP) and the few polyhedra per nucleus (FP) plaque variants of this virus were found to be infective when injected intracoelomically. When polyhedra of each plaque variant were fed to G. mellonella larvae, a difference in response was observed; the MP plaque variant was estimated to be 30 times more infective than the FP variant.     

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.     

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.     

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.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

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.     

Microbes from apiarian sources: Bacillus spp. in frass of the greater wax moth

Frass from the greater wax moth, Galleria mellonella, obtained from feral colonies of honey bees, Apis mellifera; from managed honey bee colonies; and from a laboratory culture of the wax moth was sampled for aerobic Gram-positive spore-forming rods. One hundred eighty-five strains belonging to the genus Bacillus were isolated, and most were identified. One hundred and three of the isolates were from frass from the wax moth culture, 61 were from frass from managed honey bee colonies, and 21 were from frass from feral honey bee colonies. The species most frequently isolated varied with the source. Fifty-eight isolates from frass from managed honey bee colonies were B. cereus which was isolated from this source only, but B. sphaericus was the most frequent isolate from frass from the wax moth culture. Bacillus megaterium and organisms belonging to the B. alvei-B. thiaminolyticus spectrum were the most frequent isolates from frass from feral honey bee colonies. Most strains isolated produced caprylate esterase-lipase, leucine aminopeptidase, and phosphoamidase. The numbers of isolates, the species, and the enzymatic activity of the strains varied with the source of the frass. In fact, the complete microbial complement varied with the source. These results are discussed in relation to possible roles of Bacillus spp. in the nutrition of the wax moth as well as the microbial ecology of the honey bee colony.     

Lysozymelike lytic enzyme of Streptococcus faecalis and its role in the larval development of wax moth, Galleria mellonella

The predominant type of bacteria present in the gut of larval Galleria mellonella were streptococci group D identified as Streptococcus faecalis which showed bacteriolytic activity. Young larvae usually contained mixed populations with a marked dominance of fecal streptococci while normally developed mature larvae most frequently contained large uniform populations of S. faecalis. Pupal stages were found to contain the highest percentage of individuals with pure cultures of fecal streptococci.The author suggests a hypothesis that, owing to its bacteriolytic properties, S. faecalis can be considered as a component of the natural, nonspecific defense mechanism of G. mellonella against bacterial infections. The lytic enzyme released in the exponential growth phase of S. faecalis participates in the selection process stabilizing the microbial flora of wax moth larvae; it limits the population of other forms of bacteria. Larval resistance to bacterial infections to a large extent depends on the magnitude of the populations and thus on S. faecalis muramidase concentration. Bacterial lysozyme inhibited the growth of the ingested organisms and in consequence it prevented the proliferation of undesired bacteria in the digestive tract of Galleria larvae.The lytic enzyme proved to be identical with autolysin, a β-N-acetylmuramide glycanhydrolase (EC 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates of S. faecalis by Shockman and Cheney (1969).     

Gram-positive cocci from apiarian sources

Frass from the greater wax moth, Galleria mellonella, obtained from feral colonies of honey bees, Apis mellifera; from domesticated (managed) honey bee colonies; and from a laboratory culture of the wax moth was sampled for Gram-positive cocci. One hundred twenty-three of these organisms were isolated and identified. Frass from domesticated colonies yielded only one isolate. Equal numbers of isolates (61) were obtained from frass from feral bee colonies and from the wax moth culture. Catalase-negative cocci were predominant in frass from feral colonies, whereas catalase-positive cocci were the most common isolates from frass from the wax moth culture. Catalase-positive cocci were identified as Staphylococcus epidermidis and Micrococcus sp. Catalase-negative cocci were Streptococcus faecalis var. faecalis and S. faecium. These results are discussed in relation to the rarity of Gram-positive cocci associated with honey bees, pollen, and nectar in Arizona and the frequency of association with honey bees and wax moth frass of bacteria resembling Arthrobacter spp. that appear as Gram-positive cocci during one stage of the life cycle.     

Extremely high frequency sensitivity in a ‘simple’ ear

An evolutionary war is being played out between the bat, which uses ultrasonic calls to locate insect prey, and the moth, which uses microscale ears to listen for the approaching bat. While the highest known frequency of bat echolocation calls is 212 kHz, the upper limit of moth hearing is considered much lower. Here, we show that the greater wax moth, Galleria mellonella, is capable of hearing ultrasonic frequencies approaching 300 kHz; the highest frequency sensitivity of any animal. With auditory frequency sensitivity that is unprecedented in the animal kingdom, the greater wax moth is ready and armed for any echolocation call adaptations made by the bat in the on-going bat–moth evolutionary war.     

Effect of exposure to soil on potency and spore viability of Bacillus thuringiensis

Ten-gram samples of a clay loam soil were inoculated with Bacillus thuringiensis var. galleriae (H-serotype V) and held at 25°C. Periodically the spores and δ endotoxin protein crystals of B. thuringiensis were extracted from soil samples. Numbers of viable spores were estimated by plate counts and pathogenicity determined by bioassay with larvae of Galleria mellonella. During 135 days, the number of viable spores fell slowly to 24% of the initial numbers, while pathogenicity fell rapidly to <1%, which suggests that the crystals were degraded far more rapidly than spores. Natural soil bacteria increased in numbers during the same period.     

Passive vectoring of entomopathogenic fungus Beauveria bassiana among the wax moth Galleria mellonella larvae by the ectoparasitoid Habrobracon hebetor females

Females of the ectoparasitoid Habrobracon hebetor attack and envenomate numerous host individuals during oviposition. The vectoring of the entomopathogenic fungus Beauveria bassiana during the adhesion stage by ectoparasitoid females among the wax moth larvae Galleria mellonella was explored under laboratory conditions. Vectoring occurred both from infected parasitoids to wax moth larvae and from infected to healthy wax moth larvae by parasitoids. The efficacy of vectoring in both cases was dose dependent. Parasitoid females were unable to recognize infected larvae in a labyrinth test. In addition, the presence of H. hebetor females significantly (1.5–13 fold) increased the mycoses level in clusters of G. mellonella, with 40% of the larvae infected with fungal conidia. Envenomation by H. hebetor increased conidia germination on the cuticles of the wax moth larvae by 4.4 fold. An enhanced germination rate (2 fold) was registered in the n‐hexane epicuticular extract of envenomated larvae compared to that of healthy larvae. Both envenomation and mycoses enhanced the phenoloxidase (PO) activity in the integument of G. mellonella and, in contrast, decreased the encapsulation rate in hemolymphs. We hypothesize that changes in the integument property and inhibition of cellular immunity provide the highest infection efficacy of entomopathogenic fungi with H. hebetor.     

In vivo insect hemocyte destruction by UV-irradiated Chilo iridescent virus

Large inoculum of infective or UV-irradiated Chilo iridescent virus (CIV) caused an early drastic cytopathy of the larval hemocytes of the greater wax moth, Galleria mellonella. Cytopathy of the hemocyte could be detected 2 hr after virus injection at 25°C and most hemocytes were completely destroyed within 8 hr after virus injection. Heat-treated CIV did not produce any cytopathy of Galleria hemocytes. Antiserum to CIV neutralized this cytopathic effect, while that to Tipula iridescent virus did not demonstrate any inhibitory effect. Actinomycin D, mitomycin C, and puromycin did not inhibit hemocyte destruction by UV-irradiated CIV. Galleria larva injected with a large dose of UV-irradiated CIV progressively became stunted with a decrease of body weight and did not regain its hemocyte numbers. Hemocytes infected with CIV were not destroyed by any additive injection of a large dose of UV-irradiated CIV.     

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.     

Ovarian ecdysteroids and their secretion in late-pharate adults of Galleria mellonella

Approximately two-thirds of the total amount of ecdysteroids in late—pharate adults of the wax moth, Galleria mellonella, were found in the ovaries and one-third in the ovariectomized body. Chemical analysis of these ecdysteroids by thin-layer and high-pressure liquid chromatography, coupled with an ecdysteroid radioimmunoassay, revealed the presence of 2-deoxyecdysone, ecdysone and 20-hydroxyecdysone, as well as high and low polarity unknowns. The predominant identifiable ecdysteroid in both the ovaries and ovariectomized body was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone, respectively. Incubation of late-pharate adult ovaries in culture medium revealed that they synthesize and secrete ecdysteroids in vitro. The in vitro distribution of ecdysteroids between ovaries and incubation medium was similar to that observed between ovaries and ovariectomized bodies in situ and the predominant identifiable moiety both retained and released by the ovaries in vitro was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone. Collectively, these results support the idea that the ecdysteroids synthesized by the ovaries of late-pharate adult Galleria are both stored and secreted and that the quantity of a specifically secreted ecdysteroid is precisely controlled. This apparent regulation of the distribution of ovarian ecdysteroids raises the possibility that the stored and secreted forms have distinct functions in the reproductive physiology of this insect.     

Ecology of Bacillus thuringiensis in storage moths

A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10⁴ or more spores of H serotype V Bacillus thuringiensis applied at one point on the surface of 200 g of food were required to cause epizootics, compared with 10⁷ or more when spread evenly over the surface. In infected populations, spores contaminated the surfaces of all stages of the insect. In diseased larval cadavers there were 5.6–42.2 × 10⁸ spores/g of dry insect (P. interpunctella, Ephestia cautella, Anagasta kuehniella, Ephestia elutella, and Galleria mellonella). Larvae did not cannibalize live larvae while food was present though they sometimes ate cadavers. This is the most potent means of natural spread of the disease. Occurring mainly in protected situations such as food stores, natural infections are usually light, but occasionally spectacular surface accumulations of dead larvae occur, possibly associated with stress, physiological condition of the larvae, serotype of the bacterium, or behavior pattern such as migration. Natural disease may curb infestations in debris, but it attacks too late to prevent excessive damage to stored food. A prophylactic, even admixture of 2 × 10⁹ spores/200 g of food is required for effective insect control.     

Effects of Filipin and cholesterol on housefly, Musca domestica L., and wax moth, Galleria mellonella L

The effects of filipin on insects are dependent on the molar ratio of cholesterol to filipin. The larvicidal effects of the polyene antibiotic, filipin, can be prevented by excess cholesterol (“excess” herein is defined as a molar ratio of cholesterol to filipin of greater than 2 : 1) in housefly, Musca domestica L., and wax moth, Galleria mellonella L., larvae. Excess cholesterol also prevents the chemosterilant effect of filipin in housefly adults. The filipin-induced inhibition of [¹⁴C]cholesterol uptake by wax moth larvae is prevented by excess cholesterol; cholesterol uptake is increased severalfold. Dietary filipin, in the absence of added cholesterol, caused loss of ³²P from housefly tissues and decreased the incorporation of ³²P- and [¹⁴C]methyl-labeled choline into phospholipids of wax moth tissues. Addition of excess cholesterol to filipin-containing diets enhanced incorporation of ³²P into the different classes of phospholipids, and phospholipid synthesis was nearly doubled.     

Complex of digestive proteinases of <Emphasis Type="Italic">Galleria mellonella</Emphasis> caterpillars. Composition,properties, and limited proteolysis of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> endotoxins

The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.