Greater Wax Moth Galleria Mellonella Research Articles

Food quality affects the expression of antimicrobial peptide genes upon simulated parasite attack in the larvae of greater wax moth

AbstractPredator‐prey interactions are an important evolutionary force affecting the immunity of the prey. Parasitoids and mites pierce the cuticle of their prey, which respond by activating their immune system against predatory attacks. Immunity is a costly function for the organism, as it often competes with other life‐history traits for limited nutrients. We tested whether the expression of antimicrobial peptides (AMP) of the larvae of the greater wax moth Galleria mellonella (L.) (Lepidoptera: Pyralidae) changes as a consequence of insertion of a nylon monofilament, which acts like a synthetic parasite. The treatment was done for larvae grown on a high‐quality vs. a low‐quality diet. The expression of Gloverin and 6‐tox were upregulated in response to the insertion of the nylon monofilament. The expression of 6‐tox, Cecropin‐D, and Gallerimycin were significantly higher in the ‘low‐quality diet’ group than in the ‘high‐quality diet’ group. As food quality seems to affect AMP gene expression in G. mellonella larvae, it should always be controlled for in studies on bacterial and fungal infections in G. mellonella.

Improving anti-trypanosomal activity of alkamides isolated from Achillea fragrantissima

In previous studies the aerial parts of Achillea fragrantissima were found to have substantial antileishmanial and antitrypanosomal activity. A bioassay-guided fractionation of a dichloromethane extract yielded the isolation of the essential anti-trypanosomal compounds of the plant. Seven sesquiterpene lactones (including Achillolide-A), two flavonoids, chrysosplenol-D and chrysosplenetine, and four alkamides (including pellitorine) were identified. This is the first report for the isolation of the sesquiterpene lactones 3 and 4, chrysosplenetine and the group of alkamides from this plant. Bioevaluation against Trypanosoma brucei brucei TC221 (T.b brucei) using the Alamar-Blue assay revealed the novel alkamide 13 to have an IC50 value of 40.37μM. A compound library, derived from the alkamide pellitorine (10), was synthesized and bioevaluated in order to find even more active substances. The most active compounds 26 and 27 showed activities in submicromolar concentrations and selectivity indices of 20.1 and 45.6, respectively, towards macrophage cell line J774.1. Toxicity of 26 and 27 was assessed using the greater wax moth Galleria mellonella larvae as an in vivo model. No significant toxicity was observed for the concentration range of 1.25–20mM.

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.

Activity of the Greater Wax Moth Galleria mellonella L. and the Lesser Wax Moth Achroia grisella F. in Apiary and Storage in Kafr El-Sheikh Province

Activity of the Greater Wax Moth Galleria mellonella L. and the Lesser Wax Moth Achroia grisella F. in Apiary and Storage in Kafr El-Sheikh Province

Sex-specific compensatory growth in the larvae of the greater wax moth Galleria mellonella.

Deficiency of food resources in ontogeny is known to prolong an organism's developmental time and affect body size in adulthood. Yet life-history traits are plastic: an organism can increase its growth rate to compensate for a period of slow growth, a phenomenon known as 'compensatory growth'. We tested whether larvae of the greater wax moth Galleria mellonella can accelerate their growth after a fast of 12, 24 or 72h. We found that a subgroup of female larvae showed compensatory growth when starved for 12h. Food deficiency lasting more than 12h resulted in longer development and lower mass gain. Strength of encapsulation reactions against a foreign body inserted in haemocoel was the weakest in females that showed compensatory growth, whereas the strongest encapsulation was recorded in the males and females that fasted for 24 and 72h. More specifically, we found sex-biased immune reactions so that females had stronger encapsulation rates than males in one group that fasted for 72h. Overall, rapidly growing females had a short larval development period and the shortest adult lifespan. These results suggest that highly dynamic trade-offs between the environment, life-history traits and sex lead to plasticity in developmental strategies/growth rates in the greater wax moth.

Larval instars and development of the greater wax moth Galleria mellonella (Lepidoptera, Pyralidae)

Larval instars and development of the greater wax moth Galleria mellonella (Lepidoptera, Pyralidae)

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.

Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells.

Antimicrobial proteins and peptides (AMPs) are valuable as leads in the pharmaceutical industry for the development of novel anti-infective drugs. Here we describe the efficient heterologous expression and basic characterization of a Gloverin-family AMP derived from the greater wax moth Galleria mellonella. Highly productive single-cell clones prepared by limiting dilution achieved a 100% increase in productivity compared to the original polyclonal Drosophila melanogaster S2 cell line. Comprehensive screening for suitable expression conditions using statistical experimental designs revealed that optimal induction was achieved using 600µM CuSO4 at the mid-exponential growth phase. Under these conditions, 25mg/L of the AMP was expressed at the 1-L bioreactor scale, with optimal induction and harvest times ensured by dielectric spectroscopy and the online measurement of optical density. Gloverin was purified from the supernatant by immobilized metal ion affinity chromatography followed by dialysis. In growth assays, the purified protein showed specific antimicrobial activity against two different strains of Escherichia coli.

Studies on the Virome of the Entomopathogenic Fungus Beauveria bassiana Reveal Novel dsRNA Elements and Mild Hypervirulence.

The entomopathogenic fungus Beauveria bassiana has a wide host range and is used as a biocontrol agent against arthropod pests. Mycoviruses have been described in phytopathogenic fungi while in entomopathogenic fungi their presence has been reported only rarely. Here we show that 21.3% of a collection of B. bassiana isolates sourced from worldwide locations, harbor dsRNA elements. Molecular characterization of these elements revealed the prevalence of mycoviruses belonging to the Partitiviridae and Totiviridae families, the smallest reported virus to date, belonging to the family Narnaviridae, and viruses unassigned to a family or genus. Of particular importance is the discovery of members of a newly proposed family Polymycoviridae in B. bassiana. Polymycoviruses, previously designated as tetramycoviruses, consist of four non-conventionally encapsidated capped dsRNAs. The presence of additional non-homologous genomic segments in B. bassiana polymycoviruses and other fungi illustrates the unprecedented dynamic nature of the viral genome. Finally, a comparison of virus-free and virus-infected isogenic lines derived from an exemplar B. bassiana isolate revealed a mild hypervirulent effect of mycoviruses on the growth of their host isolate and on its pathogenicity against the greater wax moth Galleria mellonella, highlighting for the first time the potential of mycoviruses as enhancers of biocontrol agents.

PATHOGENICITY, PROGENY PRODUCTION AND HISTOPATHOLOGICAL EFFECTS OF THREE ENTOMOPATHOGENIC NEMATODES IN THE GREATER WAX MOTH, GALLERIA MELLONELLA (L.)

Entomopathogenic nematodes (EPNs) of the genera Steinernema and are associated with bacterial symbionts of the genus Xenorhabdus and Photorhabdus, respectively. They are being produced commercially and used as biological control agents against many soil insect pests. The present study was carried out to investigate the effectiveness of entomopathogenic nematode species Heterorhabditis bacteriophora, Steinernema carpocapsae and Steinernema riobravis against the full-grown larvae of greater wax moth Galleria mellonella. It was found that the mortality percentage increased as the concentration of the infective juveniles (IJs) increased. Generally, H. bacteriophora was more effective than S. carpocapsae and S. riobravis. The production rate increased as the concentration of IJs increased. H. bacteriophora was the highest reproductive strain than S. carpocapsae and S. riobravis at the five tested concentrations. The three nematode species affected the muscles and fat tissues, where muscle fibers suffered destruction with some fragmentation, also complete destruction and disintegration of tissues was noticed in the treated G. mellonella larvae.

Management of the Greater Wax Moth Galleria mellonella with Neem Azal- T/S, in the Laboratory and under Semi-Field Conditions

Abstract Different concentrations of Neem Azal-T/S were used in an artificial diet, to study the mortality of the greater wax moth Galleria mellonella. A Neem formulation and different ages of natural beeswax combs were used for the effective management of the wax moth. While the diet was being prepared, Neem Azal-T/S was directly added ensure that the Neem formulation was distributed evenly in the diet at concentrations of 0.25, 0.5, 1, 2, and 4%. The obtained results showed that the different concentrations of Neem Azal-T/S mixed in the prepared artificial diet had a significant efficacy against the tested 2nd instar larvae. An outstanding elongation of the 2nd larval instar was clear in comparison with the control, even at the least tested concentration (0.25%). Neem Azal-T/S at 4, 2,1, and 0.5% caused 100% mortality for all tested larvae. When using a 4% concentration, all the tested larvae died in the 2nd instar. However, when using a 2% concentration, the larvae died in the fifth instar. When using a decreased concentration of 1.0 and 0.5%, some of the larvae were tolerant and lived till the 6th instar. Feeding the larvae on beeswax combs treated with 2% Neem Azal-T/S, caused 100% mortality when fed on very old wax. When the diet was old wax treated with 2% Neem Azal-T/S, a 91% mortality was recorded. When the diet was new wax treated with 2% Neem Azal-T/S, a 90% mortality was recorded. A 4% Neem formulation caused mortality for all larvae during the first week of treatment on the different tested ages of beeswax combs.

Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells

The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and β-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.

Effects of Antiviral Agent, Acyclovir, on the Biological Fitness of Galleria mellonella (Lepidoptera: Pyralidae) Adults.

The effects of a synthetic purine nucleoside analog, antiviral agent, acyclovir (ACV), on adult longevity, fecundity, and hatchability of a serious honeycomb pest, greater wax moth Galleria mellonella L. were investigated by adding 0.01, 0.1, 1.0, and 3.0% ACV into artificial and natural diets. Control larvae were reared on diet without ACV. The artificial diet containing the lowest level of ACV, 0.01%, raised egg production from a number of 12.9 ± 0.6 to 163.2 ± 1.3. The hatching rate of these eggs was increased from 49.2 ± 2.4% to 68.2 ± 3.2%. Higher concentrations of ACV in natural food significantly increased both egg production and egg hatching rate. Female reared on old dark combs as natural diet exposed to 1.0% of ACV produced 167.5 ± 5.8 eggs with 93.2 ± 6.8% hatched. This study emphasizes the importance of determining the dietary impact of an antimicrobial agent as a food additive to a particular species of insect before its using for dietary antimicrobial purpose.

Combination therapy with polymyxin B and netropsin against clinical isolates of multidrug-resistant Acinetobacter baumannii.

Polymyxins are last-resort antibiotics for treating infections of Gram-negative bacteria. The recent emergence of polymyxin-resistant bacteria, however, urgently demands clinical optimisation of polymyxin use to minimise further evolution of resistance. In this study we developed a novel combination therapy using minimal concentrations of polymyxin B. After large-scale screening of Streptomyces secondary metabolites, we identified a reliable polymixin synergist and confirmed as netropsin using high-pressure liquid chromatography, nuclear magnetic resonance, and mass spectrometry followed by in vitro assays using various Gram-negative pathogenic bacteria. To evaluate the effectiveness of combining polymixin B and netropsin in vivo, we performed survival analysis on greater wax moth Galleria mellonella infected with colistin-resistant clinical Acinetobacter baumannii isolates as well as Escherichia coli, Shigella flexineri, Salmonella typhimuruim, and Pseudomonas aeruginosa. The survival of infected G. mellonella was significantly higher when treated with polymyxin B and netropsin in combination than when treated with polymyxin B or netropsin alone. We propose a netropsin combination therapy that minimises the use of polymyxin B when treating infections with multidrug resistant Gram-negative bacteria.

Descriptions of four known species of the families Panagrolaimidae and Alloionematidae (Nematoda: Rhabditida) from Iran

Three species of the family Panagrolaimidae, namely: Propanagrolaimus filiformis, Panagrolaimus orientalis and P. rigidus, and one species of the family Alloionematidae, Rhabditophanes schneideri, were collected from the provinces of Kerman and Mazandaran, Iran. P. rigidus and R. schneideri were isolated from the greater wax moth Galleria mellonella. The descriptions, measurements and illustrations are provided for all species. Molecular analysis of P. rigidus based on sequences of the 18S rDNA places it close to P. davidi (HQ270131) and P. papillosus (KF011489) and two unidentified Panagrolaimus species (EU040129; KC522708). It is, however, separated from other P. rigidus populations sequenced previously. All species, except P. rigidus, are reported for the first time from Iran.http://zoobank.org/urn:lsid:zoobank.org:pub:2A83F627-73CF-4489-9A6E-B2BB52086ED4

Pathogenicity of Sudan Isolates of Bacillus spp to the Greater Wax Moth Galleria mellonella L.

Pathogenicity of Sudan Isolates of Bacillus spp to the Greater Wax Moth Galleria mellonella L.

Backbone and side chain chemical shift assignments of apolipophorin III from Galleria mellonella.

Apolipophorin III, a 163 residue monomeric protein from the greater wax moth Galleria mellonella (abbreviated as apoLp-IIIGM), has roles in upregulating expression of antimicrobial proteins as well as binding and deforming bacterial membranes. Due to its similarity to vertebrate apolipoproteins there is interest in performing atomic resolution analysis of apoLp-IIIGM as part of an effort to better understand its mechanism of action in innate immunity. In the first step towards structural characterization of apoLp-IIIGM, 99 % of backbone and 88 % of side chain (1)H, (13)C and (15)N chemical shifts were assigned. TALOS+ analysis of the backbone resonances has predicted that the protein is composed of five long helices, which is consistent with the reported structures of apolipophorins from other insect species. The next stage in the characterization of apoLp-III from G. mellonella will be to utilize these resonance assignments in solving the solution structure of this protein.

Immune reactions of the greater wax moth, Galleria mellonella L. (lepidoptera, pyralidae) larvae under combined treatment of the entomopathogens Cordyceps militaris (L.: Fr.) Link and Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota, Hypocreales)

The synergistic effect in mortality of the greater wax moth Galleria mellonella larvae was recorded after combined treatment with the entomopathogenic fungi Cordyceps militaris (L.: Fr.) Link and Beauveria bassiana (Bals.-Criv.) Vuill. Treatment with C. militaris resulted in development arrest and some changes in immune response (a sharp decrease in the total hemocyte counts and encapsulation rate and an increase in phenoloxidase activity in the hemolymph) which were accompanied by higher susceptibility to B. bassiana. The larvae killed by combined treatment with two pathogens were colonized only by B. bassiana. The mechanisms of synergism under combined treatment of the greater wax moth with C. militaris and B. bassiana are discussed.

Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model

For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R2 = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.

A Madurella mycetomatis Grain Model in Galleria mellonella Larvae.

Eumycetoma is a chronic granulomatous subcutaneous infectious disease, endemic in tropical and subtropical regions and most commonly caused by the fungus Madurella mycetomatis. Interestingly, although grain formation is key in mycetoma, its formation process and its susceptibility towards antifungal agents are not well understood. This is because grain formation cannot be induced in vitro; a mammalian host is necessary to induce its formation. Until now, invertebrate hosts were never used to study grain formation in M. mycetomatis. In this study we determined if larvae of the greater wax moth Galleria mellonella could be used to induce grain formation when infected with M. mycetomatis. Three different M. mycetomatis strains were selected and three different inocula for each strain were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 4 mg/larvae. Larvae were monitored for 10 days. It appeared that most larvae survived the lowest inoculum, but at the highest inoculum all larvae died within the 10 day observation period. At all inocula tested, grains were formed within 4 hours after infection. The grains produced in the larvae resembled those formed in human and in mammalian hosts. In conclusion, the M. mycetomatis grain model in G. mellonella larvae described here could serve as a useful model to study the grain formation and therapeutic responses towards antifungal agents in the future.