Ekaterina V. Grizanova

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.

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.

The effects of dietary nickel on the detoxification enzymes, innate immunity and resistance to the fungus Beauveria bassiana in the larvae of the greater wax moth Galleria mellonella

In this study, we tested the effects of dietary nickel on the activity of glutathione S-transferase (GST), esterases, phenoloxidase, and encapsulation in the haemolymph of larvae of the greater wax moth Galleria mellonella. We also explored the effects of dietary nickel on larval resistance to infection by the fungus Beauveria bassiana. Larvae fed a low dose of nickel (10 μg g(-1)) had significantly higher GST, phenoloxidase activity and encapsulation responses than controls fed on a nickel-free diet. We also found that larvae fed a sublethal dose of nickel (50 μg g(-1)) had increased GST, esterase activity and encapsulation rates but decreased phenoloxidase activity. Although, a sublethal dose of dietary nickel enhanced innate immunity, we found that this reduced resistance against the real pathogen. Our results suggest that enhanced immunity and detoxification enzyme activity of insects may not be beneficial to resistance to fungal infection. It appears that there is a trade off between different resistance mechanisms in insects under different metal treatments.

Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis

ABSTRACT 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.

Experimental evolution of resistance against Bacillus thuringiensis in the insect model host Galleria mellonella results in epigenetic modifications

ABSTRACT 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.

Bacterial and fungal infections induce bursts of dopamine in the haemolymph of the Colorado potato beetle Leptinotarsa decemlineata and greater wax moth Galleria mellonella

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.

Greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) as a resistant model host for Nosema pyrausta (Microsporidia: Nosematidae)

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.