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The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of Staphylococcus aureus SrtA (SaSrtA) and Streptococcus pyogenes SrtA (SpSrtA) in vitro at low micromolar concentrations. According to our X-ray crystal structure of the SpSrtAΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the thiol group of Cys208 in the active site of SpSrtA. Importantly, ML346 significantly attenuated the virulence phenotypes of S. aureus and exhibited inhibitory effects on Galleria mellonella larva infection caused by S. aureus. Collectively, our results indicate that ML346 has potential for development as a covalent antivirulence agent for treating S. aureus infections, including methicillin-resistant S. aureus.

Today, the widespread use of silicon dioxide nanoparticles (SiO2 NPs) in many consumer products (toothpaste and skin care product etc.) increases the possibility of living beings being exposed to these NPs in nature. As a result, it brings with it the risks of toxic effects that may occur in biological systems. Due to their low cost of production, lack of ethical issues, and ease of culture in lab settings, model insect species such as Galleria mellonella (Lepidoptera: Pyralidae) are used as an alternative experimental model to vertebrate experimental animals in many fields such as nanotoxicity, medicine, and pharmacy. In this study, 22 nm-sized hydrophilic amorphous SiO2 NPs concentrations (2, 10, 25, 40, and 60 µg/10 µl) were force-fed to the sixth instar of G. mellonella larvae. Control group (0) lar vae were force-fed with only ten μl of distilled water. The effects of SiO2 NPs on the pupal and adult developmental times, pupal and adult weights, adult longevity, and lifespan (pupal developmental time + adult developmental time + adult longevity) of G. mellonella were determined. Results showed that treating G. mellonella with 2 and 25 μg/10 µl SiO2 NPs significantly decreased the means of the adult developmental time. Also, the life span of G. mellonella significantly shortened in larvae exposed to the same concentrations of SiO2 NPs (2 and 25 μg/10 µl) compared to the control. Finally, statistically, no significant change was observed in the other biological parameters of the larvae (pupal developmental time, pupal and adult weights, and adult longevity) that were exposed to SiO2 NPs when compared to the control group.

International audience; The invertebrate Galleria mellonella has increasingly and widely been used in the last few years to study complex host-microbe interactions. Aspergillus fumigatus is one of the most pathogenic fungi causing life-threatening diseases in humans and animals. Galleria mellonella larvae has been proven as a reliable model for the analysis of pathogenesis and virulence factors, enable to screen a large number of A. fumigatus strains. This review describes the different uses of G. mellonella to study A. fumigatus and provides a comparison of the different protocols to trace fungal pathogenicity. The review also includes a summary of the diverse mutants tested in G. mellonella, and their respective contribution to A. fumigatus virulence. Previous investigations indicated that G. mellonella should be considered as an interesting tool even though a mammalian model may be required to complete and verify initial data.

Onychomycoses are nail infections caused primarily by dermatophytes fungi, yeasts, and other filamentous fungi, characterized by persistent infections, prolonged therapy, and high recurrence rates. In clinical practice, some of these occurrences present two or more microorganisms, and the interactions among them can change the chemical environment mediated by small diffusible molecules, producing a competitive niche. The aim of this study was to evaluate the antifungal activity of individual extracts of pure cultures of Candida albicans and C. parapsilosis against dermatophytes. To obtain the fungal extracts, cultures were filtered through a 0.2 μm membrane and submitted to liquid-liquid extraction using ethyl acetate. The Minimal Inhibitory Concentration (MIC) of each extract was evaluated by broth microdilution method and checkerboard assay with fluconazole against clinical isolates of Trichophyton rubrum and T. mentagrophytes. The invertebrate model of Galleria mellonella was used to evaluate the toxicity of the extracts. As results, the extracts of C. albicans and C. parapsilosis showed antifungal activity with MICs between 31,2 – 2000 μg/mL. In association with fluconazole, synergistic effect was detected for all combinations. The extracts presented low toxicity in G. mellonella. In the future, isolation and identification of the extract compounds may allow new therapeutic approaches in the control of fungal infections.

In an experiment conducted to evaluate the efficacy of four botanical extracts (Solvent extraction) viz., Sweet flag (Acorus calamus), Turmeric (Curcuma longa), Sweet basil (Ocimum basilicum), Medicinal Coleus (Coleus forskohlii) in comparison with three Essential oils (Steam distillation) viz., Eucalyptus (Eucalyptus globulus), Peppermint (Mentha piperita) and Lemongrass (Cymbopogan citratus) against the greater wax moth, Galleria mellonella, it was observed that Sweet basil, O. basilicum at 5% was found to be effective against G. mellonella with a maximum of 89.29% larval mortality followed by Sweet flag (A. calamus) with 65.71% mortality. Medicinal coleus, (C. forskohlii) with 56.66% mortality and Turmeric (C. longa) with 45.47% mortality in the descending order of efficacy. Among essential oils tried, Peppermint oil (M. piperita) has resulted in 80.24% larval mortality, followed by Eucalyptus oil (E. globulus) with 69.05% mortality and Lemongrass oil, C. citratus (50.48%). The botanical treatment with Sweet basil, O. basilicum (21.81%) resulted in minimum comb weight loss by G. mellonella followed by Sweet flag, A. calamus (29.04%). Among the essential oil, Peppermint oil, M. piperita (24.56%) afforded less comb weight loss, indicating the effectiveness of treatment in controlling the larval damage. Dört botanik ekstraktın (Çözücü ekstraksiyonu) etkinliğini değerlendirmek için yapılan bir deneyde, Tatlı bayrak (Acorus hint kamışı), Zerdeçal (Curcuma longa), Tatlı fesleğen (Ocimum basilicum), tıbbi Coleus (Coleus forskohlii) üç Essential ile karşılaştırıldığında yağları (Steam distilasyon) yani, Okaliptüs (Eucalyptus globulus), Nane (Mentha piperita) ve Limon otu (Cymbopogan citratus) büyük mum güvesi Galleria mellonella'ya mücadelesi için karşılaştırılmıştır. Tatlı fesleğen, O. basilicum'un %5 oranında G. mellonella'ya karşı maksimum %89.29 larva ölüm oranı ile etkili, bunu %65.71 ölüm oranı ile Tatlı bayrak (A. hint kamışı), azalan etkinlik sırasına göre %56.66 ölüm oranı ile tıbbi coleus (C. forskohlii) ve %45.47 ölüm oranı ile Zerdeçal (C. longa) takip etmektedir. Denenen uçucu yağlar arasında Nane yağı (M. piperita) %80.24 larva ölümü ile sonuçlanmıştır. Bunu %69.05 ölüm oranı ile Okaliptüs yağı (E. globulus) ve Limon otu yağı, C. citratus (%50.48) izlemiştir. Tatlı fesleğen, O. basilicum (%21.81) ile yapılan botanik tedavi, G. mellonella tarafından minimum petek ağırlığı kaybı ve ardından Sweet flag, A. kalamus (%29.04) ile sonuçlandı. Uçucu yağlar arasında, Nane yağı, M. piperita (%24.56), daha az petek ağırlığı kaybı sağladı, ve bu da tedavinin larva hasarını kontrol etmedeki etkinliğini göstermektedir.

Beauveria bassiana (Hypocreales: Cordycipitaceae) is an entomopathogenic fungus that has shown promising results as a biocontrol agent of ticks. Locally isolated B. bassiana are better acclimatised to the natural conditions of their geographical origin; therefore, they are essential in developing effective biocontrol agents for ticks. The current study aimed to isolate native strains of B. bassiana that are pathogenic to Rhipicephalus microplus ticks. The virulence of the isolates was tested against R. microplus larvae using a formulation containing 15% avocado oil, 0.05% adjuvant and 108 conidia mL-1. The two best strains were further evaluated for various biological parameters on adult engorged female ticks. Breakthru® or Ballista® (adjuvant) was mixed with the formulation to compare their effect on the isolates' virulence. In total 61 entomopathogenic fungi were isolated from the 360 greater wax moth larvae, Galleria mellonella (Lepidoptera: Pyralidae) used. The virulence test identified Bb-40 and Bb-41 to be the most virulent isolates against R. microplus larvae with mortalities of 91 and 93% and LT50 values of 5.8 and 6.2 days, respectively. Compared to the control, both strains significantly affected all the measured biological parameters. The type of adjuvant also considerably affected the susceptibility of ticks to the fungi. In conclusion, the two isolates combined with adjuvants can be used as a biocontrol agent to control R. microplus.

Since the fruits and leaves of the Olive (Olea europea) tree, which grows naturally in Mediterranean countries, are thought to be beneficial for many diseases, it is widely used in folk medicine in these countries. Olive leaf extract has been used in the treatment of many diseases such as malaria, high fever, heart diseases and cancer. The greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) is frequently used in invertebrate immunity studies due to its similarity to the mammalian natural immune system. Melanization occurs when quinone precursors are converted to melanin and inactive profenoloxidase to phenoloxidase enzyme. In this study, the phenoloxidase activity of G. mellonella hemolymph larvae injected with olive leaf extract in different doses was determined by ELISA microplate reader at 492 nm absorbance. The findings obtained from this study showed that olive leaf extract increased phenoloxidase activity at a dose of 0.001 mg mL-1. Lower doses of 0.001 mg mL-1 did not cause changes in phenoloxidase activity compared to the untreated and Dimethyl Sulfoxide (DMSO) groups. The highest olive leaf extract dose of our study is 0.01 mg mL-1 and the larvae did not survive for 24 hours over those doses. The results of our study show that olive leaf supports humoral immune responses when used in low doses.

Endoparasitoid species devoid of symbiotic viruses inject secretions derived from their reproductive glands into their hosts during parasitism in order to avoid various immune responses of their hosts. Pimpla turionellae L. (Hymenoptera: Ichneumonidae) is an endoparasitoid that lacks polydnaviruses, and its venom has previously been shown to paralyze the host Galleria mellonella (Lepidoptera: Pyralidae) and suppress its immune reactions to ensure the egg survival. The present study demonstrates that another female-injected factor calyx fluid extracted from the P. turionellae ovary is also responsible for the suppression of G. mellonella immunity. The total hemocyte counts of G. mellonella decrease after treatment with calyx fluid in a concentration-dependent manner. Significant reductions in cell viability are also observed at all calyx fluid doses both in vivo and in vitro. The analyses of the beads injected into the insects as encapsulation targets revealed that the number of encapsulated beads reduced significantly compared to controls post-calyx fluid injection. The injection of the highest calyx fluid dose (1 female equivalent calyx) is sufficient to completely inhibit the strong encapsulation and melanization reactions of the last instar larvae 24 h post-injection. These results demonstrate that P. turionellae calyx fluid is required to regulate host immunity for successful parasitization.

Summary Background Enterobacter cloacae complex contains nosocomial pathogens responsible for infection outbreaks. Identification at the species level within the E cloacae complex remains difficult. Using whole genome sequencing, our aim was to decipher the transmission routes that led to the death of six of seven neonates who had bacteraemia caused by E cloacae complex isolates in a neonatal intensive care unit (NICU) over a 13 month period. Methods In this cohort study, E cloacae complex isolates were taken from 186 newborns in an NICU: 14 were clinical samples (eg, blood cultures), 728 rectal swab samples, and 38 environmental samples (20 from siphons, 18 from incubators, and one from a mattress). The samples were analysed to decipher the possible role of manual cross transmission or environmental source in an outbreak of fatal septic shocks related to E cloacae complex bacteraemia. After the replacement of the incubators suspected to be the reservoir of some outbreak-related isolates on Feb 1, 2018, E cloacae complex strains were screened again for 10 months (503 rectal swab samples from 163 newborns). The 71 E cloacae complex isolates recovered from screening, clinical, and environmental samples across both study periods were compared by whole genome sequencing. The pathogenicity of E cloacae complex isolates responsible for fatal septic shocks was assessed using a Galleria mellonella in-vivo model. Findings From Dec 9, 2016, to Jan 31, 2018, 249 (34%) of 728 rectal swab samples were positive for E cloacae complex, with 66 (35%) of 186 newborns colonised. E cloacae complex were also recovered from four (20%) of 20 siphons and 11 (61%) of 18 incubators. During this period, whole genome sequencing identified that the isolates responsible for the six fatal septic shocks were all Enterobacter bugandensis. A G mellonella infection model showed a higher virulence of E bugandensis. Genomic analysis highlighted the role of incubators as a long-term reservoir and source of cross contamination, leading to their replacement on Feb 1, 2018. Following incubator replacement, a 10-month follow-up investigation identified a physiological gut colonisation with polyclonal E cloacae complex in 52 (34%) of 163 neonates within a median of 9 days (5–14), but no E cloacae complex-related septic shocks. Interpretation Despite around 30% of neonates being physiologically colonised with E cloacae complex, fatal sepsis was systematically linked with bacteraemia caused by E bugandensis. Our findings highlight the need for accurate identification methods to detect the hypervirulent species within the E cloacae complex recovered in neonates. Funding Assistance Publique–Hopitaux de Paris.

The occurrence of entomopathogenic nematodes (EPNs) in arable soil samples from Nigeria was investigated using Baermann extraction tray and insect-bait (White’s trap) techniques. Isolates were tested for infectivity using the larvae of Galleria mellonella (greater moth) and Tenebrio molitor (mealworm). The study revealed a new species of Heterorhabditis (MT371593) in soil samples that were randomly collected from an arable farmland cultivated with cassava TMS-30572 at the Teaching and Research Farm of Landmark University, Nigeria. Amplification of the internal transcribed spacer region (ITS) of the ribosomal DNA produced a nucleotide sequence of 933 base pairs (bp). A BLASTN search of GenBank showed that the sequence of the Nigerian isolate is identical at 99% similarity to that of Heterorhabditis sp. from Thailand. Infectivity test of the isolate showed 100% mortality against T. molitor larvae within 48 h of exposure while only 80% mortality was recorded for G. mellonella after 1 week of exposure. This is the first account of Heterorhabditis sp. in Nigeria. The varying degrees of infectivity against mealworm and greater moth observed in this study proved that the Nigerian isolate of Heterorhabditis sp. could potentially be an attractive option in the management of insect pests of cash crops.

This study determined phytochemical composition, antifungal activity and toxicity in vitro and in vivo of Syzygium cumini leaves extract (Sc). Thus, was characterized by gas chromatography coupled to mass spectrometry and submitted to determination of Minimum Inhibitory (MIC) and Fungicidal concentrations (MFC) on reference and clinical strains of Candida spp. and by growth kinetics assays. Toxicity was verified using in vitro assays of hemolysis, osmotic fragility, oxidant and antioxidant activity in human erythrocytes and by in vivo acute systemic toxicity in Galleria mellonella larvae. Fourteen different compounds were identified in Sc, which showed antifungal activity (MIC between 31.25-125 μg/mL) with fungistatic effect on Candida. At antifungal concentrations, it demonstrated low cytotoxicity, antioxidant activity and neglible in vivo toxicity. Thus, Sc demonstrated a promising antifungal potential, with low toxicity, indicating that this extract can be a safe and effective alternative antifungal agent.

In this study, 18-38 nm-sized and spherical-shaped nanopowder Fe3O4 NPs concentrations (0.4, 2, 10, 50, 250 µg/10 µl) was force-fed to sixth instar (180 ± 20 mg) Galleria mellonella (Lepidoptera: Pyralidae) Larvae under laboratory conditions. The effects of magnetic iron oxide nanoparticles (Fe3O4) on the pupal and adult developmental times, pupal and adult weights and adult longevity of G. mellonella were recorded. Results showed that treating G. mellonella with 250 μg/10 µl Fe3O4 NPs significantly increased pupal weights. Additionally, while adult developmental time increased post 250 μg/10 µl Fe3O4 NPs treatment, it was observed that pupal developmental time, pupal and adult weights, and adult longevity were not statistically significantly different when compared to the control. This study was supported by Nevşehir Hacı Bektaş Veli University Scientific Research Projects Unit with the project number ABAP21F9.

Introduction: Although, Rhizopus species, are the main causative agents of mucormycosis, we know a little about these pathogens. We investigated the virulence potential of Rhizopus arrhizus and R. microsporus strains, obtained from a wide selection of clinical and environmental sources, in Galleria mellonella larvae and a mice model. Methods: In this experiment, a total of 26 strains, 13 for each species of R. microsporus, R. arrhizus were tested in larvae with final concentration of 106/ml per strain. Eight strains were tested in mice model. Inoculated samples were monitored in insect larvae for 6 days and in mice for 14 days. Statistical data were performed using Graphpad Prism software (GraphPad Software, San Diego, CA). Survival data of the samples were plotted using Kaplen-Meyer diagrams and analyzed using log rank test P value was considered 0.05%. Results: R. arrhizus showed higher virulence compare to R. microsporus in both models. No specific difference was seen in pathogenicity between the two varieties of R. arrhizus. Virulence was not affected by source of isolation or production of toxin in some strains of R. microsporus. Virulence pattern was similar in both models. Conclusion: Considering the mortality rate which was happened with strains from environmental sources, we conclude that these fungi have an opportunistic nature, which make them pathogen in susceptible hosts in favorite conditions. Larvae model showed reliable results compare to mice model.

Liquid chromatography and MALDI mass spectrometry have been employed to study the effect of 1,1-dimethylhydrazine and its acetone hydrazone on peptide composition of hemolymph of Galleria mellonella caterpillar larvae. It has been found that the following components arise in the hemolymph: induced low-molecular-mass peptide products with masses of 1–3 kDa, fragments and methylated modifications of previously-known antibacterial peptides of Galleria mellonella, as well as six unknown peptides that exhibit bactericidal activity with respect to Gram-negative bacteria Escherichia coli.

Entomopathogenic nematodes (EPNs) are a promising alternative method for insect pest control. The idea of the work is to investigate the effect of gamma irradiation on EPNs pathogenicity under laboratory conditions aiming to enhance their lethal effect against insect pests. Heterorhabditis bacteriophora (Hb) as well as H. indica (Hi) were irradiated directly in nematode suspension and indirectly within the insect cadavers by gamma irradiation dosages of 0, 100, 250, 500, 750 and 1000 Gy.The mortality percentage of nematodes were recorded, the extracted nematodes from the irradiated insect hosts were used to investigate their pathogenicity and reproductivity against Galleria mellonella larvae. The damage of DNA was analyzed using the comet assay and the LC50 of the two nematode species was recorded. The data demonstrated that, the low dosage (100 Gy) enhanced the pathogenicity of EPNs and didn't affect the initial populations (Pi), the final population of emerged IJs (Pf) and the rate of reproduction (Rr%). Moreover, H. bacteriophora was more tolerant to gamma irradiation than H. indica which recorded more DNA damage %, DNA in tail, tail length, tail moment and olive tail moment in the all different irradiated dosages. In conclusion, low dosages of gamma irradiation can be used to enhance the pathogenicity and reproductivity of entomopathogenic nematodes in order to use as a biocontrol agent.

The objective of the present study was to investigate the impact of zinc and copper on some biomarkers in a model organism Galleria mellonella L. We investigated the effects of Cu and Zn (10, 50, and 100 mg/100 g diets) on different biomarkers such as oxidative stress parameters (SOD and CAT activities and MDA levels), energy resources (protein, lipid and glycogen levels), electrolyte contents (Ca, Na, and K levels), total hemocyte count (THC), and growth and development of G. mellonella. Additionally, the accumulation levels of the used metals were also studied. Cu caused a significant decrease in protein, lipid and glycogen levels. SOD and CAT activities significantly increased at all concentrations of Cu, while they significantly increased at only high concentrations of Zn (50 and 100 mg). Lipid peroxidation levels (MDA) significantly elevated at high concentrations of both metals. It was determined that the Cu and Zn accumulation increased depending on the increase of the concentration. Zn caused an alteration in Ca level at the concentrations of 50 and 100 mg, and K and Na levels at all concentrations. While, THC significantly reduced at all Cu concentrations, this reduction was observed only at higher Zn concentrations (50 and 100 mg). Larval and pupal development time significantly extended at the highest concentration (100 mg) of Cu, and females' lifespan significantly shortened at all concentrations of Cu. Zinc caused an extension in larval development time at the highest concentration (100 mg), and caused a shortening in females and males' lifetime at all concentrations. The observed changes in biomarkers can be used as the illustration of potential toxic effects of high levels of Cu and Zn in organisms.

In the present study, one Streptomyces species was isolated from a soil sample that was collected in Khafr El Shikh Governorate. Actinomycetes are prolific producers of antibiotics and important suppliers to the pharmaceutical industry. They can produce a wide range of secondary metabolites. The isolate was purified and characterized by morphological, physiological, biochemical methods and using Scanning electron microscope (SEM). The isolate was found to be Streptomyces catenula like microorganism and was therefore called Streptomyces catenula MY 12. The Streptomyces metabolite was extracted using ethyl acetate solvent. The purified extract was used to study the effect against 3rd instar larvae of the greater wax moth, Galleria mellonella. The study showed deleterious effects on growth and development of G. mellonella larvae. The results demonstrated that the mortality increased with increase in concentration. The extract prolonged the larval—pupal periods and reduced both pupation and percentage of adult emergence. The LC50 and LC90 values were calculated. The consumed food by unirradiated and irradiated 3rd instar larvae of G. mellonella in each concentration was studied. The larvae consumed much media in control treatment and fewer amounts in the higher concentration of the extract. The SEM revealed that different malformation or abnormalities were observed in larval cuticle and associated sensilla in the form of tear and ridged tergum cuticle. Also, broken and fallen or scarcity of trichodea sensilla. Undistinguished segments tear sternum and destroy plural membrane. The spiracles were completely closed, and prolegs crochets disappeared.

Immune responses play a key role in the survival of all living things. Intake of inorganic materials is of critical importance in living organisms because of their participation in metabolism and some component of cells. Iron (Fe), magnesium (Mg) and selenium (Se) were selected for this study because of their participation in critical constituents of cells, such as nucleic acids and enzymes in living organisms, and their indirect and direct effects on immunity. Although the separate effects of these inorganic materials are known, there is no study on their combined effects. Galleria mellonella, which is used as a model organism, is frequently used in evaluating of effects of human pathogens due to its various properties. Total hemocyte counts and encapsulation-melanization responses are the most commonly observed parameters when examining the immune responses of this model organism. In our study, the effects of the selected inorganic materials added in the ratio of 0.1%, 0.25%, 0.5% and 1% in the nutrient content of Galleria mellonella on the immune responses of this organism were investigated. As a result of the study, it was found that only 0.25% of the doses increased the hemocyte count and that the other doses significantly decreased compared to the control group. However, it was determined that 0.5% and 1% feeding caused a significant decrease in both hemocyte count and encapsulation-melanization responses. According to our findings, the nutrients which have over a certain dose by inorganic substances have negative effects on immunity.

The greater wax moth Galleria mellonella is responsible for serious economic losses to bee keepers in developing countries. Larvae feed on the wax comb in weak colonies or during the storage of wax combs in winter. The objective of the current study was to assess the effect of the Cytokinin plant growth regulator, Benzyladenine (6-BA), on the most important parameters of adult performance and reproductive potential of G. mellonella. The 3rd instar larvae were force-fed on an artificial diet supplemented with six concentrations, viz., 100, 10.0, 1.0, 0.1, 0.01 & 0.001ppm of 6-BA. No adult moths could metamorphose at the higher three concentrations because of larval and pupal deaths. The most important results could be summarized as follows. The adult emergence was blocked by 6-BA, in a dose-dependent course. Also, the tested compound displayed strong adulticidal activity, since adult mortality increased in a dose-dependent course. The total adult longevity of moths was significantly prolonged.All longevity compartments were remarkably prolonged. On the other hand, 6-BA failed to affect adult morphogenesis. The oviposition efficiency was deleteriously prohibited. Also, 6-BA exhibited a tremendous inhibitory effect on the female fecundity and reducing effect onfertility. The embryonic development in eggs laid by treated adult females was severely retarded, since the incubation period of these eggs was remarkably prolonged.

The collection of samples from a bushveld area in Brits, North West Province South Africa yielded a discovery of a possible new Steinernema species. Galleria mellonella larvae were used to bait the...

Native entomopathogenic nematodes (EPNs) were evaluated for the management of three selected pests, Sesamia calamistis, Spodoptera frugiperda and Rhynchophorus ferrugineus in laboratory bioassays. The EPNs were isolated from soils from various locations within Ibadan, Nigeria using the greater wax moth (Galleria mellonella) as insect bait. A two-factor laboratory experimental assay was laid out in a completely randomized design and replicated three times. The EPN suspension was applied on insect larvae with distilled water as control. Number of days to mortality, percentage mortality of insect larvae, and total EPN population of infective juveniles (IJs) recovered from larval cadavers were assessed and reproductive factor (RF) determined. Data were analyzed using analysis of variance, and means were separated using Tukey’s Studentized Range Test at P G. mellonella (5075.08), >S. frugiperda (3957.23), >S. calamistis (742.31). From the results, EPN had the greatest fecundity in G. mellonella and R. ferrugineus showing higher ability to be recycled. This study reveals the potential of native EPNs as a biocontrol agent of insect pests and emphasizes the need for a more environment-friendly and sustainable approach to insect pest management.

Summary The slow lethality of fungal biopesticides to insects restrains their widespread application as a strategy of pest control. In this study, unary, binary and ternary transgenic Metarhizium robertsii were created by integrating genes that encode the scorpion neurotoxin BjαIT, the cuticle‐degrading protease Pr1A, and a double‐stranded RNA (dsRNA) that targets host gnbp3, individually or collectively under a constitutive promoter to enhance virulence. Compared with the parental wild type, all unary transgenic strains had increased virulence against four insect species, Tenebrio molitor, Locusta migratoria, Plutella xylostella and Galleria mellonella, whereas the binary transgenic strain expressing both pr1A and BjαIT had increased virulence to T. molitor and L. migratoria, with no change in virulence against P. xylostella and G. mellonella. Importantly, all ternary transgenic strains simultaneously expressing pr1A, BjαIT, and the dsRNA specific to host gnbp3 exhibited the highest increase in insect‐specific virulence. This finding highlights a novel strategy for genetic engineering of dsRNAs that target genes associated with the host immune response alongside virulence genes to maximize fungal virulence and lethality against insect pests., Genetic engineering techniques have been developed to improve fungal virulence for the control of insect pests. Unary, binary and ternary transgenic strains were constructed by integrating the endo‐ or exogenous virulence genes, and the host immunity gene‐targeted individually or collectively into the fungus. The binary transgenic strain was further improved compared with unary transgenic strains in virulence, all ternary transgenic strains were most improved, which highlights a novel strategy of genetic engineering to maximize fungal virulence.

Entomopathogenic nematodes (EPNs) can be used efficiently as biological control agents against specific insects. Galleria mellonella (Gm) and Spodoptera littoralis (Sl) are considered important agricultural pests. The aim of this study was to evaluate the susceptibility of their larvae to ascending levels of four species of EPNs, Steinernema carpocapsae (Sc), S. arenarium (Sa), Heterorhabditis bacteriophora (Hb) and H. indica (Hi) as well as the ability of nematodes to penetrate and reproduce within their hosts. Fifth larval instar of Gm and Slwere used to evaluate the effect of four inoculum doses [200,400,800, and 1600 infective juveniles (IJs)] of each nematode species. Percentage of insect mortality (% M) and the nematode-rate of reproduction (Rr) were calculated after 24 and 48 hrs. The data confirmed that H. bacteriophora was the highest effective species on both insects. The % M was 75.7, 79.7, 82.3, and 83% after 24 hrs and 98.0, 99.3,100 and 100% after 48 hrs in Gm for these doses, respectively. On the other hand, the corresponding mortalities were 75.0, 78.3, 81.7, and 83.8% after 24 hrs and 97.3, 98.3, 100 and 100% after 48 hrs in Sl. The highest initial EPN population that could penetrate the insect (pi) was achieved by HbinGm (295 IJs) at 1600 IJs, while it was 221.7 IJs in Sl larvae at the same inoculum. The highest final population and Rr were obtained at the inoculum level of 200 and 400 IJs. in Gm and Sl, respectively.The study documented that Heterorhabditis and Steinernema can be used effectively in Gmand Sl management. Moreover, Hb is the most suppressive species tested against the two lepidopteran hosts.

Reducing the usage of chemical pesticides used to control pests in agriculture and stored products is an important phenomenon. Also, eco-friendly biopesticides have become highly important for use in the management of the pest control programs to overcome the negative influences of other synthetic chemicals. In this study oxidative stress changes induced by one important phytochemical compound, juglone, in Galleria mellonella larvae were investigated. For this purpose, effective concentrations (LC10: 0.5 mg; LC30: 1.5 mg; LC50: 2.3 mg) of juglone were added into the 2 g diet of the first instar larvae. Hemolymph samples from the last instar larvae were used to analyses of antioxidant enzyme activity (glutathione peroxidases – GPx, glutathione-S transferase - GST, catalase - CAT, and superoxide dismutase - SOD) and malondialdehyde (MDA) assays. Activities of antioxidant enzymes (SOD, CAT, and GST) and MDA level in the hemolymph of last instar G. mellonella larvae exposed to dietary juglone changed dose-dependently when compared to the control larvae. However, there was no change detected in GPx enzyme activity in both treated and untreated larvae. Consequently, effective doses of juglone are toxic to the model and pest insect G. mellonella.

The greater wax moth Galleria mellonella L. is a dangerous pest for the apiculture industry in the world. The objective of the present study was to evaluate the insecticidal activity of the auxin plant growth regulator, Indole-3-acetic acid (IAA), against G. mellonella and its effect on the growth, development, metamorphosis and morphogenesis of this insect pest. The 3rd instar larvae were force-fed on an artificial diet supplemented with six concentrations, viz., 100, 10.0, 1.0, 0.1, 0.01 & 0.001ppm of IAA. The most important results could be summarized as follows. IAA exhibited the strongest acute toxicity on larvae at the higher two concentrations. At other concentrations, various degrees of toxicity on larvae were found in a dose-dependent course. No pupae or adults were produced at the higher two concentrations. IAA failed to display chronic toxicity on the developed pupae. All emerged adults died at 1.00ppm of IAA but no adult mortality was observed at the three lower concentrations. LC50 value was calculated in 0.24 ppm. IAA enhanced the treated larvae to grow with increasing weight gain. Also, it promoted the treated larvae, since the growth rate increased with the increasing concentration. IAA exhibited a retarding effect on the pupation, only at the higher two concentrations. The larval and pupal durations were significantly prolonged, in a dose-dependent manner. On the other hand, IAA failed to disturb the hormonal regulation of the development program, since no intermediate creatures were produced. Also, it failed to disrupt the pupal morphogenesis, since no pupal deformities had been observed.

Background Chitin, a long-chain polymer of N-acetylglucosamine, is a major structural component of the insect exoskeleton and the peritrophic membrane (PM). Chitinases are able to effectively break down glycosidic bonds of chitin polymer thus can be used in agriculture to control plant pathogen insects. These enzymes can be synthesized by higher plants, animals, protista, bacteria, and viruses. Results In this study, viral and bacterial chitinases were compared for their potential activity on a laboratory test insect. The genes encoding chitinases of Autographa californica nucleopolyhedrovirus (AcNPV) and Cydia pomonella granulovirus (CpGV) were amplified from genomic DNAs by PCR and cloned into the pET-28a (+) expression vector. The chitinase proteins of these 2 viruses (AcNPV-Chi, CpGV-Chi) and Serratia marcescens chitinase C (ChiC) protein which was previously cloned were overexpressed in Escherichia coli. Expressed proteins were purified and confirmed by western blot analysis as 50, 63, and 68 kDa for AcNPV, CpGV, and S. marcescens chitinases, respectively. Enzyme activities of the chitinases were confirmed. Chitinases were also compared to each other in silico. The insecticidal effects of these proteins were evaluated on Galleria mellonella L. larvae. Bioassays were performed on the 3rd instar larvae for each chitinase protein in triplicate. The results showed that although there were differences in enzymatic activities and domain organizations, all 3 microbial chitinases produced almost the same level of insecticidal activity on the test insect. LC50 and LT50 values were compatible with the mortality results. These results were a preanalysis for comparing the effects of microbial chitinases. Conclusion Potential activity experiments should be carried out on more insects to provide detailed information on the insecticidal effects of bacterial and viral chitinases.

The wasp, Bracon hebetor Say, is an important potential biocontrol agent of a wide range of lepidopteran insect species. The current study was subjected to compare these ectoparasitoid fitness traits on different host species belonging to the order Lepidoptera. Moreover, we determine the population dynamic with different host densities, sex ratio patterns, super-parasitism, longevity, paralysis success, and reproductive potential under laboratory conditions. Our results revealed that oviposition increased with an increase in host density, while the adult emergence and egg hatching were decreased due to the super-parasitism on host larvae. A higher male and female-biased population were observed when virgin and mated females offered fresh hosts. Adults' longevity was recorded more in females than males when kept only with bee honey + royal jelly + host larvae. The mean duration of egg-adult development was recorded higher on Galleria mellonella and lowest on Busseola fusca. The parasitization rate, super-parasitism, and cumulative fecundity of mated and virgin female wasps with different host species were observed higher on specific host Galleria mellonella while it was lower on Phthorimaea operculella. Furthermore, the parasitoids having mating experience preferred fresh, while the parasitoids' lack of mating experiences preferred paralyzed host under olfactometer test. Besides, this research has produced novel facts on the biology of parasitic wasp, B. hebetor that may guide the advancement of sustainable biological control programs to control lepidopteran pests.

With the increasing uses of biological control methods, knowing the physiological and immunological effects of these insecticides on insects is essential for them to be used safely in agricultural areas. The aim of the study is to determine the effects of pyriproxyfen and Bacillus thuringiensis subsp. kurstaki individually and as a mixture on malondialdehyde levels (MDA), glutathione-s-transferase, acetylcholinesterase, cytochrome P450 enzyme activities in hemolymph, midgut, and fat body and total (THC) and differential hemocyte counts (DHC) of fifth instar larvae of Galleria mellonella (L., 1758) (Lepidoptera: Pyralidae) 24, 48 and 72 h after exposure under laboratory conditions (30 +/- 1 degrees C, 65 +/- 5% RH). The study was conducted in the Animal Physiology Research Laboratory of the Department of Biology, Faculty of Science and Letter, University of Cukurova between 2016-2018. Effects of these insecticides on antioxidant, detoxification enzyme activities and MDA levels were changed depends on exposure time and the differences of tissues. THC decreased after 24 h, whereas it had increased after 48 and 72 h. DHC induced depends on exposure time and applied insecticide. This study revealed that pyriproxyfen and B. thuringiensis applications caused biochemical, physiological reactions and effected the immune defense system of larvae by the alterations in hemocyte counts.

In this study, the effects of dietary CuO nanoparticles (NPs) on metabolic enzyme activity, biochemical parameters, and total (THC) and differential hemocyte counts (DHC) were determined in Galleria mellonella larvae. Using concentrations of 10, 100, 1000 mg/L and the LC10 and LC30 levels of CuO NPs, we determined that the NPs negatively impacted metabolic enzyme activity and biochemical parameters in larval hemolymph. Compared with the control, the greatest increase in THC was observed in larvae fed on diets with 100 mg L−1 of CuO NPs. Plasmatocytes and granulocytes were among the most numerous hemocytes in all treatments. These results suggest that dietary CuO NPs effects the metabolic metabolism and immune system of G. mellonella and provide indirect information regarding the toxic effects of CuO NPs in mammalian immune system given similarities between mammalian blood cells and insect hemocytes.

Balmumu güvesi, Tunus'taki arıcılar için çok fazla zarara ve kayba neden olan bal arısı zararlılarından biridir. Balmumu ve balı kirlettikleri bilinmesine rağmen böcek öldürücülerin kullanımı giderek daha fazla kullanılmaktadır. Bu çalışmada, iki Kuzey Afrika ortak bitkisi Thymbra capitata L. ve Mentha pulegium L.'nin büyük mum güvesi (GWM) G. mellonella üzerine etkisinin araştırılması amaçlanmıştır. Ayrıca büyük balmumu güvesinin gelişme aşamalarının sürelerini de belirlenmiştir. Sonuçlar, 30 ila 33°C arasında değişen bir sıcaklık altında, yumurtalardan GWM'li yetişkinlere kadar olan toplam sürenin 51 gün sürdüğünü göstermiştir. Fumigant testi, test edilen larva dönemlerinde her iki yağın da toksisitesini göstermiştir. İkinci larva dönemi, 4. evreye göre daha hassastır. Dahası, M. pulegium, 2. larva dönemine karşı, sırasıyla, 48. saatte 41.829 ve 456.276 µl / Lair'de bir LC50 ile T. capitata'dan daha toksik olduğu belirlenmiştir. Bu bulgulara göre uçucu yağlar, mum güvelerini kontrol altına almak için böcek ilaçlarına iyi bir alternatif sunmaktadır., The wax moth is one of the honeybee pests that cause a lot of damage and loss for the beekeepers in Tunisia. The use of insecticides is more and more used although they are known to contaminate wax and honey. This study aims to test the essential oils of two North African common plants Thymbra capitata L. and Mentha pulegium L. efficacy as alternative method by fumigation on instars of Galleria mellonela L. We also determined the duration of the development stages of the great wax moth (GWM) presented in Tunisia. Results showed that under a temperature ranged between 30 and 33°C, the total duration from eggs to adults of GWM lasted 51 days. The fumigant test showed the toxicity of both oils on larvae instars tested. The second larvae instar was more susceptible than the 4th instar. Moreover, M. pulegium was more toxic against the 2nd larvae instar than T. capitata with an LC50 at 48h of 41.82 and 456.27 µl/L air, respectively. The essential oils present a good alternative to the insecticides to control wax moths.

Metarhizium anisopliae and Beauveria bassiana are commonly used entomopathogenic fungi, but their non-target effects over generalist predatory insects, which can contribute to pest control, are not well known. We studied the capacity of the social wasp Polistes myersi to detect the pathogens in either a powdered form or in Galleria mellonella larvae infected with either of the two pathogens offered as prey. The effects of these treatments were compared considering wasp behaviors such as prey preference, frequency, duration and transitions of both hunting and grooming behaviors. Additionally, the effects of each entomopathogenic fungus on the wasp’s mortality were measured. Wasps seem not to detect the pathogens in powdered form but preferred healthy over infected larvae. Seventeen behavioral units for hunting and 34 for grooming were recognized. There were no differences in grooming frequency but there were significant differences on grooming duration, hunting behaviors and the patterns of transitions. Exposure of wasp colonies to either B. bassiana or M. anisopliae had no detectable impact on the mortality of adults, but mortality of larvae increased. For the first time, this study documented behavioral changes that indicated the capacity of social wasps to detect pathogens before physical contact and the display of hygienic strategies once contact occurs. The study also suggested a potential non-target effect of these entomopathogenic fungi on a generalist predator.

Klebsiella pneumoniae is an important human pathogen causing urinary tract infections and pneumonia. Due to the increase in resistant strains and being an opportunistic pathogen, it is very important to determine the virulence process, the cellular damage it causes in the host and the immunological response level of the host. In this study, invertebrate infection model Galleria mellonella larvae were used to investigate cellular damage, antioxidant response and changes in biochemical parameters due to K. pneumoniae infection. The activity of cell damage indicators alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase increased in hemolymph of G. mellonella larvae due to K. pneumoniae virulence. Creatine kinase, alkaline phosphatase, gamma glutamyl transferase and amylase activities were increased to regulate the disrupted energy metabolism due to infection. As a result of the damage caused by K. pneumoniae infection, changes occurred in the amount of non-enzymatic antioxidants, uric acid, bilirubin and albumin. Due to K. pneumoniae infection, the amount of calcium, potassium, magnesium and phosphorus altered. This study showed that G. mellonella larvae was important infection model in the investigation of infectious cell damage and physiological effects, given the opportunistic nature of the K. pneumoniae pathogen and the lack of adequate animal models.

The greater wax moth, Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) is the most destructive pest of honey bee, Apis mellifera Linnaeus (Hymenoptera: Apidae), throughout the world. The present study was conducted to determine the quantitative and qualitative impairing effects of the arthropod venoms, viz., death stalker scorpion Leiurus quinquestriatus (Hemprich & Ehrenberg) venom (SV), oriental Hornet (wasp) Vespa orientalis Linnaeus venom (WV) and Apitoxin of A. mellifera (AP) on the larval haemogram. For this purpose, the 3rd instar larvae were treated with LC50 of each of these venoms (3428.9, 2412.6, and 956.16 ppm, respectively). The haematological investigation was conducted in haemolymph of the 5th and 7th (last) instar larvae. The important results could be summarized as follows. Five basic types of the freely circulating haemocytes in the haemolymph of last instar (7th) larvae of G. mellonella had been identified: Prohemocytes (PRs), Plasmatocytes (PLs), Granulocytes (GRs), Spherulocytes (SPs) and Oenocytoids (OEs). All venoms unexceptionally prohibited the larvae to produce normal hemocyte population (count). No certain trend of disturbance in the differential hemocyte counts of circulating hemocytes in larvae of G. mellonella after treatment with the arthropod venoms. Increasing or decreasing population of the circulating hemocytes seemed to depend on the potency of the venom, hemocyte type and the larval instar. In PRs of last instar larvae, some cytopathological features had been observed after treatment with AP or WV, but SV failed to cause cytopathological features. With regard to PLs, some cytopathological features had been observed after treatment with AP while both SV and WV failed to cause cytopathological features in this hemocyte type. No venom exhibited cytopathological effects on GRs, SPs or OEs.

The greater wax moth Galleria mellonella L. is a serious destructive pest of honeybee colonies in the beekeeping industry all over the world. The larvae feed on almost all of the honeybee products and have the ability to completely destroy the bee colony, especially the weakened ones. The toxicity and biochemical effect of garden cress, Lepidium sativum seed methanolic extract as natural product against greater wax moth was evaluated under laboratory conditions. The defatted seeds (250g) of garden cress were refluxed in 500 ml methanol, filtered and then dry in a rotary evaporator. Results revealed that the toxic effect of the garden cress extract against 5th instar larvae of G. mellonella was 1.15%, (LC50) after 24h. which induced more histopathological alterations on cuticle layers, fat bodies and muscle. Moreover, it causes highly significant inhibition in α, β esterase and phenol oxidase activities as well as possesses a significant reduction in protein content. On other hand, it causes an elevation in total lipid content and acid phosphatase activity. Also, apoptotic activity with DNA fragmentation was occurred at concentrations of 3, 2 and 1%. GC-MS analysis illustrated the toxic effect of methanol extract of L. sativum seeds related to a high ratio of phenolic, flavonoid and fatty acids constituents.

The Gram-negative bacteria Photorhabdus lives in a symbiotic relationship with the insect-pathogenic Heterorhabditis nematodes and produces numerous hydrolytic enzymes, secondary metabolites and protein toxins. Seven Photorhabdus strains were previously isolated from the Heterorhabditis nematodes collected from different geographical regions of India. The strains IARI-SGMG3, IARI-SGHR2, IARI-SGHR4, IARI-SGMS1 and IARI-SGGJ2 were identified as P. akhurstii, whereas IARI-SGLDK1 and IARI-SGHP1 were identified as P. laumondii subsp. laumondii and P. laumondii subsp. clarkeii, respectively. A new and previously unreported 35 kDa molecular weight protein toxin 'Galtox' was identified from these Photorhabdus strains. The nucleotide sequences of the toxin gene from seven Photorhabdus strains were PCR amplified, sequenced, cloned into pET protein expression vector, and the protein toxin was expressed and purified. The Galtox sequence from various strains showed variations in sequence and toxicity against Galleria mellonella. The injection of purified Galtox protein into the 4th instar larvae showed median lethal dose (LD50) values of 2.39–26.08 ng toxin/g G. mellonella bodyweight after 48 h. The protein injection killed the insects quickly and exhibited a median lethal time (LT50) of 12–60 h when injected at the rate of 3.1–31.2 ng toxin/g G. mellonella bodyweight. Galtox protein sequence analysis indicated similarity to several bacterial toxin-related protein domains, such as 6rgnA domain of Bordetella membrane targeting toxin BteA, 6gy6 domain of Xenorhabdus α-Xenorhabdolysins, 4mu6A and 4xa9a domains similar to effector protein LegC3 from Legionella pneumophila and 1cv8.1 domain of staphylococcal cysteine proteinase staphopain B. The mode of action of Galtox needs to be understood to enable its use for the management of agricultural insect-pests.

Galleria mellonella is a pest of the honey bee (Apis mellifera L.) and causes significant losses to the beekeeping industry; therefore, experiments are needed to decode the effects of entomopathogenic fungi on insect physiology. The gas chromatography–mass spectrometry (GC-MS) method was successful for the determination of the organic compounds of Galleria mellonella larvae, noninfected and infected by three fungal species: M. robertsii, M. flavoviride, and I. fumosorosea. A total of 46 compounds were identified in G. mellonella, including fatty acids, other acids, fatty acid methyl esters, monoacylglycerols, amino acids, sterols, and several other organic compounds. The lipids of G. mellonella larvae after M. robertsii, M. flavoviride, and I. fumosorosea exposure contained 40, 35, and 33 organic compounds, respectively. The following organic compounds, present in the noninfected larvae, were absent from the infected larvae: fatty acids C22:0 and C24:0, glutaric acid, urocanic acid, hydroxycinnamic acid, dihydroxycinnamic acid, 10-oxodecanoic acid, glycine, aspartic acid, glutamic acid, lysine, tyrosine, tryptophan, 2-aminobutyric acid, and tyramine. These compounds can be used by fungi as an exogenous source of carbon. The following organic compounds, present in the infected larvae, were absent from the noninfected larvae: fatty acids C10:0, C11:0, C13:0, and C20:0, suberic acid, phenylacetic acid, fatty acid methyl ester (FAME) C16:0, FAME C18:2, FAME C18:1, glycerol 2-monopalmitate, norvaline, proline, sitosterol, and 2-dekanal. These compounds can be synthesized as an insect response to fungal infection.

The authors found out that the greater wax moth (Galleria mellonella) harmed bee colonies by destroying up to 30 percent of honey in a beehive. They studied the results of experiments on controlling its behavior using a developed automated microprocessor system that maintained optical radiation sources duration.(Research purpose) To develop light technologies using a microprocessor-based automated system that allows controlling Galleria mellonella behavior by realizing attractive optical radiation characteristics.(Materials and methods) Galleria mellonella behavior was controlled by an automated system that the authors worked out for maintaining the required duration of the experiment and optical radiation parameters. The ATmega328 microcontroller was chosen as the main element of the developed microprocessor automated system. The authors created a program for it using visual programming FLProg version 5.3.0.(Results and discussion) The authors found that, regardless of the experiment duration, Galleria mellonella butterflies prefered radiation with 400 nanometers wavelength.(Conclusions) The authors determined that radiation with 400 nanometers wavelength and 10 minutes duration was attractive. They showed the ability to control Galleria mellonella behavior, luring butterflies to a specific place in the beehive with comfortable spectrum parameters. An automated system for controlling the greater wax moth butterfly behavior was developed on ATmega328 microcontroller by implementing attractive optical radiation characteristics.

Among the new contaminants relevant for environment, one of the most significant roles is played by pharmaceuticals like antibiotic products for either human or veterinary use. Their presence could cause serious damage to bacteria and microfauna, like nematodes. Within the widely investigated nematodes, very little is known about the interaction between antibiotics and entomopathogenic nematodes (EPN). EPNs have been used for biological control of crops, due to their ability to penetrate arthropod pests and kill their hosts thanks to a complex symbiotic mechanism with specific gram-negative bacteria. Tetracycline is an antibiotic used in human and veterinary medicine, both for therapeutic purposes and for the growth of livestock. Since its action against gram-negative bacteria is documented, we verified in this study the survival, growth and pathogenicity of two species of EPNs, Steinernema vulcanicum and S. feltiae. All tests were performed with tetracycline in 1% ethanol solution and up to 300 mg/L. Apparently, this incubation did not harm the vitality of EPNs. Both S. vulcanicum as S. feltiae recovered their vitality and entomopathogenic ability after 48 h. Moreover, the latter EPN species did not grow nor reproduce in the hemolymph of the Greater Wax Moth, Galleria mellonella, and their endosymbionts did not grow on MacConkey Agar. Our results suggest that the first EPN species has always retained all its abilities and that endosymbionts have acquired resistance to tetracycline, while experiments with the second EPN species provided some contrasting results in time that will require further investigations.

Nanomaterials of different sizes and diameters are frequently used in various industrial areas, due to the rapid development of nanotechnology. Hence, it leads to toxic effects on the environment and non-target organisms, and adverse effects such as oxidative stress and membrane damage in cells and tissues are occurred. Some biomarkers such as the accumulation of intermediate products, detoxification of the immune system, or xenobiotic are used in the detection of toxic effects of exogenous substances in living organisms. In this study, the effects on catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-s-transferase (GST) enzyme activities to determine the toxic effects of TiO2 NPs on antioxidant defense system, acetylcholinesterase (AChE) activity to determine their neurotoxic effects, and total hemocyte count (THC) to determine their effect on the immune system were investigated in model organism Galleria mellonella larvae. It was determined that major amounts of Ti were mostly eliminated through the Malpighian tubules. Moreover, TiO2 NPs in different concentrations caused the formation of reactive oxygen species in G. mellonella and lead to an increase in antioxidant enzymes. Decreases were found in THC due to the TiO2 NP application. As a result, it was concluded that TiO2 NPs caused accumulation in tissues of the model organism G. mellonella, resulting in oxidative stress and has adverse effects on the immune system.