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Bacillus thuringiensis (Bt) is one of the most common entomopathogenic bacteria used as a biopesticide, and source of endotoxin genes for generating insect-resistant transgenic plants. The mechanisms underpinning an insect's susceptibility or resistance to B. thuringiensis are diverse. The bacterial lifecycle does not end with the death of a host, they continue to exploit the cadaver to reproduce and sporulate. Herein, we studied the progression of B. thuringiensis subsp. galleriae infection in two populations of wax moth larvae (Galleria mellonella) to gain further insight into the "arms race" between B. thuringiensis virulence and insect defences. Two doses of B. thuringiensis subsp. galleriae (spore and crystalline toxin mixtures) were administered orally to compare the responses of susceptible (S) and resistant to Bt (R) populations at ∼30% mortality each. To investigate B. thuringiensis-insect antibiosis, we used a combination of in vivo infection trials, bacterial microbiome analysis, and RNAi targeting the antibacterial peptide gloverin. Within 48 hours post-inoculation, B. thuringiensis-resistant insects purged the midgut of bacteria, i.e., colony forming unit numbers fell below detectable levels. Second, B. thuringiensis rapidly modulated gene expression to initiate sporulation (linked to quorum sensing) when exposed to resistant insects in contrast to susceptible G. mellonella. We reinforce earlier findings that elevated levels of antimicrobial peptides, specifically gloverin, are found in the midgut of resistant insects, which is an evolutionary strategy to combat B. thuringiensis infection via its main portal of entry. A sub-population of highly virulent B. thuringiensis can survive the enhanced immune defences of resistant G. mellonella by disrupting the midgut microbiome and switching rapidly to a necrotrophic strategy, prior to sporulation in the cadaver.

The efficacy of the fungus Lecanicillium lecanii and two bacteria, Bacillus thuringiensis and Streptomyces avermitilis against the two-spotted spider mite Tetranychus urticae Koch and side effects on its predatory mite Phytoseiulus persimilis A.-H. was studied under laboratory conditions. Both S. avermitilis and B. thuringiensis based biopesticides resulted in maximum mortality rates of 90–100% and 91–99% for spider mite adults and larvae, respectively. The mortality of spider mite larvae under fungus L. lecanii treatment was around 60%. These bacteria and fungus also had toxic effects against P. persimilis on the same day of applying insecticides and releasing the predatory mite. The release of predatory mites one day post-treatment of plants with L. lecanii and 7 days post-treatment with B. thuringiensis or S. avermitilis did not negatively affect the survival of predators released. These findings support the potential use of entomopathogenic fungi and bacteria in combination with predatory mites in spider mite biocontrol.

In order for entomopathogenic fungi to colonize an insect host, they must first attach to, and penetrate, the cuticle layers of the integument. Herein, we explored the interactions between the fungal pathogen Metarhizium brunneum ARSEF 4556 and two immunologically distinct morphs, melanic (M) and non-melanic (NM), of the greater wax moth Galleria mellonella. We first interrogated the cuticular compositions of both insect morphs to reveal substantial differences in their physiochemical properties. Enhanced melanin accumulation, fewer hydrocarbons, and higher L-dihydroxyphenylalanine (DOPA) decarboxylase activity were evident in the cuticle of the M larvae. This “hostile” terrain proved challenging for M. brunneum – reflected in poor conidial attachment and germination, and elevated expression of stress-associated genes (e.g., Hsp30, Hsp70). Lack of adherence to the cuticle impacted negatively on the speed of kill and overall host mortality; a dose of 107 conidia killed ~30% of M larvae over a 12-day period, whereas a 100-fold lower dose (105 conidia) achieved a similar result for NM larvae. Candidate gene expression patterns between the insect morphs indicated that M larvae are primed to “switch-on” immunity-associated genes (e.g., phenoloxidase) within 6–12 h of conidia exposure and can sustain a “defense” response. Critically, M. brunneum responds to the distinct physiochemical cues of both hosts and adjusts the expression of pathogenicity-related genes accordingly (e.g., Pr2, Mad1, Mad2). We reveal previously uncharacterized mechanisms of attack and defence in fungal-insect antibiosis.

The co-evolutionary arms race between disease-causing agents and their insect victims is ancient and complex - leading to the development of specialised attack and defence strategies. Among such strategies is the capacity of fungal and oomycete pathogens to deploy degradative enzymes, notably proteases, to facilitate infection directly across the integument. To counter these proteases, insects such as the greater wax moth Galleria mellonella release metalloprotease inhibitors and other immune factors to thwart the invading fungus. To date, molecular-based confirmation of insect metalloprotease inhibitor's incontrovertible role in antifungal defence has been lacking. We targeted the IMPI gene for suppression using RNAi and exposed those insects to the entomopathogenic fungus Metarhizium brunneum ARSEF4556. Levels of IMPI were reduced significantly in the integument (10-fold) and fat body (5-fold) of RNAi-treated insects when compared to control larvae, and displayed a significantly higher mortality rate. We also surveyed candidate immune/detoxification gene expression levels (e.g., DOPA decarboxylase, galiomycin) in three tissues (integument, midgut, fat body) in order to gauge any potential non-target effects of RNAi. The loss of IMPI via RNAi compromises antifungal defences and leaves G. mellonella vulnerable to infection.

Dopamine (DA) is known as a hormone neurotrasnmitter molecule involved in several stress reactions in both vertebrates and invertebrates. Following infections with the fungi Metarhizium robertsii or Beauveria bassiana and the bacterium Bacillus thuringiensis, dopamine the concentration was measured at different time points in the haemolymph of the Colorado potato beetle, Leptinotarsa decemlineata and the larvae of the greater wax moth Galleria mellonella. The infection with M. robertsii increased (4 to 12-fold) DA concentrations in the haemolymph of the potato beetle larvae and the oral infection by B. thuringiensis also lead to a 30 and 45-fold increase. During infection of the greater wax moth larvae with Beauveria bassiana and B. thuringiensis DA increased 4 to 20-fold and about 2 to 2,5-fold respectively, compared to non-infected insects. The relative DA concentrations varied between the two insects and depended on the pathogens and post infection time.

Epigenetic mechanisms have been proposed to translate environmental stimuli into heritable transgenerational phenotypic variations that can significantly influence natural selection. An intriguing example is exposure to pathogens, which imposes selection for host resistance. To test this hypothesis, we used larvae of the greater wax moth Galleria mellonella as model host to experimentally select for resistance to Bacillus thuringiensis (Bt), the most widely used bacterial agent for the biological control of pest insects. To determine whether epigenetic mechanisms contribute to the evolution of resistance against pathogens, we exposed G. mellonella larvae over 30 generations to spores and crystals mix of Bt and compared epigenetic markers in this selected line, exhibiting almost 11-fold enhanced resistance against Bt, to those in a non-selected control population. We found that experimental selection influenced acetylation of specific histones and DNA methylation as well as transcription of genes encoding the enzymatic writers and erasers of these epigenetic mechanisms. Using microarray analysis, we also observed differences in the expression of conserved miRNAs in the resistant and susceptible larvae, resulting in the repression of candidate genes that confer susceptibility to Bt. By combining in silico minimum free energy hybridization with RT-PCR experiments, we identified the functions and biological processes associated with the mRNAs targeted by these miRNAs. Our results suggest that epigenetic mechanisms operating at the pre-transcriptional and post-transcriptional levels contribute to the transgenerational inherited transcriptional reprogramming of stress and immunity-related genes, ultimately providing a mechanism for the evolution of insect resistance to pathogen.

Galleria mellonella fed 3 million Nosema pyrausta spores per larva showed 0 and 5% infestation rate at 30 °C and 24 °C, respectively. N. pyrausta virulence did not increase after passage through G. mellonella for three generations. When larvae were pretreated with phenylthiourea, Bacillus thuringiensis or combination of both, infection rates were 11%, 15% and 22%, respectively. Injection of untreated and potassium hydroxide-primed spores resulted in approximately 10% and 50% infection, respectively. G. mellonella is resistant to high dosages of N. pyrausta spores, serving as a prospective model of insect resistance to microsporidia, while host immunosuppression and/or spore activation increases success of the pathogen.

Some mechanisms of resistance to Bacillus thuringiensis, including esterase activity in the midgut, can appear in laboratory and field insect populations. We found that B. thuringiensis infection significantly reduced the esterase activity in the midgut of greater wax moth Galleria mellonella 48 h post infection. Esterase activity was inhibited 1.5-fold by triphenyl phosphate to test the impact of the enzyme in resistance to the bacteria. Bioassays demonstrated that pre-exposure of the insects to triphenyl phosphate followed by bacterial infection resulted in two-fold elevated susceptibility of the greater wax moth larvae to B. thuringiensis.

Recent concepts in evolutionary biology suggest that epigenetic mechanisms can translate environmental selection pressures into heritable changes in phenotype. To determine whether experimental selection for a complex trait in insects involves epigenetic modifications, we carried out a generation-spanning experiment using larvae of the greater wax moth Galleria mellonella as a model host to investigate the role of epigenetics in the heritability of resistance against the parasitic fungus Metarhizium robertsii. We investigated differences in DNA methylation, histone acetylation and microRNA (miRNA) expression between an experimentally resistant population and an unselected, susceptible line, revealing that the survival of G. mellonella larvae infected with M. robertsii correlates with tissue-specific changes in DNA methylation and histone modification and the modulation of genes encoding the corresponding enzymes. We also identified miRNAs differentially expressed between resistant and susceptible larvae and showed that these regulatory molecules target genes encoding proteinases and proteinase inhibitors, as well as genes related to cuticle composition, innate immunity and metabolism. These results support our hypothesis that epigenetic mechanisms facilitate, at least in part, the heritable manifestation of parasite resistance in insects. The reciprocal adaptations underlying host–parasite coevolution therefore extend beyond the genetic level to encompass epigenetic modifications.

Microevolutionary mechanisms of resistance to a bacterial pathogen were explored in a population of the Greater wax moth, Galleria mellonella, selected for an 8.8-fold increased resistance against the entomopathogenic bacterium Bacillus thuringiensis (Bt) compared with a non-selected (suspectible) line. Defense strategies of the resistant and susceptible insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management and immune defense in the fat body. Following oral infection with Bt, the expression of these genes was further elevated in the fat body and midgut of both lines and to a greater extent some of them in resistant line than the susceptible line. This gene expression analysis reveals a pattern of resistance mechanisms targeted to sites damaged by Bt with the insect placing greater emphasis on tissue repair as revealed by elevated expression of these genes in both the fat body and midgut epithelium. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affects the viability of other gut bacteria. Remarkably the resistant line employs multifactorial adaptations for resistance to Bt without any detected negative trade off since the insects exhibited higher fecundity.

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione-S-transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15 min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione-S-transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.

Activities of enzymatic antioxidants—superoxide dismutase, glutathione-S-transferase, and catalase—as well as generation of reactive oxygen species (ROS) in lymph of the honeycomb moth Galleria mellonella L. were studied at development of the process of encapsulation of nylon implants. It has been established that as soon as 15 min after piercing of cuticle with the implant the capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. A statistically significant increase of the ROS generation in lymph and a decrease of the enzymatic antioxidant activities in the insect hemocytes have been revealed after the implant incorporation. The authors suggest that the key role in maintenance of the oxidation-reduction balance in hemolymph at development of the encapsulation process is played by non-oxidative antioxidants.

Prophenoloxidase system, which is represented by awhole cascade of enzymatic reactions, one of the finalproducts of which is melanin, plays a major role ininsect immunity [1]. Reactive intermediates producedduring melanogenesis may participate in cytotoxicreactions in the course of granule formation and incap-sulation of a foreign agent in an insect organism. Thisgroup primarily involves o-semiquinones formed dur-ing enzymatic oxidation of o-hydroquinones (in partic-ular, 3,4-dihydroxyphenylalanine, DOPA) [1, 2]. Suchcompounds can interact with oxygen to form superox-ide anion (), which, after a number of transforma-tions (dismutation and Fenton reaction), is eventuallyconverted to other reactive particles, such as hydrogenperoxide and hydroxyl radical [1]. Earlier, we appliedEPR to demonstrate the formation of DOPA o-semi-quinones in the hemolymph of Galleria mellonella lar-vae during melanogenesis [3, 4]. Studies of the genera-tion of superoxide and hydrogen peroxide in insecthemolymph are represented by a few papers. It shouldbe noted that the formation of has not yet beenrecorded directly. Some authors tested generation of in insect hemolymph using the method based onreduction of nitro blue tetrazolium [1, 5, 6]. However,the nonspecificity of this method [7] does not allow thedemonstration of formation in insects' organism tobe regarded as reliable data. Today, the most informa-tive method of recording short-lived oxygen radicals inbiological systems is the spin trap method in combina-tion with EPR spectroscopy. The goal of this study wasO

The name of a funding organization for the first, second and last authors is incorrect. The Funding statement should read: "Funding from both the Royal Society International Joint Project and the Russian Foundation for Basic Research (awarded to TB, MW, ID), presidium SBRAS (VG, ID, VK) and the DFG Priority Program 1399 'Host-Parasite-Coevolution – rapid reciprocal adaptation and its genetic basis' to AV (VI 219/3-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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.

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, 25(th) 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.

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.