Purpureocillium jiangxiense sp. nov.: Entomopathogenic Effects on Ostrinia furnacalis and Galleria mellonella.

The genus Purpureocillium is renowned for its role in biocontrol and biotechnological applications. The identification of new species within this genus is crucial for broadening our understanding of its ecological roles and potential utility in sustainable agriculture. This study aimed to characterize a new species of Purpureocillium, isolated from soil in eastern China, and to evaluate its bioactivity against Ostrinia furnacalis (corn moth) and Galleria mellonella (greater wax moth). We utilized morphological characterization; molecular phylogenetic analysis employing ITS, nrLSU, and tef1 genes; and bioactivity assays to identify and characterize the new species. The newly identified species, Purpureocillium jiangxiense sp. nov., displays unique morphological and genetic profiles compared to known species. Bioactivity tests showed that this species exhibits inhibitory effects against O. furnacalis and G. mellonella, highlighting its potential in biocontrol applications. By the ninth day at a spore concentration of 1 × 108 spores/mL, the mortality rate of the corn moth and greater wax moth reached 30% to 50% respectively. The discovery of P. jiangxiense sp. nov. adds to the genetic diversity known within this genus and offers a promising candidate for the development of natural biocontrol agents. It underscores the importance of continued biodiversity exploration and the potential for natural solutions in pest and disease management.

Analysis of comb-gnawing behavior in Apis cerana cerana (Hymenoptera: Apidae).

Apis cerana cerana exhibits a prominent biological trait known as comb gnawing. In this study, gnawed combs from colonies during different seasons were collected, investigating the comb age and locations of gnawing. Patterns of comb gnawing were recorded, and the effects of 2 factors, namely, comb type and season, on the mass of wax residues and the gnawed surface area were measured. The results revealed that A. c. cerana predominantly gnaws combs that have been used for over 6 months, with gnawing concentrated in the brood-rearing area. In the first 3 seasons, significantly higher masses of wax residues and larger gnawed surface areas were found in greater wax moth larvae (GWML)-infested combs compared to newly built and old combs. Also, there were significantly higher masses and areas gnawed by A. c. cerana in old combs compared to newly built combs in all 4 seasons. Compared to other seasons, it exhibited significantly higher masses and areas resulting from comb-gnawing in newly built or old combs in winter. However, there were no significant differences in the masses of wax residues and surface areas gnawed in GWML-infested combs across the first 3 seasons. In conclusion, this study documented the impact of comb type and season on the comb-gnawing behavior of A. c. cerana, contributing to beekeeping management practices and the current understanding of bee biology.

Humoral immune response of Galleria mellonella after mono- and co-injection with Hypericum perforatum extract and Candida albicans.

Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co-injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co-injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono-injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono-injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.

Honey Environmental DNA Can Be Used to Detect and Monitor Honey Bee Pests: Development of Methods Useful to Identify Aethina tumida and Galleria mellonella Infestations.

Environmental DNA (eDNA) contained in honey derives from the organisms that directly and indirectly have been involved in the production process of this matrix and that have played a role in the hive ecosystems where the honey has been produced. In this study we set up PCR-based assays to detect the presence of DNA traces left in the honey by two damaging honey bee pests: the small hive beetle (Aethina tumida) and the greater wax moth (Galleria mellonella). DNA was extracted from 82 honey samples produced in Italy and amplified using two specific primer pairs that target the mitochondrial gene cytochrome oxidase I (COI) of A. tumida and two specific primer pairs that target the same gene in G. mellonella. The limit of detection was tested using sequential dilutions of the pest DNA. Only one honey sample produced in Calabria was positive for A. tumida whereas about 66% of all samples were positively amplified for G. mellonella. The use of honey eDNA could be important to establish early and effective measures to contain at the local (e.g., apiary) or regional scales these two damaging pests and, particularly for the small hive beetle, to prevent its widespread diffusion.

Galleria mellonella (greater wax moth) as an eco-friendly in vivo approach for the assessment of the acute toxicity of medicinal plants: Application to some plants from Cameroon.

BACKGROUND: The evaluation of medicinal plants' toxicity is a prerequisite prior to their usage. The vertebrate models used for this purpose are often the object of ethical consideration. Though invertebrate models including Galleria mellonella (GM) have demonstrated the ability to be used to assess the toxicity of various products. To the authors' knowledge, GM has never been exploited to determine the toxicity of medicinal plants. AIM: The aim of this study was to demonstrate the potential of GM larvae as a simple, inexpensive, and rapid model for the evaluation of the toxicity of herbal medicines. METHOD: In this study, the toxicity of aqueous and ethanolic (80%, v/v) extracts of seven well known plants from Cameroon namely leaves of Cymbopogon citratus (DC.) Stapf, Moringa oleifera Lam and Vernonia amygdalina Delile; barks of Cinchona officinalis and Enantia chlorantha Oliv; barks and seeds of Garcinia lucida Vesque and leaves and seeds of Azadirachta indica (Neem) was evaluated using the larval form of GM. The median lethal doses (LD50), 90% (LD90), and 100% (LD100) were determined using the spline cubic survival curves and equations from the data obtained on the survival rate of GM 24 hours after the injection with the extracts. RESULTS: We found that distilled water extracted a more important mass of phytochemical compounds (7.4%-21.2%) compared to ethanolic solution (5.8%-12.4%). LD varied depending on the plant materials and ethanolic extracts (hydroalcoholic extract, (HAE)) were more toxic to GM than aqueous ones. The LD50 (mg/ml) of the tested extracts varied from 4.87 [3.90 g/kg body weight (bw)] to >200 (> 166.67 g/kg bw), the LD90 (mg/ml) from 25.00 (18.52 g/kg bw) to >200 (> 181.82 g/kg bw) and LD100 (mg/ml) from 45.00 (40.91 g/kg bw) to > 200 (>181.82 g/kg bw). The HAE of A. indica seed and C. officinalis bark exhibited the highest toxicity with LD50 (g/kg bw) of 3.90 and 4.81, respectively. CONCLUSION: The results obtained in this study suggest that GM can be used as a sensitive, reliable, and robust eco-friendly model to gauge the toxicity of medicinal plants. Thus, avoid the sacrifice of vertebrate models often used for this purpose to limit ethical concerns.

In vitro degradation of low-density polyethylene by new bacteria from larvae of the greater wax moth, Galleria mellonella.

Three bacterial species isolated from whole body extracts of the greater wax moth larvae, Galleria mellonella, were evaluated for their ability to utilize low-density polyethylene (LDPE) as a sole carbon source in vitro. These bacteria were identified as Lysinibacillus fusiformis, Bacillus aryabhattai, and Microbacterium oxydans. Their ability to biodegrade LDPE was assessed by growth curves, cell biomass production, polyethylene (PE) weight loss, and the presence of LDPE hydrolysis products in the growth media. Consortia of these bacteria with three other bacteria previously shown to degrade LDPE (Cupriavidus necator H16, Pseudomonas putida LS46, and Pseudomonas putida IRN22) were also tested. Growth curves of the bacteria utilizing LDPE as a sole carbon source revealed a peak in cell density after 24 h. Cell densities declined by 48 h but slowly increased again to different extents, depending on the bacteria. Incubation of LDPE with bacteria isolated from greater wax moth larvae had significant effects on bacterial cell mass production and weight loss of LDPE in PE-containing media. The bacterial consortia were better able to degrade LDPE than were the individual species alone. Gas chromatographic analyses revealed the presence of linear alkanes and other unknown putative LDPE hydrolysis products in some of bacterial culture media.

Overview of rearing and testing conditions and a guide for optimizing Galleria mellonella breeding and use in the laboratory for scientific purposes.

The greater wax moth Galleria mellonella is an increasingly popular and consolidated alternative infection model to assess microbial virulence and the effectiveness of antimicrobial compounds. The lack of G. mellonella suppliers aiming at scientific purposes and a lack of well-established protocols for raising and testing these animals may impact results and reproducibility between different laboratories. In this review, we discuss the state of the art of rearing the larvae in situ, providing an overview of breeding and testing conditions commonly used and their influence on larval health and experiments results, from setting up the environment, providing the ideal diet, understanding the effects of pretreatments, choosing the best testing conditions, to exploring the most from the results obtained. Meanwhile, we guide the reader through the most practical ways of dealing with G. mellonella to achieve successful experiments.

Impact of polyethylene on salivary glands proteome in Galleria melonella.

Polyethylene pollutions are considered inert in nature and adversely affect the entire ecosystem. Larvae of greater wax moth (Galleria mellonella) have the ability to masticate and potentially biodegrade polyethylene films at elevated rates. The wax moth has been thought to metabolize PE independently of gut flora, however the role of the microbiome is poorly understood and degradation by the wax moth might be involved. To determine whether the salivary glands of the wax moth were potentially involved in the PE degradation, it was investigated how surface changes of polyethylene were affected by mastication and consumption. Formation of pitting and degradation intermediates including carbonyl groups, indicated that salivary glands could assist in polyethylene degradation. We investigated the biochemical effect of exposure by PE on the composition of the salivary gland proteome. The expression of salivary proteins was found to be affected by PE exposure. The proteins that were significantly affected by the exposure to PE revealed that the wax moth are undergoing general changes in energy levels, also enzymatic pathways associated to fatty acid beta oxidation during consumption to PE were induced.

Immune response of Galleria mellonella after injection with non-lethal and lethal dosages of Candida albicans.

The immune response of Galleria mellonella to injection with non-lethal and lethal dosages of Candida albicans was compared. Larvae infected with the non-lethal dosage (2 × 104 cells/larva) did not show significant morphological changes, while those infected with the lethal dosage (2 × 105 cells/larva) showed inhibition of motility and cocoon formation and became darker around the area of injection after 24 h. While the administration of the lower dosage caused approx. 5- and 20-fold induction of genes for gallerimycin and galiomycin, respectively, the injection with the higher dosage induced approx. 25 and 120-fold expression of the respective genes. Similar differences were obtained for the insect metalloproteinase inhibitor (IMPI) and hemolin gene transcripts. The relatively low level of immune gene expression was confirmed by an assay of hemolymph antifungal activity, which was detected only in larvae infected with lethal dosage of C. albicans. Furthermore, greater amounts of immune-inducible peptides were detected in the hemolymph extracts in the same group of larvae. The stronger humoral immune response was not correlated with survival. Phenol oxidase (PO) activity was induced only in the hemolymph of larvae infected with the non-lethal dose; injection of the lethal dose resulted in strong inhibition of this enzyme after 24 h. We showed that PO is susceptible to regulation by immune priming with the non-lethal dose of C. albicans. The activity of this enzyme was enhanced in primed larvae at the time of re-injection. When both primed and non-primed larvae received 2 × 105 cells, the inhibition of PO was stronger in the primed group. G. mellonella infected with the lethal dose of C. albicans died despite the strong induction of humoral defence mechanisms. The priming-enhanced activity of PO was correlated with increased resistance to subsequent infection.

Host-pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana.

The filamentous fungus Beauveria bassiana is a natural pathogen of the greater wax moth Galleria mellonella. Infection with this fungus triggered systemic immune response in G. mellonella; nevertheless, the infection was lethal if spores entered the insect hemocel. We observed melanin deposition in the insect cuticle and walls of air bags, while the invading fungus interrupted tissue continuity. We have shown colonization of muscles, air bags, and finally colonization and complete destruction of the fat body-the main organ responsible for the synthesis of defense molecules in response to infection. This destruction was probably not caused by simple fungal growth, because the fat body was not destroyed during colonization with a human opportunistic pathogen Candida albicans. This may mean that the infecting fungus is able to destroy actively the insect's fat body as part of its virulence mechanism. Finally, we were unable to reduce the extremely high virulence of B. bassiana against G. mellonella by priming of larvae with thermally inactivated fungal spores.

Galleria mellonella Infection Model Identifies Both High and Low Lethality of Clostridium perfringens Toxigenic Strains and Their Response to Antimicrobials.

Research progress into mechanisms of the anaerobe Clostridium perfringens and associated diseases has been frustrated by the lack of reliable infection models. Wax moth larvae (Galleria mellonella) have emerged as a viable alternative to other models of infection since they are economic, survive at 37°C and require no specialist equipment. This study aims to establish to what extent G. mellonella larvae can be used to study the virulence of C. perfringens strains and its suitability for studying novel treatment strategies by an improved time-lapse approach to data collection. Mortality and morbidity rates of larvae challenged with 105 CFU of C. perfringens isolates from various sources were observed over 72 h and dose response data obtained. Phenoloxidase enzyme activity was investigated as a marker for immune response and tissue burden assessed by histopathological techniques. Results demonstrate that C. perfringens is pathogenic toward G. mellonella although potency varies dramatically between C. perfringens isolates and the reference strain ATCC 13124 was shown to be avirulent. Infection with C. perfringens strains activated the melanisation pathway resulting in melanin deposition but no increase in enzyme activity was observed. Efficacy of antibiotic therapy (penicillin G, bacitracin, neomycin, and tetracycline) administered parenterally to some extent correlates with that of in vitro analysis. The findings suggest G. mellonella might be a useful in vivo model of infection and convenient as a pre-screening assay for virulence of C. perfringens strains or as a simple, cheap and rapid in vivo assay in the first stage development of novel therapeutics against anaerobes. HIGHLIGHTS: -Potential novel in vivo model for the study of Clostridium perfringens infection.-Novel time-lapse approach to data collection.-First report of the pathogenicity of C. perfringens toward G. mellonella.-First report of the efficacy of antibiotic therapy in response to C. perfringens infection in G. mellonella.

The microbiome of the invertebrate model host Galleria mellonella is dominated by Enterococcus.

BACKGROUND: The popularity of Galleria mellonella as invertebrate model is increasing rapidly, because it forms an attractive alternative to study bacterial, fungal and viral infections, toxin biology, and to screen antimicrobial drugs. For a number of vertebrate and invertebrate animal and plant models, it has been established that the commensals present within the microbial communities on various host surfaces will influence the host's immune and growth development state and the colonization capacity of newly introduced micro-organisms. The microbial communities of Galleria mellonella larvae have, however, not yet been well characterized. RESULTS: In this study, we present the bacterial communities that were found by 16S rRNA amplicon sequencing on different body sites of G. mellonella larvae. These communities showed very little diversity and were mostly dominated by one Enterococcus taxon. In addition, we found that the production conditions (as 'bait' for fishing or under more controlled 'research grade' conditions - with or without hormones and antibiotics) appear to have little impact on the microbiota of the larvae. CONCLUSIONS: Establishment of the simplicity of the microbiota of G. mellonella larvae underlines the potential of the larvae as a model host system for microbiome-host interactions.

Oxytetracycline reduces the diversity of tetracycline-resistance genes in the Galleria mellonella gut microbiome.

BACKGROUND: Clinically-relevant multidrug resistance is sometimes present in bacteria not exposed to human-made antibiotics, in environments without extreme selective pressures, such as the insect gut. The use of antibiotics on naïve microbiomes often leads to decreased microbe diversity and increased antibiotic resistance. RESULTS: Here we investigate the impact of antibiotics on the insect gut microbiome by identifying tetracycline-resistance genes in the gut bacteria of greater wax moth (Galleria mellonella) larvae, feeding on artificial food containing oxytetracycline. We determined that G. mellonella can be raised on artificial food for over five generations and that the insects tolerate low doses of antibiotics in their diets, but doses of oxytetracycline higher than sub-inhibitory lead to early larval mortality. In our experiments, greater wax moth larvae had a sparse microbiome, which is consistent with previous findings. Additionally, we determined that the microbiome of G. mellonella larvae not exposed to antibiotics carries a number of tetracycline-resistance genes and some of that diversity is lost upon exposure to strong selective pressure. CONCLUSIONS: We show that G. mellonella larvae can be raised on artificial food, including antibiotics, for several generations and that the microbiome can be sampled. We show that, in the absence of antibiotics, the insect gut microbiome can maintain a diverse pool of tetracycline-resistance genes. Selective pressure, from exposure to the antibiotic oxytetracycline, leads to microbiome changes and alteration in the tetracycline-resistance gene pool.

The complete mitochondrial genome sequence of the greater wax moth Galleria mellonella (Insecta, Lepidoptera, Pyralidae): sequence and phylogenetic analysis comparison based on whole mitogenome.

The larva of Galleria mellonella is widely used as a model organism for in vivo toxicology and pathogenicity testing. Here, we report complete sequence of the mitochondrial genome (mitogenome) from G. mellonella, which is comprised of 15,320 base pairs encoding 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and an A + T rich region. The overall base composition was G + C: 19.6%, A + T: 80.4%, with an apparent AT bias. Phylogenetic analysis using whole mitogenome revealed that G. mellonella was closely related to Corcyra cephalonica, which is in the same Pyralidae family.

Galleria mellonella: An invertebrate model to study pathogenicity in correctly defined fungal species.

The high mortality rates and economic burden associated with fungal infections, plus the emergence of fungal strains resistant to antifungal drugs, make it necessary to get a deeper understanding of fungal pathogenesis, as well as to identify new target structures for antifungal drug development. Still, murine models are considered as the gold standard for studying pathogenesis, quantifying virulence, and analysing the efficacy of antifungal drugs. However, invertebrates, such as the larvae of the greater wax moth Galleria mellonella, are promising alternative hosts to address some of these questions, especially when a large number of fungal strains need to be evaluated. The purpose of this review is to summarize the benefits and drawbacks, explain the utilization of the invertebrate model host G. mellonella, and compare the virulence potential of the most important human fungal pathogens, with the focus on different virulence potential of closely related species.

Morphology and its underlying genetic regulation impact the interaction between Cryptococcus neoformans and its hosts.

Cryptococcus neoformans is a fungus that causes the majority of fatal cryptococcal meningitis cases worldwide. This pathogen is capable of assuming different morphotypes: yeast, pseudohypha, and hypha. The yeast form is the most common cell type observed clinically. The hyphal and pseudohyphal forms are rarely observed in the clinical setting and are considered attenuated in virulence. However, as a ubiquitous environmental pathogen, Cryptococcus interacts with various organisms, and it is known to be parasitic to different hosts. Capitalizing on recent discoveries, morphogenesis regulators were manipulated to examine the impact of cell shape on the cryptococcal interaction with three different host systems: the soil amoeba Acanthamoeba castellanii (a protist), the greater wax moth Galleria mellonella (an insect), and the murine macrophage cell line J774A.1 (mammalian cells). The regulation of Ace2 and morphogenesis (RAM) pathway is a highly conserved pathway among eukaryotes that regulates cytokinesis. Disruption of any of five RAM components in Cryptococcus renders cells constitutively in the pseudohyphal form. The transcription factor Znf2 is the master activator of the yeast to hyphal transition. Deletion of ZNF2 locks cells in the yeast form, while overexpression of this regulator drives hyphal growth. Genetic epistasis analyses indicate that the RAM and the Znf2 pathways regulate distinct aspects of cryptococcal morphogenesis and independently of each other. These investigations using the Cryptococcus RAM and ZNF2 mutants indicate that cell shape, cell size, and likely cell surface properties weigh differently on the outcome of cryptococcal interactions with different hosts. Thus, certain traits evolved in Cryptococcus that are beneficial within one host might be detrimental when a different host is encountered.

Mass Production of the Entomogenous Nematode Heterorhabditis heliothidis (Nematoda: Heterorhabditidae) on Artificial Media.

Heterorhabditis heliothidis is reared monoxenically on an artificial medium consisting of commercially available nutrient broth, yeast extract, and vegetable oil. These components are cooked with flour and coated onto polyether polyurethane sponge, autoclaved, inoculated with a suspension of the bacterial symbiont of the nematode, and incubated at 25 C for 3 d. The bacterial garden on sponge provides an excellent rearing medium. Up to 10 million infective juveniles are produced per 250 ml rearing flask in one month.