Development of Bacillus thuringiensis in Galleria mellonella larvae exposed to gamma radiation

A suspension of Bacillus thuringiensis was inoculated at 24 and 72 hr into the oral cavity of Galleria mellonella larvae following exposure to 20, 50, and 70 Kr of gamma radiation, respectively. The cytopathology was conducted after B. thuringiensis had developed for 3, 5, and 7 hr and after radiation damage had developed for 27, 29, 31, 75, 77, and 79 hr in the larvae exposed to 20, 50, and 70 Kr, respectively.B. thuringiensis spores appeared in the midgut lumen from 3 to 7 hr after inoculation of 20 Kr irradiated larvae. At 7 hr after B. thuringiensis infection, and 79 hr after 20 Kr irradiation, the following changes were seen: B. thuringiensis rods appeared adsorbed onto the walls of epithelial cells, a few spores appeared in hemolymph, epithelial cells developed vacuoles, and villi appeared detached from the basement membrane.Within a period ranging from 3 to 5 hr after infection, B. thuringiensis rods attacked vacuolated epithelial cells of most of the 50 and 70 Kr irradiated larvae. At 7 hr after infection and at 31 hr after 70 Kr irradiation, the spores reached the interior of some epithelial cells and were also seen concentrated near the basement membrane.In general, the midgut epithelial cells of the 70 Kr-irradiated groups of larvae appeared highly vacuolated, badly disrupted, and in most cases undistinguishable as a result of attack of B. thuringiensis.In short, B. thuringiensis did not show a characteristic pattern of pathology on 20 and 50 Kr-irradiated midgut cells. The problem of permeability of B. thuringiensis toxin into the irradiated cells needs further investigation.     

Recovery of Bacillus thuringiensis and other bacteria from larvae of Spodoptera littoralis previously fed B. thuringiensis-treated leaves

Exposure of a spore-crystal suspension of Bacillus thuringiensis to UV irradiation for (200 lx) 8.5 min killed most of the spores ($\text{P}\text{P}{}_0\text{= 2.6 × 10}{}^{\mathrm{?4}}$), while the insecticidal activity of the suspension to larvae of Spodoptera littoralis was only slightly affected. Numbers of colony-forming units (CFU) of B. thuringiensis recovered from larvae after ingestion of spores decreased with time as long as the larvae lived and several hours after larval death. Only 3–6 hr after larval death, the spores germinated and multiplied, reaching up to 100-fold after 24 hr. When UV-irradiated suspensions were used, numbers of CFU per larva were too scarce to be recovered from living larvae. However, 1.5 × 10⁶ CFU/larva were recovered 24 hr after death. It seems that the disruption of the gut epithelium by the endotoxin caused a change in the unfavorable conditions for endospore germination, thus providing the suitable ambient for germination and multiplication of B. thuringiensis. Numbers of other bacteria present per milligram of healthy larva increased with larval weight, predominantly Streptococcus sp. and Erwinia sp. In dead larvae, the increase of Erwinia sp. was higher than that of Streptococcus sp. Other bacterial species isolated were: Corynebacterium sp., Micrococcus sp., Serratia marcescens, and Bacillus sp.     

Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

The time course of clearance of an injected dose of 10⁶ CFU ml^?1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.     

Lack of absorption and rapid elimination of Rifampicin as factors precluding tests against nuclear polyhedrosis in Spodoptera littoralis larvae

Rifampicin administered per os in artificial medium to first-instar larvae of Spodoptera littoralis together with nuclear polyhedrosis virus did not affect virus infection and resulted in total larval mortality. A pharmacokinetic study of this antibiotic was carried out by means of a microbiological assay. Using Bacillus thuringiensis var. thuringiensis as the test organism for assaying the antibiotic, as little as 0.5 μg of Rifampicin could be detected in the presence of hemolymph, and no such detectable amounts could be found in the hemolymph of sixth instar larvae which had been fed the antibiotic. Most if not all of the antibiotic was in the feces, excreted in active form. When given parenterally, Rifampicin was detected in the hemolymph 30 min after injection but not 24 hr thereafter, when active antibiotic was found concentrated in the first fecal pellets evacuated after injection. This disappearance is ascribed to excretion via the Malpighian tubules. The larval responses detailed above preclude the testing of Rifampicin as an antiviral drug against nuclear polyhedrosis.     

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 10⁶ cells/ml (n = 15, range 2–7 × 10⁶ cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

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.     

Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(−) Vibrio anguillarum and V. splendidus, Gram(+) Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus > V. anguillarum > M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+ 35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+ 16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus > V. anguillarum > M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.     

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 specificity of immune priming in silkworm,Bombyx mori,is mediated by the phagocytic ability of granular cells

In the past decade, the phenomenon of immune priming was documented in many invertebrates in a large number of studies; however, in most of these studies, behavioral evidence was used to identify the immune priming. The underlying mechanism and the degree of specificity of the priming response remain unclear. We studied the mechanism of immune priming in the larvae of the silkworm, Bombyx mori, and analyzed the specificity of the priming response using two closely related Gram-negative pathogenic bacteria (Photorhabdus luminescens TT01 and P. luminescens H06) and one Gram-positive pathogenic bacterium (Bacillus thuringiensis HD-1). Primed with heat-killed bacteria, the B. mori larvae were more likely to survive subsequent homologous exposure (the identical bacteria used in the priming and in the subsequent challenge) than heterologous (different bacteria used in the priming and subsequent exposure) exposure to live bacteria. This result indicated that the B. mori larvae possessed a strong immune priming response and revealed a degree of specificity to TT01, H06 and HD-1 bacteria. The degree of enhanced immune protection was positively correlated with the level of phagocytic ability of the granular cells and the antibacterial activity of the cell-free hemolymph. Moreover, the granular cells of the immune-primed larvae increased the phagocytosis of a previously encountered bacterial strain compared with other bacteria. Thus, the enhanced immune protection of the B. mori larvae after priming was mediated by the phagocytic ability of the granular cells and the antibacterial activity of the hemolymph; the specificity of the priming response was primarily attributed to the phagocytosis of bacteria by the granular cells.     

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.     

Galleria mellonella infected with Bacillus thuringiensis involves Hsp90

Insects are good models for studying the innate immune response. We report that Galleria mellonella larvae infected with entomopathogenic bacteria Bacillus thuringiensis kurstaki show changes in the level of Hsp90. Our experimental approach was to pre-treat larvae with the Hsp90-binding compound, 17-DMAG, before infection with B. thuringiensis. We show that pre-treated animals display a higher level of immune response. This was mainly manifested by enhanced action of their hemolymph directed toward living bacteria as well as lysozyme activity digesting bacterial peptidoglycan. The observed phenomenon was due to the higher activity of antimicrobial peptides which, in contrast to healthy animals, was detected in the hemolymph of the immunestimulated larvae. Finally, the physiological significance of our observation was highlighted by the fact that G. mellonella pre-treated with 17-DMAG showed a prolonged survival rate after infection with B. thuringiensis than the control animals. Our report points to a role for Hsp90 in the immune response of G. mellonella after infection with B. thuringiensis at the optimal growth temperature.     

Ultrastructural studies on the fungus,Nomuraea rileyi,infecting the velvetbean caterpillar,Anticarsia gemmatalis

Combined scanning and transmission electron microscopy was used to study the fine structure of the developmental stages of Nomuraea rileyi infecting host larvae of Anticarsia gemmatalis. Larvae were dusted with large numbers of fungal conidia, which germinated and penetrated the cuticle within 6 hr post-treatment. Within 24 hr, penetration hyphae had reached the cuticular epidermis and, via a budding process, initiated the hyphal body stage in the hemocoel. The hyphal bodies, suspended in hemolymph, multiplied and spread throughout the host larvae. By 6–7 days post-treatment, the majority of larvae were mummified. Within 12 hr postmortem numerous conidiophores emerged producing a confluent mycelial mat over the entire cuticular surface. Numerous hydrophobic conidia were formed on phialides present on the aerial conidiophores.     

Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simuliidae)

The pathogenesis and midgut histopathology which resulted when larvae of the blackfly, Simulium vittatum, were exposed to Bacillus thuringiensis at various temperatures and periods of exposure were investigated. The onset of mortality was studied at 10°, 15°, 19°, and 24°C. For each 4–5°C increase in temperature above 15°C, the onset of mortality was shortened by 24 hr. Exposures as brief as 15 min to 10 ppm of a whole spore preparation resulted in an average mortality of 29% in late-instar larvae. Mortality increased sharply for exposures up to 3 hr, approaching a maximum of 80%.The gross signs of disease included cessation of feeding and tetany with brachytosis. The tissue most affected was the midgut epithelium in the regions of the gastric caeca and posterior stomach. The formation of cytoplasmic vacuoles followed by cell lysis and/or sloughing were very apparent in moribund larvae. Death resulted without bacteremia.     

Bacillus thuringiensis parasporal crystal toxin: Dissociation into toxic low molecular weight peptides

Parasporal crystals of Bacillus thuringiensis can be dissociated into low molecular weight peptides (< 5000 daltons) by dissolving them in 0.1 M N-morpholinopropane sulfonic acid buffer pH 7.8 containing 0.05 M dithiothreitol and 2M–4M KSCN, or by performic acid oxidation. The peptides obtained by dissolving in KSCN were still toxic to silkworm larvae.     

Susceptibility of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigmata, to Bacillus thuringiensis: Chemical insecticide combinations

Bacillus thuringiensis mixed with the organophosphate insecticides, fenitrothion (Sumithion), Gardona^®, and Orthene^®, or the synthetic pyrethroid, SBP 1382, was incorporated into synthetic diet and fed larvae of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigma. Mortality was highest when larvae were fed combinations of low concentrations of the insecticides and low to moderate concentrations of the pathogen. The data indicated that applications of a B. thuringiensis dosage expected to produce about 45% mortality of third and fourth instar larvae of the spruce budworm combined with a dosage of fenitrothion causing about 40% mortality or a dosage of Orthene causing from 5 to 25% mortality should result in low budworm survival. With a B. thuringiensis dosage causing 20–60% mortality combined with a fenitrothion dosage causing 15–50% mortality or a sublethal dosage of Gardona, a low survival rate of young white-marked tussock moth larvae may be expected.     

Effect of cobra venom factor on the in vivo immune response in Galleria mellonella to Pseudomonas aeruginosa

Wax moth larvae receiving 0.2 or 0.4 units of cobra venom factor (CVF) per insect, 6 hr after vaccination, have a significantly reduced resistance to Pseudomonas aeruginosa when compared with the normal immune response. Curiously, lower doses of CVF (0.1 units per insect) had a tendency to enhance the resistance of the vaccinated larvae to the pathogen. The CVF had no observable effect on the LD₅₀ in unvaccinated larvae. The in vitro bactericidal capacity of immune hemolymph, with respect to P. aeruginosa, was lowered significantly by prior incubation of the hemolymph with CVF. The involvement of an invertebrate analog to the vertebrate complement system is discussed.