Hemocyte responses of the Colorado potato beetle, Leptinotarsa decemlineata, and the greater wax moth, Galleria mellonella, to the entomopathogenic nematodes, Steinernema feltiae and Heterorhabditis bacteriophora.

Hemocyte encapsulation reactions of infective juveniles of two Iranian isolates of the entomopathogenic nematodes, Heterorhabditis bacteriophora Poinar (Rhabditina: Heterorhabditidae) and Steinernema feltiae Filipjev (Tylenchina: Steinernematidae), were compared in the economic pest Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), and the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae). The former was a more responsive host than the latter and the hemocyte responses occurred sooner and more extensively. Complete encapsulation of some of the nematodes occurred by 4 h post injection for H. bacteriophora in both L. decemlineata and G. mellonella, and by 2 h pi for S. feltiae in L. decemlineata. The percentage of encapsulation from 24 h to 72 h pi in L. decemlineata was 86.2% for S. feltiae and 39% for H. bacteriophora. In G. mellonella there were no encapsulation or melanization responses against S. feltiae, whereas when H. bacteriophora was encapsulated and melanized (16.7%) the encapsulation level was lower than in L. decemlineata. This study may contribute to effectively selecting entomopathogenic nematode species active against significant economic pests based on the latter's cellular immune response.

Apolipophorin-III in the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae).

The level of apolipophorin-III reached a maximum in the haemolymph of Galleria mellonella at the end of the feeding phase of the seventh larval instar and declined to a plateau value in the pupal and the adult stages. Apolipophorin-III was detected immunologically in fat body tissue, haemocyte lysates, and plasma. In its native state, apolipophorin-III may be associated with another protein with an apparent molecular mass of 77 kDa, possibly apolipophorin-II. Injections of octopamine did not cause lipid loading of high density lipophorin.

Apolipophorin-III and the interactions of lipoteichoic acids with the immediate immune responses of Galleria mellonella.

We investigated the effects of lipoteichoic acids, surface components of Gram-positive bacteria, on the hemocytes and phenoloxidase activity in last instar Galleria mellonella larvae, as well as the binding of apolipophorin-III, an insect lipid-binding protein, to lipoteichoic acids. Binding of apolipophorin-III to lipoteichoic acid was studied using an assay based on 1,9-dimethylmethylene blue. Apolipophorin-III bound the lipoteichoic acids from Bacillus subtilis, Enterococcus hirae, and Streptococcus pyogenes and to intact cells of E. hirae. E. hirae lipoteichoic acid promoted the binding of apolipophorin-III to the cells of this species. All lipoteichoic acids tested caused a dose- and time-dependent drop in the total counts of hemocytes and, depending on the species of lipoteichoic acid, partial or complete depletion of plasmatocytes. Granulocyte counts were not affected. Apolipophorin-III prevented partially the loss of plasmatocytes due to B. subtilis lipoteichoic acid. All three lipoteichoic acids studied activated phenoloxidase in vitro; injections of B. subtilis lipoteichoic acid into the larvae elevated the phenoloxidase activity, whereas injections of E. hirae or S. pyogenes lipoteichoic acid, or apolipophorin-III alone, suppressed it. Apolipophorin-III decreased the activation of phenoloxidase by B. subtilis lipoteichoic acid.

Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus.

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.

Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria.

The hemocytes to which bacteria adhere were defined and the contribution of the prophenoloxidase system of fifth instar nymphs of Acheta domesticus to adhesion were examined. The physicochemical parameters affecting hemocyte and phenoloxidase activity were determined. Both plasmatocytes and granular cells responded to bacteria, the latter cells entrapping the microorganisms on filopodial extensions. The optimum pH for hemocyte adhesion to glass slides was 6.5, the granular cells being the most sensitive hemocyte type. Although hydrophobic resin beads and positively-charged beads favoured hemocyte attachment, these parameters did not contribute to differential bacterial adhesion to hemocytes. Activation of phenoloxidase was neither enhanced nor inhibited by 0.1 and 1 mg/ml of laminarin or zymosan nor by dead Bacillus subtilis. However, live B. subtilis activated the enzyme and dead Xenorhabdus nematophilus inhibited enzyme activation. Serine protease components of the prophenoloxidase system had opsonic properties for B. subtilis but not for X. nematophilus. Phenoloxidase activity was enhanced by Ca(2+) and Mg(2+) and inhibited by SO(2-)(4).

Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity.

Heat-inactivated serum of non-immune Galleria mellonella larvae enhanced the lytic activity of larval cell-free hemolymph against Micrococcus lysodeikticus. The increase in bacterial lysis was due to a 17.2 kDa protein known previously to bind to bacterial lipopolysaccharides. The protein enhanced the lytic activity of insect cell-free hemolymph and hen lysozyme in vitro and insect hemolymph in vivo. The hydrophobic protein, which adhered to M. lysodeikticus, was identified by its amino acid sequence homology as apolipophorin-III. The titer of apolipophorin-III in 200-250 mg last instar larvae was 8.7 mg/ml of hemolymph. Apolipophorin-III did not bind to lysozyme. A possible mode of action of apolipophorin-III with lysozyme in the insect is proposed.

Serum composition of Aedes aegypti (Diptera: Culicidae) larvae and the production of an oviposition repellent are influenced by infection with the entomopathogenic digenean Plagiorchis elegans (Trematoda: Plagiorchiidae), starvation, and crowding.

Subjecting Aedes aegypti (L.) larvae to conditions that induced the production of oviposition repellency also reduced their wet and dry weights and the concentration of total serum carbohydrates, amino acids, and proteins. Thus, infection with metacercariae of the entomopathogenic digenean Plagiorchis elegans (Rudolphi), starvation for 7 d, or crowding for 2 d reduced larval dry weights by as much as 32, 20, and 23%, respectively, and wet weights by 20, 14, and 11%, respectively. Total serum carbohydrates declined by as much as 36, 21, and 29% for infected, starved, and crowded larvae, respectively, amino acids by 39, 48, and 44%, and protein concentrations by 72, 63, and 62%, respectively. Repellency dilution titers were correlated inversely with movement of the mouth parts and gut. Incubation of infected, starved, and crowded larvae in 0.01 g/liter glucose greatly reduced the level of repellency of their waters, whereas adding glucose to repellent waters had only minor effects. Results indicate that the induction of repellency is associated strongly with nutritional depletion effects.

Chitinase activity of Xenorhabdus and Photorhabdus species, bacterial associates of entomopathogenic nematodes.

Xenorhabdus nematophilus (three strains), Xenorhabdus bovienii (one strain), and Photorhabdus luminescens (one strain) showed both exo- and endochitinase activity using p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-beta-D-N,N',N'-triacetylchitotriose, respectively, as substrates. One to three bands were detected on PAGE gel with glycol chitin after electrophoresis. Variation in exo- and endochitinase activity among different species and strains was detected with the strongest activity in X. nematophilus and the weakest in P. luminescens. The primary form of X. bovienii had significantly greater chitinase activity than the secondary form, whereas their growth rate and total protein released into culture medium were similar. The partially purified chitinase of X. bovienii showed significant antimycotic activity against conidial germination and germ tube elongation of Botrytis cinerea.

Entomopathogenic fungi of the order Entomophthorales (Zygomycotina) in adult black fly populations (Diptera: Simuliidae) in Quebec.

Infections by insect-pathogenic fungi of the order Entomophthorales in two adult black fly populations were studied in the Réserve Faunique du Saint-Maurice (Quebec). Entomophaga near limoniae infected Simulium verecundum/rostratum in June whereas Erynia curvispora predominantly parasitized Simulium decorum from the 2nd week of July to September and Erynia conica attacked the Simulium venustum complex, Simulium verecundum/rostratum, and the Simulium vittatum complex from May to September. The fungi did not infect the adult stage of Prosimulium species. Possible evidence of host specificity for Erynia conica is discussed.

Interaction of mutants of Xenorhabdus nematophilus (Enterobacteriaceae) with antibacterial systems of Galleria mellonella larvae (Insecta: Pyralidae).

Xenorhabdus nematophilus mutants that took longer to kill insects than did the wild type were used to determine the relationship of the physicochemical properties and outer membrane composition to bacterial interaction with the antibacterial systems of Galleria mellonella larvae and to bacterial virulence. Insect serum slowed the growth of the wild-type and mutant bacteria. This was attributed to increased spheroplast formation for the mutants. Spheroplast formation was associated with an increased sensitivity to insect lysozyme and a reduction in overall bacterial cationic charge. Increasing bacterial hydrophobicity was correlated with both increased bacterial attachment to the insect's haemocytes and the accelerated removal of the bacteria from the haemolymph. Attachment of the mutants to the insect haemocytes also increased as the bacterial lipopolysaccharide content increased, the level of prophenoloxidase activation increased, and cationic charge declined. Bacterial emergence into the haemolymph occurred in parallel with haemocyte damage but neither the total lipopolysaccharide levels in the bacteria nor the rate of bacterial emergence were associated with virulence. The rate of lipopolysaccharide release into the haemolymph influenced the rate of haemocyte damage. The contribution of outer membrane proteins to lipopolysaccharide release, bacterial adhesion to haemocytes, and virulence is discussed. Virulence reflects bacterial tolerance to the host's antibacterial defences, favouring an increase in bacteria and toxic lipopolysaccharides.

Stability and Activities of Antibiotics Produced during Infection of the Insect Galleria mellonella by Two Isolates of Xenorhabdus nematophilus.

Xenorhabdus nematophilus subsp. dutki, an entomopathogenic bacterium, is vectored by steinernematid nematodes into insects, where it produces broad-spectrum antibiotics. The use of the nematode-bacterium complex against soil-dwelling pest insects could introduce antibiotics into the soil via the dead insect fragments during the emergence phase of the nematodes. Studies on the stability and activities of these antibiotics produced in the insect Galleria mellonella may contribute to assessing the possible impact of antibiotics on soil bacteria. Two isolates of X. nematophilus subsp. dutki (isolates GI and SFU) produced xenocoumacins 1 and 2 in cadavers of G. mellonella larvae in a 1:1 ratio. Total xenocoumacin 1 and 2 production was 800 ng/200 mg (wet weight) of insect tissue for the GI isolate. Antibiotic activity of water extracts from insects that had been infected with X. nematophilus was stable at 60 degrees C for 1 h and after repeated freeze-thaw cycles. The antibiotic titer of extracts held at 27 degrees C declined by day 10. The spectrum of bacterial species killed by antibiotics produced in insect cadavers varied with the isolate of X. nematophilus. Levels of antibiotic activity were greater in vivo than in tryptic soy broth, which may represent a nutrient effect. The bacterial isolate, culture condition, and presence of nematodes influenced the total antibiotic production in vivo. However, the levels of activity were not correlated with bacterial levels in the different growth environments. Insect cadavers with antibiotic activity transiently lowered the numbers of the bacteria in the soil, the extent of decline varying with the strain of X. nematophilus and the time of sampling.

Effects of selected carbohydrates and the contribution of the prophenoloxidase cascade system to the adhesion of strains of Pseudomonas aeruginosa and Proteus mirabilis to hemocytes of nonimmune larval Galleria mellonella.

The adhesion of strains of Pseudomonas aeruginosa and Proteus mirabilis to the plasmatocytes and granular cells of nonimmune larval Galleria mellonella was influenced by and varied with the type of carbohydrate. Laminarin enhanced prophenoloxidase activation and bacterial adhesion to the hemocytes whereas sucrose suppressed both activities. For all other sugars there was no correlation between bacterial adhesion to the hemocytes and phenoloxidase activity. It is proposed that bacterial adhesion to the hemocytes may be mediated by both lectinlike binding and components of the prophenoloxidase activating system acting like opsonins.

Reactions of hemocytes of immune and non-immune Galleria mellonella larvae to Proteus mirabilis.

Immune larval Galleria mellonella removed live Proteus mirabilis from the hemolymph less effectively than did non-immune larvae. This was attributed to a decline in total hemocyte counts, levels of plasmatocytes and granulocytes and hemocyte adhesion capacity. Immune serum possessed factors which reduced bacterial adhesion to the hemocytes. This was not due to altering bacterial surfaces but rather to irreversible suppression of hemocyte activity.

Growth and Virulence of Steinernema glaseri Influenced by Different Subspecies of Xenorhabdus nematophilus.

Three Xenorhabdus nematophilus subspecies influenced Steinernema glaseri growth profiles and growth rates, but this was not necessarily because of different bacterial growth rates. Virulence of dauer nematodes in larval Galleria mellonella varied with the number of dauers retaining bacteria and the bacterial subspecies. Virulence was least for dauers grown on X. nematophilus subsp. bovienii because of the lack of retained bacteria. Virulence was subsequently restored by culturing these nematodes on X. nematophilus subsp. poinari.

The in vitro generation of an antibacterial activity from the fat body and hemolymph of non-immunized larvae of Galleria mellonella.

A state of immunity in Galleria mellonella against the pathogen Pseudomonas aeruginosa is known to be induced by the injection of lipopolysaccharide (LPS), isolated from the homologous organism. An in vitro mixture of the LPS and whole or cell-free hemolymph from non-immunized larvae is not antibacterial. In vitro mixtures of fat body and cell-free hemolymph from non-immunized larvae, incubated at 25 degrees C for 20 hours generated a proteinaceous antibacterial activity. The generation of this activity was enhanced by the presence in the incubation mixture of LPS and/or hemocytes from non-immunized larvae. It is suggested that LPS causes the release of a hemocyte factor(s) which acts in conjunction with or directly on the fat body resulting in an enhanced production of antibacterial factors.

Lipid level and total fatty acid composition for selected developmental stages of Entomophthora egressa.

The major fatty acids (greater than 10%) of Entomophthora egressa were C16:0 and C18:1 . Minor fatty acids, which varied with the stage of fungal development, included C11:0, C12:0, C13:0, C14:0, C15:0, C16;1, C17:0, C18:0, C18:2, C18:3 C:201, C20:2, C20:3, C20:4, C20:5 and two unidentified unsaturated fatty acids. Differences were observed between the total fatty acid levels of C12:0, C14:0, C17:0, C18:0, and C20:5 and the degree of unsaturation of the fatty acids of 37-h protoplasts grown in modified Grace's medium and a simplified growth medium (SGM). The levels of C12:0, C14:0, C18:1, C20:4, and C20:5 decreased and the levels of C18:0 and C20:2 increased with the formation of spherical hyphal body (shb)initials. With the production of mature shb increased levels of C12:0, C14:0, C15:0, C18:1, C20:4, and C20:5 were detected. During the germination of the shb the levels of C14:0, C16:1, C18;1, and C20:4 increased, whereas C15:0 and C20:5 levels declined. The fatty acid levels, except for C12:0, C13:0, and C20:2, remained constant during the mycelial stage. The degree of fatty acid unsaturation decreased during early stages of development (protoplasts through shb initials). In SGM the degree of fatty acid unsaturation was lowest during the shb initial stage and highest during the shb stage. The total lipid level increased during shb maturation and declined during shb germination.