V. V. Glupov

Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae)

Bacillus thuringiensis is one of the most widely used sources of biorational pesticides, as well as a key source of genes for transgenic expression to provide pest resistance in plants. In this study the effect of Bacillus thuringiensis ssp. galleriae (Bt) infection on the activity of superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), concentrations of oxidated and reduced thiols (RSSR/RSH) and malondialdehyde (MDA) was tested in the midgut of Galleria mellonella larvae. We found that Bt infection resulted in increased activities of SOD, GST, malondialdehyde and RSSR/RSH ratio the first day after inoculation. However, catalase activity decreased on the first and following days after bacterial infection by Bt. Our results confirm the hypothesis that Bt infection increases the level of oxidative stress in the larval midgut. In light of this study, it seems possible that oxidative damage contributes to cell death in the midgut during bacteriosis.

More than a colour change: insect melanism, disease resistance and fecundity

A ‘dark morph’ melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.

Can insects develop resistance to insect pathogenic fungi

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25th generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses.

Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. After the peroral inoculation of Galleria mellonella by the Bt in 5% sublethal concentration (LC(5)), a 1.5-fold increase in the phagocytic activity of infected larvae has been registered on the second and third days after the inoculation. With the increase of Bt-inoculum amount to 15% of sublethal concentration (LC(15)), a further increase of the phagocytic activity and enhanced encapsulation rates in the haemolymph of infected larvae has been observed. The enhanced cellular immunity during the bacteriosis seems to have resulted from the destruction of midgut epithelium cells followed by the subsequent exposure of gut content to lymph factors activating the immune system of haemocoel.

Activity and heavy metal resistance of non-specific esterases in leaf beetle Chrysomela lapponica from polluted and unpolluted habitats

We compared the general activity and heavy metal resistance of non-specific esterases in two populations of the leaf beetle Chrysomela lapponica from habitats severely contaminated by heavy metals (mostly Ni and Cu) and two populations from unpolluted habitats. Concentrations of Ni and Cu in adult beetles from the most polluted site were 7.7 and 3.6 times higher that in beetles from unpolluted habitats. Larval esterases showed higher activity and lower susceptibility to heavy metals than esterases of adults. Larval esterase activity did not differ between populations from polluted and unpolluted sites, but adult beetles from polluted localities had lower esterase activity than beetles from unpolluted habitats. Both Cu and Ni sulfates in millimolar concentrations in vitro suppressed esterase activity of larvae from unpolluted habitats, but caused no negative effect on esterases of larvae from polluted sites. Similarly, inhibition of adult esterase activity by Ni was stronger in beetles from unpolluted localities than in beetles from polluted localities. This indicates that resistance of non-specific esterases to heavy metals is higher in leaf beetle populations from contaminated environment.

Influence of fungal infection on the DOPA-semiquinone and DOPA-quinone production in haemolymph of Galleriamellonella larvae.

The formation of reactive oxygen metabolites in haemolymph of Galleria mellonella larvae was studied by ESR spectroscopy. The inhibition of the production of the reactive oxygen metabolites of DOPA in haemolymph under the action of fungal infection was shown using spin trap 1-hydroxy-3-carboxy-pyrrolidine. This inhibition correlated with decrease of phenoloxidase activity in haemolymph of infected insects. Simultaneously, the decrease of production of DOPA-semiquinone was detected using method of spin stabilization by diamagnetic metal ions. Moreover, it was shown that the formation of DOPA-quinone was slowed down in haemolymph of infected insects. Our results suggest that the DOPA-derived quinones/semiquinones may be involved in immune response of insects as part of its defense mechanism.

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase.

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-dl-alanine (DOPA) has been studied. The ability of DOPA to react with has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and being equal to (3.4±0.6)×105 M−1 s−1. The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with

Contributions of cellular and humoral immunity of Galleria mellonella larvae in defence against oral infection by Bacillus thuringiensis

In this study the cellular and humoral immune reactions of the Greater wax moth Galleria mellonella have been investigated during bacterial infection caused by oral administration of Bacillus thuringiensis. Two different dose strengths were investigated to assess the contribution of immune parameters to induced Bt resistance. Low-dose (sublethal LC15) infection resulted in significantly elevated haemolymph phenoloxidase and lysozyme-like activity, enhanced phagocytic activity of haemocytes, and increased encapsulation responses in infected larvae at 48 and 72 h post infection. Higher doses of Bt (half-lethal LC50) also triggered significantly elevated haemolymph phenoloxidase and lysozyme-like activity, but decreased the coagulation index and activity of phenoloxidase in haemocytes of infected larvae. In both types of infection, the pool of circulating haemocytes became depleted. The importance of cellular and humoral immune reactions in induced insect resistance to intestinal bacterial infection Bt is herein discussed.

Synergistic action of entomopathogenic hyphomycetes and the bacteria Bacillus thuringiensis ssp. morrisoni in the infection of Colorado potato beetle Leptinotarsa decemlineata

A synchronous coinfection of the Colorado potato beetle Leptinotarsa decemlineata (Say) with the entomopathogenic bacteria Bacillus thuringiensis ssp. morrisoni Bonnifoi & de Barjak var. tenebrionis Krieg et al. and hyphomycete Metarhizium anisopliae (Metsch.) Sorokin or Beauveria bassiana (Bals.) Vuill leads to the rapid death of 95–100% of larvae. The bacteria arrest the nutrition of insects, while the fungal spores kill the weakened larvae. The synergistic effect of two pathogens is recorded at a relatively low hyphomycete titer (1–5 × 106 conidia/ml) and is evident in the mortality dynamics at all larval ages. These bacterial and fungal pathogens display no antagonism on artificial nutrient media. This microbial complex is highly efficient under natural conditions (80–90% larval mortality rate and no plant defoliation).

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.