Interactions of alumina and polystyrene nanoparticles with the innate immune system of Galleria mellonella.

Nowadays, particularly metallic, and polymeric nanoparticles (NPs) are widely produced and used in many fields. Due to the increase in both their usage and diversity, their release and accumulation in the environment are also accelerating. Therefore, their interactions with cells, especially immune cells, and their health risks are not fully understood. The impacts of metallic alumina (Al) NPs and polystyrene (PS) NPs obtained after the polymerization of carcinogenic styrene on living organisms have not yet been elucidated. Galleria mellonella larvae can biodegrade plastics. While biodegradation and solving the waste problem have attracted much attention, the interactions of this distinctive property of G. mellonella larvae in the immune system and ecosystem are not yet completely understood. Al and PS NPs were applied to G. mellonella separately. Al NPs were purchased and PS NPs were prepared from PS by single-emulsion technique and characterized. Then LC50 values of these NPs on G. mellonella were determined. The interactions of these NPs with encapsulation, melanization, and phenoloxidase activity, which express innate immune responses in G. mellonella larvae, were revealed. NP exposure resulted in suppression of the immune response, probably because it affects the functions of hemocytes such as enzymatic activation, hemocyte division, and populations. In this context, our data suggest that Al and PS NPs induce toxic impacts and negatively alter the physiological status of G. mellonella. It is also shown that G. mellonella has the potential to be an impactful alternative model for biosafety and nanotoxicology studies.

Immunosuppressive influence of parasitoid wasp Pimpla turionellae calyx fluid on host Galleria mellonella cell-mediated immune response and hemocyte viability.

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.

Reprograming of epigenetic mechanisms controlling host insect immunity and development in response to egg-laying by a parasitoid wasp.

Parasitoids are insects that use other insects as hosts. They sabotage host cellular and humoral defences to promote the survival of their offspring by injecting viruses and venoms along with their eggs. Many pathogens and parasites disrupt host epigenetic mechanisms to overcome immune system defences, and we hypothesized that parasitoids may use the same strategy. We used the ichneumon wasp Pimpla turionellae as a model idiobiont parasitoid to test this hypothesis, with pupae of the greater wax moth Galleria mellonella as the host. We found that parasitoid infestation involves the suppression of host immunity-related effector genes and the modulation of host genes involved in developmental hormone signalling. The transcriptional reprogramming of host genes following the injection of parasitoid eggs was associated with changes in host epigenetic mechanisms. The introduction of parasitoids resulted in a transient decrease in host global DNA methylation and the modulation of acetylation ratios for specific histones. Genes encoding regulators of histone acetylation and deacetylation were mostly downregulated in the parasitized pupae, suggesting that parasitoids can suppress host transcription. We also detected a strong parasitoid-specific effect on host microRNAs regulating gene expression at the post-transcriptional level. Our data therefore support the hypothesis that parasitoids may favour the survival of their offspring by interfering with host epigenetic mechanisms to suppress the immune system and disrupt development.

Pathological and ultrastructural changes in cultured cells induced by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

The ectoparasitic wasp Nasonia vitripennis produces a proteinaceous venom that induces death in fly hosts by non-paralytic mechanisms. Previous in vitro assays have suggested that the primary cause of cell and tissue death is oncosis, a non-programmed cell death (PCD) pathway characterized by cellular swelling and lysis. However, ultrastructural analyses of BTI-TN-5B1 cells exposed to LC(99) doses of wasp venom revealed cellular changes more consistent with apoptosis and/or non-apoptotic PCD than oncosis or necrosis: By 3h after incubation with venom, susceptible cells displayed indentations in the nuclear membranes, large nucleoli, and extensive vacuolization throughout the cytoplasm. In the vast majority of venom treated cells, annexin V bound to the plasma membrane surface within 15 min after treatment, a characteristic consistent with translocation of phosphatidylserine to the cell surface during the early stages of apoptosis. Likewise, mitochondrial transmembrane potential was depressed in cells within 15 min in venom-treated cells, an event that occurred in the absence of mitochondrial swelling or rupturing of cristae. Active caspase 3 was detected by fluorescent labeling in nearly all venom treated cells 3h after exposure to venom, and in turn, the potent caspase 3 inhibitor Z-VAD-FMK attenuated the morphological changes elicited by wasp venom and afforded protection to BTI-TN-5B1-4 cells.

Age and diet influence the composition of venom from the endoparasitic wasp Pimpla turionellae L. (Hymenoptera: Ichneumonidae).

Venom from the endoparasitic wasp Pimpla turionellae L. (Hymenoptera: Ichneumonidae) was found to contain a complex mixture of biogenic amines, noradrenalin, phospholipase B, and several proteins and peptides. The amount of noradrenalin and serotonin was found to be highest in venom from newly emerged wasps and decreased with age. Histamine was detected in minute amounts in comparison to the other venom components, and declined with increasing age of the parasitoids. Total peptides and proteins detected by reversed-phase HPLC increased with host age. Old-aged (30-33 days after emergence) wasps contained 2-fold more phospholipase B than young (<10 days [d] old) or medium-aged (10-22-d-old) females. Increases in phospholipase B alone, however, did not account for all changes in total venom protein because by 40 days after emergence, the levels of this enzyme began to decline while the amount of total protein was higher than in younger wasps. For all venom components detected, the amount present in the venom sharply decreased following host exposure. This was presumed to be the result of venom depletion associated with envenomation. Consistent with this view were the modest increases in venom components in wasps displaying a decreased rate of parasitization. When adult females were offered honey alone or in combination with feeding on hosts, no significant changes in venom composition were observed, with the exception of noradrenalin, which was found to be 5 times higher in concentration in wasps fed honey only. These results suggest that wasp age and incidence of parasitism are more important features influencing the composition of venom than the diet of adult females.

In vivo and in vitro activity of venom from the endoparasitic wasp Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae).

The biological activity of venom from Pimpla turionellae L. (Hymenoptera: Ichneumonidae) was examined in vivo toward larvae and pupae of Galleriae mellonella L. (Lepidoptera: Pyralidae), and in vitro toward bacterial and fungal cultures, as well as cultured insect cells. Pupae of G. mellonella were far more susceptible to the venom than larvae. At low doses of venom [0.1 venom reservoir equivalents (VRE)], pupal abdominal mobility was inhibited within 30 min, and by 24 h, all pupae injected with venom concentrations >0.5 VRE were completely paralyzed. These same doses of venom resulted in an inhibition of adult emergence. Host larvae were far less sensitive to wasp venom as evidenced by all venom injected larvae remaining responsive to mechanical stimulation by 1 h post injection, even at concentrations equivalent to 1 venom reservoir. Eventually (>2 h at 25 degrees C), venom-injected larvae became immobile, then flaccid, and all died within 24 h post-injection. At lower concentrations of wasp venom, the onset of paralysis was delayed by comparison to that evoked by 1 VRE, and few host larvae were able to pupate. Development of host larvae to adult emergence was also reduced in a dose-dependent manner, with eclosion completely prevented at high concentrations (>0.5 VRE) of venom. Venom doses <0.5 VRE did not appear to induce paralysis or alter larval development. When venom was incubated with bacterial or fungal cultures, no antimicrobial activity was detected. However, wasp venom was found to be cytotoxic and cytolytic to cultured cells derived from the cabbage looper Trichoplusia ni Hubner (Lepidoptera: Noctuidae) and the yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culcidae). Though both cell types displayed similar susceptibility in terms of LC50s, the lepidopteran cells responded much more rapidly with regard to the onset of morphological changes and the timing of cell death. A possible mode of action for the venom is discussed.

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.