Sensitive bioluminescence imaging of cryptococcosis in Galleria mellonella improves antifungal screening under in vivo conditions.

Cryptococcus neoformans is an environmental yeast that primarily affects immunocompromised individuals, causing respiratory infections and life-threatening meningoencephalitis. Treatment is complicated by limited antifungal options, with concerns such as adverse effects, dose-limiting toxicity, blood-brain barrier permeability, and resistance development, emphasizing the critical need to optimize and expand current treatment options against invasive cryptococcosis. Galleria mellonella larvae have been introduced as an ethical intermediate for in vivo testing, bridging the gap between in vitro antifungal screening and mouse studies. However, current infection readouts in G. mellonella are indirect, insensitive, or invasive, which hampers the full potential of the model. To address the absence of a reliable non-invasive method for tracking infection, we longitudinally quantified the cryptococcal burden in G. mellonella using bioluminescence imaging (BLI). After infection with firefly luciferase-expressing C. neoformans, the resulting bioluminescence signal was quantitatively validated using colony-forming unit analysis. Longitudinal comparison of BLI to health and survival analysis revealed increased sensitivity of BLI in discriminating cryptococcal burden during early infection. Furthermore, BLI improved the detection of treatment efficacy using first-line antifungals, thereby benchmarking this model for antifungal testing. In conclusion, we introduced BLI as a real-time, quantitative readout of cryptococcal burden in G. mellonella over time, enabling more sensitive and reliable antifungal screening.

Mechanistic insights on the antibacterial action of the kyotorphin peptide derivatives revealed by in vitro studies and Galleria mellonella proteomic analysis.

OBJECTIVES: The selected kyotorphin derivatives were tested to improve their antimicrobial and antibiofilm activity. The antimicrobial screening of the KTP derivatives were ascertained in the representative strains of bacteria, including Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa. METHODS: Kyotorphin derivatives, KTP-NH2, KTP-NH2-DL, IbKTP, IbKTP-NH2, MetKTP-DL, MetKTP-LD, were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. Peptides were synthesized by standard solution or solid-phase peptide synthesis and purified using RP-HPLC, which resulted in >95 % purity, and were fully characterized by mass spectrometry and 1H NMR. The minimum inhibitory concentrations (MIC) determined for bacterial strains were between 20 and 419 µM. The direct effect of IbKTP-NH2 on bacterial cells was imaged using scanning electron microscopy. The absence of toxicity, high survival after infection and an increase in the hemocytes count was evaluated by injections of derivatives in Galleria mellonella larvae. Proteomics analyses of G. mellonella hemolymph were performed to investigate the underlying mechanism of antibacterial activity of IbKTP-NH2 at MIC. RESULTS: IbKTP-NH2 induces morphological changes in bacterial cell, many differentially expressed proteins involved in DNA replication, synthesis of cell wall, and virulence were up-regulated after the treatment of G. mellonella with IbKTP-NH2. CONCLUSION: We suggest that this derivative, in addition to its physical activity on the bacterial membranes, can elicit a cellular and humoral immune response, therefore, it could be considered for biomedical applications.

Safety attributes of Pseudomonas sp. P26, an environmental microorganism with potential application in contaminated environments.

Currently, the selection of non-pathogenic microorganisms that lack clinically relevant antimicrobial resistance is crucial to bioaugmentation strategies. Pseudomonas sp. P26 (P26) is an environmental bacterium of interest due to its ability to remove aromatic compounds from petroleum, but its safety characteristics are still unknown. The study aimed to: a) determine P26 sensitivity to antimicrobials, b) investigate the presence of quinolone and ß-lactam resistance genes, c) determine the presence of virulence factors, and d) evaluate the effect of P26 on the viability of Galleria mellonella (an invertebrate animal model). P26 antimicrobial sensitivity was determined in vitro using the Kirby-Bauer agar diffusion method and the VITEK 2 automated system (BioMerieux®). Polymerase Chain Reaction was employed for the investigation of genes associated with quinolone resistance, extended-spectrum ß-lactamases, and carbapenemases. Hemolysin and protease production was determined in human blood agar and skimmed-milk agar, respectively. In the in vivo assay, different doses of P26 were injected into Galleria mellonella larvae and their survival was monitored daily. Control larvae injected with Pseudomonas putida KT2440 (a strain considered as safe) and Pseudomonas aeruginosa PA14 (a pathogenic strain) were included. Pseudomonas sp. P26 was susceptible to most evaluated antimicrobials, except for trimethoprim-sulfamethoxazole. No epidemiologically relevant genes associated with quinolone and ß-lactam resistance were identified. Hemolysin and protease production was only evidenced in the virulent strain (PA14). Furthermore, the results obtained in the in vivo experiment demonstrated that inocula less than 108 CFU/mL of P26 and P. putida KT2440 did not significantly affect larval survival, whereas larvae injected with the lowest dose of the pathogenic strain P. aeruginosa PA14 experienced instant mortality. The results suggest that Pseudomonas sp. P26 is a safe strain for its application in environmental bioremediation processes. Additional studies will be conducted to ensure the safety of this bacterium against other organisms.

Entomopathogenic pseudomonads can share an insect host with entomopathogenic nematodes and their mutualistic bacteria.

A promising strategy to overcome limitations in biological control of insect pests is the combined application of entomopathogenic pseudomonads (EPPs) and nematodes (EPNs) associated with mutualistic bacteria (NABs). Yet, little is known about interspecies interactions such as competition, coexistence, or even cooperation between these entomopathogens when they infect the same insect host. We investigated the dynamics of bacteria-bacteria interactions between the EPP Pseudomonas protegens CHA0 and the NAB Xenorhabdus bovienii SM5 isolated from the EPN Steinernema feltiae RS5. Bacterial populations were assessed over time in experimental systems of increasing complexity. In vitro, SM5 was outcompeted when CHA0 reached a certain cell density, resulting in the collapse of the SM5 population. In contrast, both bacteria were able to coexist upon haemolymph-injection into Galleria mellonella larvae, as found for three further EPP-NAB combinations. Finally, both bacteria were administered by natural infection routes i.e. orally for CHA0 and nematode-vectored for SM5 resulting in the addition of RS5 to the system. This did not alter bacterial coexistence nor did the presence of the EPP affect nematode reproductive success or progeny virulence. CHA0 benefited from RS5, probably by exploiting access routes formed by the nematodes penetrating the larval gut epithelium. Our results indicate that EPPs are able to share an insect host with EPNs and their mutualistic bacteria without major negative effects on the reproduction of any of the three entomopathogens or the fitness of the nematodes. This suggests that their combination is a promising strategy for biological insect pest control.

Abietic Acid as a Novel Agent against Ocular Biofilms: An In Vitro and Preliminary In Vivo Investigation.

Biofilm-related ocular infections can lead to vision loss and are difficult to treat with antibiotics due to challenges with application and increasing microbial resistance. In turn, the design and testing of new synthetic drugs is a time- and cost-consuming process. Therefore, in this work, for the first time, we assessed the in vitro efficacy of the plant-based abietic acid molecule, both alone and when introduced to a polymeric cellulose carrier, against biofilms formed by Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in standard laboratory settings as well as in a self-designed setting using the topologically challenging surface of the artificial eye. These analyses were performed using the standard microdilution method, the biofilm-oriented antiseptic test (BOAT), a modified disk-diffusion method, and eyeball models. Additionally, we assessed the cytotoxicity of abietic acid against eukaryotic cell lines and its anti-staphylococcal efficacy in an in vivo model using Galleria mellonella larvae. We found that abietic acid was more effective against Staphylococcus than Pseudomonas (from two to four times, depending on the test applied) and that it was generally more effective against the tested bacteria (up to four times) than against the fungus C. albicans at concentrations non-cytotoxic to the eukaryotic cell lines and to G. mellonella (256 and 512 µg/mL, respectively). In the in vivo infection model, abietic acid effectively prevented the spread of staphylococcus throughout the larvae organisms, decreasing their lethality by up to 50%. These initial results obtained indicate promising features of abietic acid, which may potentially be applied to treat ocular infections caused by pathogenic biofilms, with higher efficiency manifested against bacterial than fungal biofilms.

Biodegradation of Polystyrene by Galleria mellonella: Identification of Potential Enzymes Involved in the Degradative Pathway.

Galleria mellonella is a lepidopteran whose larval stage has shown the ability to degrade polystyrene (PS), one of the most recalcitrant plastics to biodegradation. In the present study, we fed G. mellonella larvae with PS for 54 days and determined candidate enzymes for its degradation. We first confirmed the biodegradation of PS by Fourier transform infrared spectroscopy- Attenuated total reflectance (FTIR-ATR) and then identified candidate enzymes in the larval gut by proteomic analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Two of these proteins have structural similarities to the styrene-degrading enzymes described so far. In addition, potential hydrolases, isomerases, dehydrogenases, and oxidases were identified that show little similarity to the bacterial enzymes that degrade styrene. However, their response to a diet based solely on polystyrene makes them interesting candidates as a potential new group of polystyrene-metabolizing enzymes in eukaryotes.

Bbhox2 is a key regulator for conidiation and virulence in Beauveria bassiana.

Beauveria bassiana, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field and forest pests. To explore the potential factors involved in the fungal pathogenicity, Bbhox2, an important and conserved functional transcription factor containing homeodomain was carried out by functional analysis. Homologous recombination was used to disrupt the Bbhox2 gene in B.bassiana. The conidia yield of the deletant fungal strain was significantly reduced. The conidial germination was faster, and stress tolerance to Congo red and high osmotic agents were decreased compared with that in the wildtype. Additionally, ΔBbhox2 showed a dramatic reduction in virulence no matter in topical inoculations or in intra-hemolymph injections against Galleria mellonella larvae, which is likely due to the failure of appressorium formation and the defect in producing hyphal body. These results indicate that the Bbhox2 gene markedly contributes to conidiation and pathogenicity in B. bassiana.

Bacteriophage-mediated decolonization of Klebsiella pneumoniae in a novel Galleria mellonella gut colonization model with Enterobacteriaceae.

Galleria mellonella larvae have emerged as an invertebrate model for investigating bacterial pathogenesis and potential therapies, addressing ethical concerns related to mammalian models. This model has the advantage of having a simple gut microbiome, which is suitable for gut colonization studies. Intestinal colonization by Enterobacteriaceae significantly contributes to the spread of antibiotic resistance. This study aimed to establish a novel Enterobacteriaceae gut colonization larval model and assess its suitability for evaluating distinct antimicrobial efficacies. Larvae were force-fed sequentially with bacterial doses of K. pneumoniae and E. coli at 0, 24, and 48 h, with survival monitoring at 24 h intervals. Bacterial counts were assessed after 48 h and 120 h of force-feeding. Successfully colonized larvae were subjected to one-time force feeding of a bacteriophage cocktail (107 PFU/larvae) or MIC-based meropenem and ciprofloxacin. The colonized bacterial load was quantified by CFU count. Three doses of 106 CFU/larvae resulted in stable gut colonization, independent of the K. pneumoniae or E. coli strain. Compared with the control, force-feeding of the bacteriophage reduced the colonization of the strain Kp 419614 by 5 log10 CFU/larvae, while antibiotic treatment led to a 3 log10 CFU/larval reduction. This novel G. mellonella model provides a valuable alternative for gut colonization studies, facilitating proof-of-concept investigations and potentially reducing or replacing follow-up experiments in vertebrate models.

Conserved infections and reproductive phenotypes of Wolbachia symbionts in Asian tortrix moths.

Wolbachia is a ubiquitous endosymbiotic bacterium that manipulates insect reproduction. A notable feature of Wolbachia is male killing (MK), whereby sons of infected females are killed during development; however, the evolutionary processes by which Wolbachia acquired the MK ability remain unclear. The tea tortrix moth Homona magnanima (Tortricidae) harbours three non-MK Wolbachia strains (wHm-a, wHm-b and wHm-c) and an MK strain wHm-t. Although wHm-t and wHm-c are closely related, only wHm-t has an MK-associated prophage region. To understand the evolutionary processes underlying the emergence of MK wHm-t, we examined Wolbachia infections and phenotypes in 62 tortricid species collected from 39 localities across Japan, Taiwan, Vietnam and Indonesia. PCR assays detected wHm-c relatives in 51 species and triple infection of wHm-a, wHm-b and wHm-c in 31 species. Apart from Taiwanese H. magnanima, no species exhibited the MK phenotype and were positive for the wHm-t-specific prophage. While wHm-t infection was dominant in Taiwanese H. magnanima, wHm-a, wHm-b and wHm-c were dominant in Japanese H. magnanima populations. These results suggest that wHm-a, wHm-b and wHm-c strains descended from a common ancestor with repeated infection loss and that wHm-t evolved from the wHm-c acquiring MK ability in allopatric populations of H. magnanima.

A novel Galleria mellonella experimental model for zoonotic pathogen Brucella.

Brucellosis is a major threat to public health and animal husbandry. Several in vivo vertebrate models, such as mice, guinea pigs, and nonhuman primates, have been used to study Brucella pathogenesis, bacteria-host interactions, and vaccine efficacy. However, these models have limitations whereas the invertebrate Galleria mellonella model is a cost-effective and ethical alternative. The aim of the present study was to examine the invertebrate G. mellonella as an in vivo infection model for Brucella. Infection assays were employed to validate the fitness of the larval model for Brucella infection and virulence evaluation. The protective efficacy of immune sera was evaluated by pre-incubated with a lethal dose of bacteria before infection. The consistency between the mouse model and the larval model was confirmed by assessing the protective efficacy of two Brucella vaccine strains. The results show that G. mellonella could be infected by Brucella strains, in a dose- and temperature-dependent way. Moreover, this larval model can effectively evaluate the virulence of Brucella strains in a manner consistent with that of mammalian infection models. Importantly, this model can assess the protective efficacy of vaccine immune sera within a day. Further investigation implied that haemolymph played a crucial role in the protective efficacy of immune sera. In conclusion, G. mellonella could serve as a quick, efficient, and reliable model for evaluating the virulence of Brucella strains and efficacy of immune sera in an ethical manner.

Investigating bacterial infections in Galleria mellonella larvae: Insights into pathogen dissemination and behavior.

The insect Galleria mellonella is an alternative animal model widely used for studying bacterial infections. It presents a wide range of advantages, including its low cost, easy maintenance and lack of ethical constraints. Among other features, their innate immune system is very similar to that of mammals. In this study, we dissected several larvae infected with important human pathogens: Mycobacterium abscessus, Staphylococcus aureus and Pseudomonas aeruginosa. By observing the fat body, gut, trachea, and hemolymph under the microscope, we were able to describe where bacteria tend to disseminate. We also quantified the number of bacteria in the hemolymph throughout the infection course and found significant differences between the different pathogens. With this work, we aimed to better understand the behavior and dissemination of bacteria in the infected larvae.

Biofilm Inhibition of Inula viscosa (L.) Aiton and Globularia alypum L. Extracts Against Candida Infectious Pathogens and In Vivo Action on Galleria mellonella Model.

The increasing importance of fungal infections has fueled the search for new beneficial alternatives substance from plant extracts. The current study investigates the antifungal and antibiofilm activity of Inula viscosa (L.) Aiton and Globularia alypum (L.) leaves extracts against Candida both in vitro and in vivo. The inhibition of planktonic and sessile Candida albicans and Candida glabrata growth using both leaf extracts are evaluated. Moreover; an in vivo infection model using Galleria mellonella larvae; infected and treated with the extracts are performed. All extracts show fungicidal activity; with a minimum fungicidal concentration (MFC) ranging from 128 to 512 µg mL-1 against the two selected strains of Candida. In particular, the best results are obtained with methanolic extract of I. viscosa and G. alypum with an MFC value of 128 µg mL-1 . The extracts are capable to prevent 90% of biofilm development at minor concentrations ranging from 100.71 ± 2.49 µg mL-1 to 380.4 ± 0.92 µg mL-1 . In vivo, tests on Galleria mellonella larvae show that the extracts increase the survival of the larvae infected with Candida. The attained results reveal that I. viscosa and G. alypum extracts may be considered as new antifungal agents and biofilm inhibiting agents for the pharmaceutical and agro-food field.

Effects of passages through an insect or a plant on virulence and physiological properties of the fungus Metarhizium robertsii.

Species of the genus Metarhizium are characterized by a multitrophic lifestyle of being arthropod parasites, rhizosphere colonizers, endophytes, and saprophytes. The process of adaptation to various organisms and substrates may lead to specific physiological alterations that can be elucidated by passaging through different hosts. Changes in virulence and cultivation properties of entomopathogenic fungi subcultured on different media or passaged through a live insect host are well known. Nevertheless, comparative in-depth physiological studies on fungi after passaging through insect or plant organisms are scarce. Here, virulence, plant colonization, hydrolytic enzymatic activities, toxin production, and antimicrobial action were compared between stable (nondegenerative) parent strain Metarhizium robertsii MB-1 and its reisolates obtained after eight passages through Galleria mellonella larvae or Solanum lycopersicum or after subculturing on the Sabouraud medium. The passaging through the insect caused similar physiological alterations relative to the plant-based passaging: elevation of destruxin A, B, and E production, a decrease in protease and lipase activities, and lowering of virulence toward G. mellonella and Leptinotarsa decemlineata as compared to the parent strain. The reisolates passaged through the insect or plant showed a slight trend toward increased tomato colonization and enhanced antagonistic action on tomato-associated bacterium Bacillus pumilus as compared to the parental strain. Meanwhile, the subculturing of MB-1 on the Sabouraud medium showed stability of the studied parameters, with minimal alterations relative to the parental strain. We propose that the fungal virulence factors are reprioritized during adaptation of M. robertsii to insects, plants, and media.

A Falciformispora senegalensis grain model in Galleria mellonella larvae.

Eumycetoma is a subcutaneous implantation mycosis often found in the foot. One of the hallmarks of eumycetoma is the formation of grains. These grains are either black or white, and the consistency and morphology differs per causative agent. The two most common causative agents of black-grain eumycetoma are Madurella mycetomatis and Falciformispora senegalensis. Since grains cannot be formed in vitro, in vivo models are needed to study grain formation. Here, we used the invertebrate Galleria mellonella to establish an in vivo grain model for F. senegalensis. Three different F. senegalensis strains were selected, and four different inocula were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 10 mg/larvae. Larval survival was monitored for 10 days. Grain formation was studied macroscopically and histologically. The efficacy of antifungal therapy was determined for itraconazole, amphotericin B, and terbinafine. A concentration of 10 mg F. senegalensis per larva was lethal for the majority of the larvae within 10 days. At this inoculum, grains were formed within 24 h after infection. The grains produced in the larvae resembled those formed in human patients. Amphotericin B given at 1 mg/kg 4 h, 28 h, and 52 h after infection prolonged larval survival. No enhanced survival was noted for itraconazole or terbinafine. In conclusion, we developed a F. senegalensis grain model in G. mellonella larvae in which grains were formed that were similar to those formed in patients. This model can be used to monitor grain formation over time and study antifungal efficacy.

Within eumycetoma lesions, the causative agents are embedded in grains. However, the grains differ per causative agent. In this study, we developed a grain model of Falciformispora senegalensis in the larvae of Galleria mellonella. This model can be used in the future to study the efficacy of novel antifungal agents.

Negative interactions and virulence differences drive the dynamics in multispecies bacterial infections.

Bacterial infections are often polymicrobial, leading to intricate pathogen-pathogen and pathogen-host interactions. There is increasing interest in studying the molecular basis of pathogen interactions and how such mechanisms impact host morbidity. However, much less is known about the ecological dynamics between pathogens and how they affect virulence and host survival. Here we address these open issues by co-infecting larvae of the insect model host Galleria mellonella with one, two, three or four bacterial species, all of which are opportunistic human pathogens. We found that host mortality was always determined by the most virulent species regardless of the number of species and pathogen combinations injected. In certain combinations, the more virulent pathogen simply outgrew the less virulent pathogen. In other combinations, we found evidence for negative interactions between pathogens inside the host, whereby the more virulent pathogen typically won a competition. Taken together, our findings reveal positive associations between a pathogen's growth inside the host, its competitiveness towards other pathogens and its virulence. Beyond being generalizable across species combinations, our findings predict that treatments against polymicrobial infections should first target the most virulent species to reduce host morbidity, a prediction we validated experimentally.

Powerful and Real-Time Quantification of Antifungal Efficacy against Triazole-Resistant and -Susceptible Aspergillus fumigatus Infections in Galleria mellonella by Longitudinal Bioluminescence Imaging.

Aspergillus fumigatus is an environmental mold that causes life-threatening respiratory infections in immunocompromised patients. The plateaued effectiveness of antifungal therapy and the increasing prevalence of triazole-resistant isolates have led to an urgent need to optimize and expand the current treatment options. For the transition of in vitro research to in vivo models in the time- and resource-consuming preclinical drug development pipeline, Galleria mellonella larvae have been introduced as a valuable in vivo screening intermediate. Despite the high potential of this model, the current readouts of fungal infections in G. mellonella are insensitive, irreproducible, or invasive. To optimize this model, we aimed for the longitudinal quantification of the A. fumigatus burden in G. mellonella using noninvasive bioluminescence imaging (BLI). Larvae were infected with A. fumigatus strains expressing a red-shifted firefly luciferase, and the substrate dosage was optimized for the longitudinal visualization of the fungal burden without affecting larval health. The resulting photon flux was successfully validated for fungal quantification against colony forming units (CFU) analyses, which revealed an increased dynamic range from BLI detection. Comparison of BLI to survival rates and health index scores additionally revealed improved sensitivity for the early discrimination of differences in fungal burdens as early as 1 day after infection. This was confirmed by the improved detection of treatment efficacy against triazole-susceptible and -resistant strains. In conclusion, we established a refined G. mellonella aspergillosis model that enables the noninvasive real-time quantification of A. fumigatus by BLI. This model provides a quick and reproducible in vivo system for the evaluation of treatment options and is in line with 3Rs recommendations. IMPORTANCE Triazole-resistant Aspergillus fumigatus strains are rapidly emerging, and resistant infections are difficult to treat, causing mortality rates of up to 88%. The recent WHO priority list underscores A. fumigatus as one of the most critical fungal pathogens for which innovative antifungal treatment should be (urgently) prioritized. Here, we deliver a Galleria mellonella model for triazole-susceptible and -resistant A. fumigatus infections combined with a statistically powerful quantitative, longitudinal readout of the A. fumigatus burden for optimized preclinical antifungal screening. G. mellonella larvae are a convenient invertebrate model for in vivo antifungal screenings, but so far, the model has been limited by variable and insensitive observational readouts. We show that bioluminescence imaging-based fungal burden quantification outperforms these readouts in reliability, sensitivity, and time to the detection of treatment effects in both triazole-susceptible and -resistant infections and can thus lead to better translatability from in vitro antifungal screening results to in vivo confirmation in mouse and human studies.

Expressing Parasitoid Venom Protein VRF1 in an Entomopathogen Beauveria bassiana Enhances Virulence toward Cotton Bollworm Helicoverpa armigera.

Despite entomopathogenic fungi being used in various insect pest control, it is recognized that they could replace more chemical insecticides if they were more efficient. We have found that cotton bollworm Helicoverpa armigera responded to the infection of entomopathogenic fungus Beauveria bassiana by activating the Toll pathway. Koinobiont wasps also regulate host immunity and development to ensure the survival of their progeny. Previously, venom protein VRF1 was identified in Microplitis mediator. It enters H. armigera hemocytes, suppresses the expression of antimicrobial peptides (AMPs) by inhibiting the Toll pathway, and prevents parasite offspring from being encapsulated. With this in mind, we thought that it might be feasible to increase the virulence of B. bassiana by embedding VRF1 into its genome. Compared with that of wild-type (WT) B. bassiana, the median lethal dose (LD50) of the transformant expressing VRF1 (named BbVRF1) decreased approximately 2.36-fold, and the median time to lethality (LT50) was shortened to 84% when infecting H. armigera (a natural host of M. mediator). The AMP expression level of hemocytes in H. armigera infected with BbVRF1 strain was significantly downregulated compared to that in the control group infected with the WT. In addition, the LD50 of BbVRF1 against the fall armyworm Spodoptera frugiperda (an unnatural host of M. mediator) was decreased 3.45-fold and the LT50 was shortened to 73%, showing a greater virulence. Our research indicated that BbVRF1, an engineered strain of B. bassiana, has greater efficacy against pest insects both within and outside its host range (M. mediator), expanding the utilization of parasitoid wasp virulence effectors. IMPORTANCE Mycoinsecticides are essential for the development of integrated pest management as substitutes to chemical insecticides, but their usage is limited by their inferior virulence. Thus, genetically engineered bioinsecticides, including recombinant entomopathogenic fungi, have been regarded as a breakthrough to rapidly control pests. Deep knowledge of parasitoid wasps allows us to take advantage of this natural enemy of pest insects beyond raising them for field release. Our transformant BbVRF1 (Beauveria bassiana integrated with a venom protein VRF1 from Microplitis mediator) showed a higher virulence in H. armigera and S. frugiperda, demonstrating its potential for managing natural or unnatural hosts of M. mediator. This result provides a new strategy regarding which venom protein of parasitoid wasps can become part of the arsenal with which to equip entomopathogenic fungi. Utilizing parasitoid wasps with this approach could easily overcome the difficulties of artificial culture and enhance the virulence of other biocontrol agents.

Increased Production of Pathogenic, Airborne Fungal Spores upon Exposure of a Soil Mycobiota to Chlorinated Aromatic Hydrocarbon Pollutants.

Organic pollutants are omnipresent and can penetrate all environmental niches. We evaluated the hypothesis that short-term (acute) exposure to aromatic hydrocarbon pollutants could increase the potential for fungal virulence. Specifically, we analyzed whether pentachlorophenol and triclosan pollution results in the production of airborne fungal spores with greater virulence than those derived from an unpolluted (Control) condition. Each pollutant altered the composition of the community of airborne spores compared to the control, favoring an increase in strains with in vivo infection capacity (the wax moth Galleria mellonella was used as an infection model). Fungi subsisting inside larvae at 72 h postinjection with airborne spore inocula collected in polluted and unpolluted conditions exhibited comparable diversity (mainly within Aspergillus fumigatus). Several virulent Aspergillus strains were isolated from larvae infected with the airborne spores produced in a polluted environment. Meanwhile, strains isolated from larvae injected with spores from the control, including one A. fumigatus strain, showed no virulence. Potential pathogenicity increased when two Aspergillus virulent strains were assembled, suggesting the existence of synergisms that impact pathogenicity. None of the observed taxonomic or functional traits could separate the virulent from the avirulent strains. Our study emphasizes pollution stress as a possible driver of phenotypic adaptations that increase Aspergillus pathogenicity, as well as the need to better understand the interplay between pollution and fungal virulence. IMPORTANCE Fungi colonizing soil and organic pollutants often meet. The consequences of this encounter constitute an outstanding question. We scrutinized the potential for virulence of airborne fungal spores produced under unpolluted and polluted scenarios. The airborne spores showed increased diversity of strains with higher infection capacity in Galleria mellonella whenever pollution is present. Inside the larvae injected with either airborne spore community, the surviving fungi demonstrated a similar diversity, mainly within Aspergillus fumigatus. However, the isolated Aspergillus strains greatly differ since virulence was only observed for those associated with a polluted environment. The interplay between pollution and fungal virulence still hides many unresolved questions, but the encounter is costly: pollution stress promotes phenotypic adaptations that may increase Aspergillus pathogenicity.

The Changes in Mitochondrial Morphology and Physiology Accompanying Apoptosis in Galleria mellonella (Lepidoptera) Immunocompetent Cells during Conidiobolus coronatus (Entomophthorales) Infection.

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, their role in insects is not fully understood; thus, more indepth studies of insect cell apoptosis are necessary. The present study investigates mitochondrial involvement during Conidiobolus coronatus-induced apoptosis in Galleria mellonella hemocytes. Previous research has shown that fungal infection could induce apoptosis in insect hemocytes. Our findings indicate that mitochondria undergo several morphological and physiological changes during fungal infection, e.g., loss of mitochondrial membrane potential, megachannel formation, disturbances in intracellular respiration, increased nonrespiratory oxygen consumption in mitochondria, decreased ATP-coupled oxygen consumption and increased non-ATP-coupled oxygen consumption, decreased extracellular and intracellular oxygen consumption, and increased extracellular pH. Our findings confirm that G. mellonella immunocompetent cells demonstrate Ca2+ overload in mitochondria, translocation of cytochrome c-like protein from mitochondrial to cytosol fraction, and higher activation of caspase-9-like protein after C. coronatus infection. Most importantly, several of the changes observed in insect mitochondria are similar to those accompanying apoptosis in mammalian cells, suggesting that the process is evolutionarily conserved.

Using the Galleria mellonella burn wound and infection model to identify and characterize potential wound probiotics.

Burn wound infection is the leading cause of mortality among burn wound patients. One of the most commonly isolated bacterial burn wound pathogens is Pseudomonas aeruginosa, a notorious nosocomial multidrug-resistant pathogen. As a consequence of its recalcitrance to frontline antibiotic therapy, there is an urgent need to develop alternative treatment avenues to tackle this pathogen. One potential alternative infection prevention measure is to seed the wound bed with probiotic bacteria. Several species of Lactobacillus, a common commensal bacterium, have been previously reported to display growth inhibition activity against wound pathogens. Various species of this genus have also been shown to augment the wound healing process, which makes it a promising potential therapeutic agent. Due to the complexity of the burn wound trauma and burn wound infection, an in vivo model is required for the development of novel therapeutics. There are multiple in vivo models that are currently available, the most common among them being the murine model. However, mammalian burn wound infection models are logistically challenging, do not lend themselves to screening approaches and come with significant concerns around ethics and animal welfare. Recently, an invertebrate burn wound and infection model using G. mellonella has been established. This model addresses several of the challenges of more advanced animal models, such as affordability, maintenance and reduced ethical concerns. This study validates the capacity of this model to screen for potential wound probiotics by demonstrating that a variety of Lactobacillus spp. can limit P. aeruginosa burn wound infection and improve survival.