Larvae Of Wax Moth Galleria Mellonella Research Articles

Debilitation of Galleria mellonella hemocytes using CytCo a cytolytic-like protein derived from the entomopathogen Conidiobolus obscurus

Cytolytic (Cyt)-like genes are present in both pathogenic bacteria and fungi. Bacterial Cyt proteins can destroy insect midgut epithelial cells after ingestion by hosts and some of them have been developed as biopesticides; however, few studies have investigated their functions in fungal pathogens. This study investigated the effects of a Cyt-like protein (CytCo) derived from Conidiobolus obscurus (Entomophthoromycotina) on the hemocytes of the greater wax moth Galleria mellonella larvae. The results showed a significant decline in hemocyte viability after treatment with CytCo in vivo or in vitro. The hemocyte density in the hemolymph was reduced by 65.2% and 50.2% after 12 h in vivo and 6 h in vitro treatments, respectively. Apoptosis/necrosis tests using fluorescence microscopy demonstrated that CytCo-treated hemocytes displayed apoptosis, and many of them also showed necrosis after 6 h in vitro treatment. Based on transcriptome analysis, several genes involved in the programmed cell death signaling pathway were upregulated in the CytCo-treated hemocytes. Meanwhile, the differentially expressed genes related to energy production, signal transduction, transcription regulation, and melanization were upregulated, demonstrating activated immune responses; those putatively related to hemocyte adhesion were downregulated, possibly in response to the reduction of hemocytes in hemolymph. In conclusion, CytCo as a virulence factor, could irreversibly incapacitate host hemocytes, playing an important role in debilitating insect immunity. This novel insecticidal protein holds a potential to develop biopesticide for controlling agroforestry pests.

Evaluation of the Effectiveness of Some Entomopathogenic Fungi on the Greater Wax Moth Larvae, Galleria mellonella (L.) (Lepidoptera: galleriidae)

The present study designed to investigate the effect of three entomopathogenic fungi (EPF), Beauveria bassiana (Biovar), Trichoderma album (Biozed) and Metarhizium anisopliae (Bioranza) on greater wax moth Galleria mellonella larvae. The results revealed that the LC25 values of T. album, M. anisopliae and B. bassiana against G. mellonella larvae were 14.08, 44.20 and 57.82 ppm, respectively. The LC50 were 104.03, 252.55 and 389.05 ppm, respectively for the same biocides. T. album was the most toxic biocide for controlling G. mellonella larvae, While B. bassiana was the least toxic one. The slope of the tested compounds was 0.78, 0.89 and 0.81 for T. album, M. anisopliae and B. bassiana, respectively. The toxicity index of the tested compounds was 24.35 & 26.74 & 31.85 and 41.18, respectively. On the other hand, the relative potency of the three tested compounds at LC25 and LC50 were 1.31 & 1.54 and 4.11 & 3.74 fold for both M. anisopliae and T. album compared to 1.00 fold in case of B. bassiana. The results showed that the cumulative mortality percentages of G. mellonella larvae treated with the three products can be arranged according to the most efficient compounds 47.61, 41.52 and 39.67%, respectively for T. album, M. anisopliae and B. bassiana compared to control. Also, the results indicated that three tested biocides have a significant effect on some biological aspects of G. mellonella larvae. B. bassiana was the most effective biocide on larval and pupal duration, pupal mortality, pupal weight and sex ratio percentages. While, treatment with M. anisopliae reduced male longevity, and the malformation percentages recorded as 27.35% compared to control. But, T. album had the most toxic effect on accumulative larval mortality, pupation percentages, pupal duration and significantly decreased the female sex ratio than control. The three tested biocides had an effect on some biochemical parameters of G. mellonella larvae. The activity of amylase enzyme had a significantly decreased effect after five periods of treatment compared to control. A significant increase in relative activity in protease enzyme was recorded after treatment with the three EPF. A fluctuation in the enzyme activity of transaminase enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was estimated. There was also a significant reduction in total protein content. Results cleared that, significantly increased gradually in total lipid up to ten days after treatments. The results indicated that the tested bio-products can be used in honey bee colonies as a strategy to control G. mellonella larvae as one of the components of biological control programs.

Using the wax moth larva Galleria mellonella infection model to detect emerging bacterial pathogens.

Climate change, changing farming practices, social and demographic changes and rising levels of antibiotic resistance are likely to lead to future increases in opportunistic bacterial infections that are more difficult to treat. Uncovering the prevalence and identity of pathogenic bacteria in the environment is key to assessing transmission risks. We describe the first use of the Wax moth larva Galleria mellonella, a well-established model for the mammalian innate immune system, to selectively enrich and characterize pathogens from coastal environments in the South West of the UK. Whole-genome sequencing of highly virulent isolates revealed amongst others a Proteus mirabilis strain carrying the Salmonella SGI1 genomic island not reported from the UK before and the recently described species Vibrio injenensis hitherto only reported from human patients in Korea. Our novel method has the power to detect bacterial pathogens in the environment that potentially pose a serious risk to public health.

Effects of Oxfendazole on Some Biochemical Parameters in Hemolymph of Galleria mellonella L. (Lepidoptera: Pyralidae) Larvae Reared on Artificial Diet

Oxfendazole is used as an antihelminthic drug in parasite infections of animals. Oxfendazole was added at different concentrations (0, 0.0015, 0.015, 0.15, and 1.5%) to artificial diet and the wax moth Galleria mellonella larvae were reared to 7th stage. The changes in metabolic enzymes activity in G. mellonella hemolymph tissue were investigated. It was found that the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was increased which are cell damage biomarkers. The highest concentration of this antihelmintic was increased the enzme activity (ALT) from 329 ± 4.33 to 529. 5 ± 3.17 U/L. Gamma glutamyl transferase (GGT) enzyme activity was increased from 0.87 ± 0.1 to 4.32 ± 0.22 U/L. Creatine kinase (CK) and amylase (AMYL) activities were significantly increased in hemolymph of G. mellonella larvae fed with the highest oxfendazole concentration (1.5%) when compared to the control group. Our results indicate that oxfendazole can be used as an insecticide with a well-adjusted concentrations because of its low acute toxicity to humans and other non-target organisms.

Entomopathogenic Nematodes Survey, Persistence in Soil, Reproductive Potential and their Effects on Meloidogyne incognita

The increasing cost of nematicides and environmental deterioration are the great challenges to agriculture. Entomopathogenic nematodes (EPNs) could be a choice as biocontrol agent in suppressing the root knot nematode, Meloidogyne incognita on tomato and other crops. A total number of 75 soil samples were collected from different locations of Vehari and Faisalabad, Pakistan. In Vehari out of 14 sites, 13 sites were found positive for EPNs, Nematodes were recovered from 19 (54 %) of 35 soil samples obtained from ecologically diverse habitats. From University of Agriculture Faisalabad, EPNs were recovered from 6 (40 %) of 15 soil samples. While, out of 25 samples collected from Ayub Agriculture Research Institute 9(36%) were found positive for EPNs. Both Steinernema and Heterorhabditis species were found in these locations. The soil samples were also analyzed to indicate the effect of soil texture. Maximum EPNs (36 %) were recovered from sandy loam soil followed by loamy soil. A series of experiments was carried out to unearth EPNs’ soil persistence, reproductive potential and their effects on root knot nematodes. All species and strains of EPNs had potential to persist in almost all type of soils. Persistence was observed in sterilized soil sand and clay (1:1). Maximum persistence was found in S. glaseri (29.11 %) followed by S. pakistanese (28.41 %), H. indica (26.93 %), S. asiaticum (24.42 %), S. feltiae (21.52 %) and H. bacteriophora (21.11 %) which showed the least persistence in sterilized soil. All species of both the families Heterorhabditidae and Steinernematidae did not persist more than 30 % in sterilized soil. Reproductive potential of three EPNs was recorded using the greater wax moth Galleria mellonella larvae. H. indica showed very high reproductive potential within wax moth followed by S. asiaticum and S. pakistanese. On the other hand, maximum emerged infective juveniles per day were recovered from H. indica followed by S. pakistanese and S. asiaticum. Suppressive effect of EPNs against root knot nematodes was observed on susceptible tomato cultivars at different concentration. S. pakistanese suppress M. incognita population more efficiently than H. indica at all the levelsi.e. 1250, 2500 and 5000 nematodes pot-1 . After 35days egg masses (EM) recovered from S. pakistanese treated pots at level 1250 were 232/pot, at level 2500 were 207 and at level 5000 were 135 respectively. Similarly, egg masses recovered from H. indica treated plants at level 1250 were 276, at level 2500 were 212 and at level 5000 were 154 EM pot-1 respectively. No EM were recovered from clean plants on contrary328 EM were recovered from plants with root knot nematodes only.

Maintenance of redox balance by antioxidants in hemolymph of the greater wax moth Galleria mellonella larvae during encapsulation response.

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione-S-transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione-S-transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.

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.

Effect of plant secondary metabolites on susceptibility of insects to entomopathogenic microorganisms

The effect of a number of plant extracts on the susceptibility of experimental insects to enthomopathogenic microorganisms has been studied. It is shown that the weight of the wax moth Galleria mellonella larvae tends to decrease by 30–50% under the treatments of polar and nonpolar extracts from the ledum sprouts and the reindeer lichen, while the crude hemlock extract had the opposite effect, contributing to the larva weight gain by approximately 30%. The treatment with the reindeer lichen extract causes synergistic effects on mortality from both the nuclear polyhedrosis virus and the fungal infection in the gypsy moth Lymantria dispar and the wax moth, respectively. It has been determined that the main components of this extract are perlatolic acid, usnic acid, and a third component whose exact chemical identity is still unknown. The usnic acid is the most prospective additive component to entomopathogenic microorganisms. The treatment with the usnic acid caused the increase in mortality from the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana in the Colorado potato beetle Leptinotarsa decemlineata and the wax moth. However, the maximum effect occurs only after the treatment with the crude extract, which can be explained by either the combined effects of all the extract components or the change in a range of the properties of the components in the presence of the other crude extract components.

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.

The effect of Habrobracon hebetor venom on the activity of the prophenoloxidase system, the generation of reactive oxygen species and encapsulation in the haemolymph of Galleria mellonella larvae

The cellular and humoral immune reactions in haemolymph of the wax moth Galleria mellonella larvae naturally injected by venom of ectoparasitic wasp Habrobracon hebetor were analyzed. A strong decline of phenoloxidase (PO) activity in the haemolymph and the number of haemocytes with PO activity of envenomated wax moth was observed. In addition, it has been shown that the rate of capsule melanization in the envenomated larvae was half that of the control. Also production of reactive oxygen species (ROS) in the haemolymph of envenomated larvae decreased. The obtained data casts light on the suppression of the main immune reactions in G. mellonella larvae during natural envenomation by H. hebetor.

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria.

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live gram-positive bacteria Micrococcus lysodeikticus and gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae.

Changes of the Antioxidant Status and System of Generation of Free Radicals in Hemolymph of Galleria mellonella Larvae at Microsporidiosis

oChanges of superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities as well as of the content of SH-containing compounds were revealed in hemolymph of the native and the Vairimorpha ephestiae microsporidian-infected greater wax moth Galleria mellonella larvae. The SOD and GST activities in hemolymph of infected insects decreased at the stage of merogony, whereas during massive sporulation the enzymatic antioxidant activity in host tissues was higher than in control. By the ESR spectroscopy method, using the 1-hydroxy-3-carboxy-pyrrolidinespin-trap, generation of free radicals in hemolymph of infected insects was shown to decrease only at the stage of sporogony. The phenoloxidase activity in lymph was lower at acute microsporidiosis than in native larvae. The hemolymph concentration of thiol-containing proteins in infected insects did not differ from that in control. We suggest that decrease of generation of free radicals in hemolymph of the greater wax moth larvae at the stage of sporogony is due to a suppression of the prophenoloxidase system and an elevation of the antioxidant activity.

Acclimation of entomopathogenic nematodes to novel temperatures: trehalose accumulation and the acquisition of thermotolerance

The effect of thermal acclimation on trehalose accumulation and the acquisition of thermotolerance was studied in three species of entomopathogenic nematodes adapted to either cold or warm temperatures. All three Steinernema species accumulated trehalose when acclimated at either 5 or 35°C, but the amount of trehalose accumulation differed by species and temperature. The trehalose content of the cold adapted Steinernema feltiae increased by 350 and 182%, of intermediate Steinernema carpocapsae by 146 and 122% and of warm adapted Steinernema riobrave by 30 and 87% over the initial level (18.25, 27.24 and 23.97 μg trehalose/mg dry weight, respectively) during acclimation at 5 and 35°C, respectively. Warm and cold acclimation enhanced heat (40°C for 8 h) and freezing (−20°C for 4 h) tolerance of S. carpocapsae and the enhanced tolerance was positively correlated with the increased trehalose levels. Warm and cold acclimation also enhanced heat but not freezing tolerance of S. feltiae and the enhanced heat tolerance was positively correlated with the increased trehalose levels. In contrast, warm and cold acclimation enhanced the freezing but not heat tolerance of S. riobrave, and increased freezing tolerance of only warm acclimated S. riobrave was positively correlated with the increased trehalose levels. The effect of acclimation on maintenance of original virulence by either heat or freeze stressed nematodes against the wax moth Galleria mellonella larvae was temperature dependent and differed among species. During freezing stress, both cold and warm acclimated S. carpocapsae (84%) and during heat stress, only warm acclimated S. carpocapsae (95%) maintained significantly higher original virulence than the non-acclimated (36 and 47%, respectively) nematodes. Both cold and warm acclimated S. feltiae maintained significantly higher original virulence (69%) than the non-acclimated S. feltiae (0%) during heat but not freezing stress. In contrast, both warm and cold acclimated S. riobrave maintained significantly higher virulence (41%) than the non-acclimated (14%) nematodes during freezing, but not during heat stress. Our data indicate that trehalose accumulation is not only a cold associated phenomenon but is a general response of nematodes to thermal stress. However, the extent of enhanced thermal stress tolerance conferred by the accumulated trehalose differs with nematode species.

Calreticulin enriched as an early-stage encapsulation protein in wax moth Galleria mellonella larvae

To investigate the molecular mechanism of the early-stage encapsulation reaction in insects, we purified a 47 kDa protein from injected beads into Galleria mellonella larvae. When a cDNA clone was isolated, the 47 kDa protein showed high homology with Drosophila and human calreticulin. Western blotting analysis showed that the 47 kDa protein was present in the hemocytes, but not in the plasma. When the early-stage encapsulated beads were coated with 47 kDa protein antibody and reinjected into G. mellonella larvae, any further encapsulation reaction was inhibited. These results suggest that calreticulin is involved in non-self recognition in invertebrate cellullar defense reactions.

Changes in the activity and pattern of hemolymph esterases in the larvae of wax moth Galleria mellonella L. (Lepidoptera, Pyralidae) during mycosis

The extended pattern of multiple esterase forms has been revealed in the hemolymph of wax moth Galleria mellonella larvae infected by the fungi Metarhizium anisopliae, Beauveria bassiana or Paecilomyces fumoso-roseus. The total esterase activity of the hemolymph also increases during mycosis. Mechanical damage of the cuticle, treatment with deltamethrin, and chilling of the caterpillars induced similar changes in the hemolymph pattern of esterase activity. Presumably, the changed spectrum and activity of the hemolymph esterases during mycosis is due to the damaged cuticle and epidermis cells.

Influence of Production and Bioassay Methods on Infectivity of Two Ambush Foragers (Nematoda: Steinernematidae)

The influence of production and bioassay methods on infectivity of two ambush foragers,Steinernema carpocapsaeandSteinernema scapterisci,was evaluated. Both species were mass-producedin vitroin liquid culture andin vivousing live insects:S. carpocapsaein last instar wax mothGalleria mellonellalarvae andS. scapterisciin adult house cricketsAcheta domesticus.Infectivity was assessed at 28°C against last-instarG. mellonellalarvae using a filter-paper bioassay and a newly developed “sand-well” bioassay. The infectivity ofS. carpocapsaewas not influenced by the method of production or bioassay, whereas the infectivity ofS. scapterisciwas influenced by both factors. Bothin vivoandin vitroproducedS. carpocapsaecaused >60% larval mortality at one nematode per larva (1:1) in the filter-paper bioassay, butS. scapteriscielicited less than 10% mortality. At 50 nematodes per larva,in vitro S. scapteriscicaused 41.7% mean larval mortality in the filter-paper bioassay, whereasin vivo S. scapteriscielicited only 28.5% mortality. The replacement of filter paper with a 2.5-mm-deep layer of sand (termed sand well) resulted in 2.5-fold increase in infectivity ofS. scapterisci.In the new sand-well procedure, 15S. scapterisciper larva (15:1) caused an overall mean larval mortality of 47.5% and the pattern of mortality showed a normal distribution. The infectivity ofS. carpocapsaewas not different in the 1:1 filter paper or 1:1 sand-well bioassay. These results demonstrate that nematode infectivity could be strongly influenced by both the production and bioassay methods, and there are no universal assays even when nematodes have similar foraging strategies.

Enhanced cold tolerance of the entomopathogenic nematode Steinernema feltiae through genetic selection

Summary.Cold sensitivity of entomopathogenic nematodes severely restricts their biological control potential in some environments. We selected the SN strain of Steinernema feltiae together with its bacterial symbiont, Xenorhabdus bovenii, for improved cold tolerance by repeated passage through the wax moth Galleria mellonella larvae at 15°C. Nematode virulence (total insect mortality and speed of kill) and establishment (initiation of nematode development following penetration) were evaluated after six (= 12–24 generations) and 12 passages (= 24–36 generations). Cold selection enhanced nematode virulence at the cooler temperatures. Virulence measured as total insect‐mortality at 8°C improved by 5.3‐ and 6.6‐fold after six and 12 passages, respectively. Only small improvements (1.2–1.5‐fold) were observed in speed of kill. Nematode establishment improved at all temperatures after 12 passages; the highest increase of 9‐fold was observed at 8°C. Our results lend support to the hypotheses that functional traits along a continuously distributed environmental variable are genetically correlated and that the area under the fitness function is not always constant. Trade‐offs in percentage mortality and speed of kill by the selected nematodes were observed at the warmer extreme after six passages of selection only. The implications of rapid changes in thermal sensitivity for economic mass‐production of nematodes are discussed.

Plaque assay and replication of Tipula iridescent virus in Spodoptera frugiperda ovarian cells

A plaque assay was developed for the study of Tipula iridescent virus (TIV) replication using a cell line derived from the fall army worm Spodoptera frugiperda (Sf9). Infection and plaque formation were monitored with time by phase contrast microscopy, video and fluorescent light microscopy. Structure of virions, viroplasmic centres and organelles of infected cells were examined by transmission electron microscopy (TEM). After 4 h postinfection, plaques were visibly detected within the cell monolayer by the presence of localized cell damage and production of numerous vesicular-like cytoplasmic structures. Quantitation of virions present per A260 unit of TIV preparation was determined by TEM. The number of visible plaques corresponded to virus concentration and 1 A260 produced approximately 10(5) plaques. DNA hybridization analysis revealed no gross differences in genomic DNA from TIV propagated in either Sf9 cells or wax moth Galleria mellonella larvae. These findings indicate that Sf9 is permissive for replication of TIV and superior by some parameters to other cell lines currently in use for the study of host cell/TIV interactions.