RESUMEN
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 (R) populations at â¼30% 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 h 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.
Asunto(s)
Bacillus thuringiensis , Mariposas Nocturnas , Animales , Bacillus thuringiensis/metabolismo , Mariposas Nocturnas/microbiología , Insectos/microbiología , Larva/microbiología , Sistema Digestivo/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Three types of modified silicon dioxide nanoparticles (SiO2, 10−20 nm) with additives of epoxy, silane and amino groups, used independently and in combination with the entomopathogenic bacteria Bacillus thuringiensis subsp. morrisoni and fungus Metarhizium robertsii were tested against Colorado potato beetle (Leptinotarsa decemlineata) and cabbage beetles (Phyllotreta spp.). All three nanoparticles were found to have an entomocidal effect on Colorado potato beetle larvae and crucifer flea beetles when ingested. Increased susceptibility of insects to B. thuringiensis or M. robertsii blastospores and their metabolites was shown after exposure to the modified silicon dioxide nanoparticles. The potential of modified silicon dioxide nanoparticles to enhance the efficiency of biopesticides based on the bacteria B. thuringiensis and fungi M. robertsii is considered in the paper.
RESUMEN
The insect integument (exoskeleton) is an effective physiochemical barrier that limits disease-causing agents to a few portals of entry, including the gastrointestinal and reproductive tracts. The bacterial biopesticide Bacillus thuringiensis (Bt) enters the insect host via the mouth and must thwart gut-based defences to make its way into the body cavity (haemocoel) and establish infection. We sought to uncover the main antibacterial defences of the midgut and the pathophysiological features of Bt in a notable insect pest, the Colorado potato beetle Leptinotarsa decemlineata (CPB). Exposing the beetles to both Bt spores and their Cry3A toxins (crystalline δ-endotoxins) via oral inoculation led to higher mortality levels when compared to either spores or Cry3A toxins alone. Within 12 h post-exposure, Cry3A toxins caused a 1.5-fold increase in the levels of reactive oxygen species (ROS) and malondialdehyde (lipid peroxidation) within the midgut - key indicators of tissue damage. When Cry3A toxins are combined with spores, gross redox imbalance and 'oxidation stress' is apparent in beetle larvae. The insect detoxification system is activated when Bt spores and Cry3A toxins are administered alone or in combination to mitigate toxicosis, in addition to elevated mRNA levels of candidate defence genes (pattern-recognition receptor, stress-regulation, serine proteases, and prosaposin-like protein). The presence of bacterial spores and/or Cry3A toxins coincides with subtle changes in microbial community composition of the midgut, such as decreased Pseudomonas abundance at 48 h post inoculation. Both Bt spores and Cry3A toxins have negative impacts on larval health, and when combined, likely cause metabolic derangement, due to multiple tissue targets being compromised.
Asunto(s)
Toxinas de Bacillus thuringiensis/fisiología , Bacillus thuringiensis/fisiología , Escarabajos/microbiología , Endotoxinas/fisiología , Proteínas Hemolisinas/fisiología , Control de Insectos , Control Biológico de Vectores , Esporas Bacterianas/fisiología , Animales , Escarabajos/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Larva/microbiología , LongevidadRESUMEN
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.
Asunto(s)
Inmunidad Innata/genética , Proteínas de Insectos/antagonistas & inhibidores , Metarhizium/crecimiento & desarrollo , Mariposas Nocturnas/inmunología , Animales , Susceptibilidad a Enfermedades/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/inmunología , Proteínas de Insectos/genética , Larva/inmunología , Larva/microbiología , Metarhizium/inmunología , Mariposas Nocturnas/genética , Mariposas Nocturnas/microbiología , Micosis/inmunología , Micosis/prevención & control , Interferencia de ARNRESUMEN
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.
Asunto(s)
Interacciones Huésped-Patógeno , Integumento Común/microbiología , Metarhizium/patogenicidad , Mariposas Nocturnas/microbiología , Esporas Fúngicas/inmunología , Animales , Antibiosis , Susceptibilidad a Enfermedades , Expresión Génica , Proteínas de Insectos , Insectos/microbiología , Larva/microbiología , Melaninas/metabolismo , Metarhizium/genética , Metarhizium/inmunología , Control Biológico de Vectores , Esporas Fúngicas/patogenicidadRESUMEN
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.
Asunto(s)
Resistencia a la Enfermedad/fisiología , Modelos Animales , Mariposas Nocturnas/microbiología , Micosis/veterinaria , Nosema/patogenicidad , Animales , Microsporidiosis/veterinaria , VirulenciaRESUMEN
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.
Asunto(s)
Infecciones Bacterianas/metabolismo , Escarabajos/parasitología , Dopamina/biosíntesis , Mariposas Nocturnas/parasitología , Micosis/metabolismo , Animales , Bacillus thuringiensis , Beauveria , Escarabajos/metabolismo , Hemolinfa/metabolismo , Mariposas Nocturnas/metabolismo , Control Biológico de Vectores/métodosRESUMEN
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.
Asunto(s)
Antioxidantes/metabolismo , Hemolinfa/metabolismo , Mariposas Nocturnas/inmunología , Especies Reactivas de Oxígeno/metabolismo , Animales , Antígenos/inmunología , Larva/inmunología , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/metabolismo , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Bacillus thuringiensis/patogenicidad , Mariposas Nocturnas/inmunología , Mariposas Nocturnas/microbiología , Tejido Adiposo/inmunología , Animales , Microbioma Gastrointestinal/fisiología , Perfilación de la Expresión Génica , Inflamación/genética , Larva/genética , Larva/inmunología , Larva/microbiología , Mariposas Nocturnas/genéticaRESUMEN
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.