Induction of antimicrobial peptides from Bacillus thuringiensis challenged Spodoptera litura larvae and investigation of the antimicrobial properties of hemolymph samples

Antimicrobial peptides constitute key factors in insect humoral immune response against invading microorganisms. In this study, biochemical approach was identified antimicrobial peptides which appeared in larval hemolymph of Spodoptera litura after bacterial challenge. HPLC profile showed two major peaks in two samples, Brassica oleracea and Ricinus communis fed S. litura that were collected at 5 min interval. It was shown to be active against Gram-positive and Gram-negative bacteria. The highest zone of inhibition was observed in Staphylococcus aureus and Escherichia coli in B. oleracea fed S. litura hemolymph fraction II and R. communis fed S. litura hemolymph fraction I and it also contributes the increased antioxidant, lysozyme, and less hemolytic activity were increase in treated groups. TLC activity was tested with hemolymph extract samples, pink color pots was identified the protein present in the samples. An SDS-PAGE result shows that high expression of antimicrobial peptide present in the treated sample. The appearance of peptides with such different properties in insect hemolymph in response to immune challenge indicates the complexity of the insect immune system.

In Vivo Efficacy of Voriconazole in a Galleria mellonella Model of Invasive Infection Due to Azole-Susceptible or Resistant Aspergillus fumigatus Isolates

Aspergillus fumigatus is an environmental filamentous fungus responsible for life-threatening infections in humans and animals. Azoles are the first-line treatment for aspergillosis, but in recent years, the emergence of azole resistance in A. fumigatus has changed treatment recommendations. The objective of this study was to evaluate the efficacy of voriconazole (VRZ) in a Galleria mellonella model of invasive infection due to azole-susceptible or azole-resistant A. fumigatus isolates. We also sought to describe the pharmacokinetics of VRZ in the G. mellonella model. G. mellonella larvae were infected with conidial suspensions of azole-susceptible and azole-resistant isolates of A. fumigatus. Mortality curves were used to calculate the lethal dose. Assessment of the efficacy of VRZ or amphotericin B (AMB) treatment was based on mortality in the lethal model and histopathologic lesions. The pharmacokinetics of VRZ were determined in larval hemolymph. Invasive fungal infection was obtained after conidial inoculation. A dose-dependent reduction in mortality was observed after antifungal treatment with AMB and VRZ. VRZ was more effective at treating larvae inoculated with azole-susceptible A. fumigatus isolates than larvae inoculated with azole-resistant isolates. The concentration of VRZ was maximal at the beginning of treatment and gradually decreased in the hemolymph to reach a Cmin (24 h) between 0.11 and 11.30 mg/L, depending on the dose. In conclusion, G. mellonella is a suitable model for testing the efficacy of antifungal agents against A. fumigatus.

Comparative Transcriptome Analysis of Bombyx mori (Lepidoptera) Larval Hemolymph in Response to Autographa californica Nucleopolyhedrovirus in Differentially Resistant Strains

Bombyx mori nucleopolyhedrovirus (BmNPV) is a kind of pathogen that causes huge economic losses to silkworm production. Although Autographa californica nucleopolyhedrovirus (AcMNPV) and BmNPV are both baculoviruses, the host domains of these two viruses have almost no intersection in nature. Recently, it has been found that some silkworms could be infected by recombinant AcMNPV through a puncture, which provided valuable material for studying the infection mechanism of baculovirus to silkworm. In this study, comparative transcriptomics was used to analyse the hemolymph of two differentially resistant strains following AcMNPV inoculation. There were 678 DEGs in p50 and 515 DEGs in C108 following viral infection. Among them, the upregulation and downregulation of DEGs were similar in p50; however, the upregulated DEGs were nearly twice as numerous as the downregulated DEGs in C108. The DEGs in different resistant strains differed by GO enrichment. Based on KEGG enrichment, DEGs were mainly enriched in metabolic pathways in p50 and the apoptosis pathway in C108. Moreover, 13 genes involved in metabolic pathways and 11 genes involved in the apoptosis pathway were analysed. Among the DEGs involved in apoptosis, the function of BmTex261 in viral infection was analysed. The BmTex261 showed the highest expression in hemolymph and a significant response to viral infection in the hemolymph of C108, indicating that it is involved in anti-AcMNPV infection. This was further validated by the significantly decreased expression of viral gene lef3 after overexpression of BmTex261 in BmN cells. The results provide a theoretical reference for the molecular mechanism of resistance to BmNPV in silkworms.

Enhanced Antioxidant and Cytotoxic Potentials of Lipopolysaccharides-Injected Musca domestica Larvae

The usage of insects as a sustainable and functional natural products resource is a new promise in complementary and alternative medicine. The present study aimed to investigate the ability of Musca domestica (housefly) larval hemolymph (insect blood) to display the enhanced in vitro antioxidant and cytotoxic effects. The oxidative stress (OS) was elicited by inducing lipopolysaccharides (LPS) treatment as an exogenous stressor. Determination of superoxide dismutase 1 (SOD1), glutathione (GSH), malondialdehyde (MDA) and total antioxidant capacity (TAC), and mRNA and protein expressions of SOD1, was investigated as confirmatory markers of oxidative stress induction. Cytotoxicity on cancerous MCF-7 and normal Vero cells were also evaluated using an MTT assay at 24 h post-injection. The injection of LPS induced a significant (p < 0.05) increase in SOD, GSH and TAC, whereas, the MDA was diminished. Hemolymph was collected from normal and treated larvae after 6, 12 and 24 h. The M. domestica superoxide dismutase (MdSOD1) transcripts were significantly (p < 0.05) upregulated 6 and 12 h post-treatment, while a significant downregulation was observed after 24 h. Western blot analysis showed that MdSOD1 was expressed in the hemolymph of the treated larvae with an increase of 1.2 folds at 6 and 12 h and 1.6 folds at 24 h relative to the control group. LPS-treated larval hemolymphs exhibited significant cytotoxicity with respect to the untreated ones against MCF-7 while Vero cells showed no cytotoxicity for both hemolymphs. The DPPH free radical scavenging activity was examined and a significant antioxidant potential potency was observed at 6 h (50% maximal inhibitory concentration (IC50): 63.3 ± 3.51 µg/mL) when compared to the control M. domestica larval hemolymph (IC50: 611.7 ± 10.41 µg/mL). Taken together, M. domestica larval hemolymph exhibited enhanced antioxidant and consequently increased cytotoxic capacities under stressed conditions.

THE EFFECTS OF PYRETHRUM EXTRACT ON Galleria mellonella HEMOLYMPH PHENOLOXIDASE ENZYME

Pyrethrum is a natural neurotoxic insecticide which is obtained from the flowers of Chrysanthemum cinerariaefolium plant. Pyrethrum extract causes DNA damage, genotoxic effect, induction of autophagy and apoptosis, mitochondrial dysfunction, oxidative stress, inhibition of biochemical processes. The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is gaining increasing attention in immunity studies as an invertebrate model organism. Melanization, which is the most important response of invertebrate humoral immunity, occurs when inactive prophenoloxidase turns into phenoloxidase enzyme. Changes in phenoloxidase enzyme activity are an important marker for humoral immunity. In our study, the phenoloxidase enzyme activity of hemolymph collected from G. mellonella larvae treated with different doses of pyrethrum extract was determined by reading against a certain absorbance in an ELISA microplate reader. The findings obtained from this study showed that 0.6 mg/ml pyrethrum extract increased phenoloxidase enzyme activity. Doses above and below this dose did not cause a significant change in phenoloxidase activity compared to control groups. In the evaluation made in terms of the change of enzyme activity over time, while the enzyme activity increased rapidly in the first 15 minutes, the enzyme activity rate decreased after the 20th minute. The effect of pyrethrum extract on phenoloxidase enzyme activity in G. mellonella larval hemolymph at a certain dose is consistent with the literature. The reason for this effect of the extract is closely related to its genotoxic and cytotoxic effects.

Immune Response and Hemolymph Microbiota of Apis Mellifera and Apis Cerana After the Challenge With Recombinant Varroa Toxic Protein

Background: The honeybee is a significant crop pollinator and key model insect for understanding social behavior, disease transmission, and development. The ectoparasitic Varroa destructor mite put threats on the honeybee industry. A Varroa toxic protein (VTP) from the saliva of Varroa mites contributes to the toxicity toward Apis cerana and the DWV elevation in Apis mellifera. However, the immune response and hemolymph microbiota of honeybee species after the injection of recombinant VTP has not yet been reported.Methods: In this study, both A. cerana and A. mellifera worker larvae were injected with the recombinant VTP. Then the expressions of the honeybee immune genes abaecin, defensin and domeless at three time points were determined by qRT-PCR, and hemolymph microbial community were analyzed by culture-dependent method, after recombinant VTP injection. Results: The mortality rates of A. cerana larvae were much higher than those of A. mellifera larvae after VTP challenge. VTP injection induced the up-regulation of defensin gene expression in A. mellifera larvae, and higher levels of abaecin and domeless mRNAs response in A. cerana larvae, compared with the control (without any injection). PBS injection also upregulated the expression levels of abaecin, defensin, and domeless in A. mellifera and A. cerana larvae. Three bacterial species (Enterococcus faecalis, Staphylococcus cohnii and Bacillus cereus) were isolated from the hemolymph of A. cerana larvae after VTP injection and at 48 h after PBS injections. Two bacterial species (Stenotrophomonas maltophilia and Staphylococcus aureus) were isolated from A. mellifera larvae after VTP challenge. No bacterial colonies were detected from the larval hemolymph of both honeybee species treated by injection only and the control. Conclusion: The result indicates that abaecin, defensin, and domeless genes and hemolymph microbiota respond to the VTP challenge. VTP injection might induce the dramatic growth of different bacterial species in the hemolymph of the injected larvae of A. mellifera and A. cerana, which provide cues for further studying the interactions among the honeybee, VTP and hemolymph bacteria.

Ecdysone regulates the Drosophila imaginal disc epithelial barrier, determining the duration of regeneration checkpoint delay

Regeneration of Drosophila imaginal discs, larval precursors to adult tissues, produces a systemic response, a regeneration checkpoint that coordinates regenerative growth with developmental progression. This regeneration checkpoint is coordinated by the release of the relaxin-family peptide Dilp8 from regenerating tissues. Secreted Dilp8 protein can be detected within the imaginal disc lumen. The disc epithelium separates from the lumen from the larval hemolymph and the targets for Dilp8 activity in the brain and prothoracic gland. Here we demonstrate that the imaginal disc epithelial barrier limits Dilp8 signaling and checkpoint delay. We also observe that the wing imaginal disc barrier becomes more restrictive during development, becoming impermeable only at end of the final larval instar. This change in barrier permeability is driven by the steroid hormone ecdysone and correlates with changes in localization of Coracle, a component of the septate junctions that is required for the late, impermeable epithelial barrier. Based on these observations, we propose that the imaginal disc epithelial barrier regulates the duration of the regenerative checkpoint, providing a mechanism by which tissue function can signal the completion of regeneration.Summary StatementEcdysone signaling directs the Drosophila third instar imaginal disc epithelial barrier to mature, becoming more restrictive. This mature barrier limits Dilp8 signaling and determines the duration of the regeneration checkpoint.