Antimicrobial potential of a ponericin-like peptide isolated from Bombyx mori L. hemolymph in response to Pseudomonas aeruginosa infection.

The main effectors in the innate immune system of Bombyx mori L. are antimicrobial peptides (AMPs). Here, we infected B. mori with varied inoculum sizes of Pseudomonas aeruginosa ATCC 25668 cells to investigate changes in morpho-anatomical responses, physiological processes and AMP production. Ultraviolet-visible spectra revealed a sharp change in λmax from 278 to 285 nm (bathochromic shift) in the hemolymph of infected B. mori incubated for 24 h. Further, Fourier Transform InfraRed studies on the hemolymph extracted from the infected B. mori showed a peak at 1550 cm-1, indicating the presence of α-helical peptides. The peptide fraction was obtained through methanol, acetic acid and water mixture (90:1:9) extraction, followed by peptide purification using Reverse Phase High Performance Liquid Chromatography. The fraction exhibiting antibacterial properties was collected and characterized by Matrix-Assisted Laser Desorption/Ionization-Time of Flight. A linear α-helical peptide with flexible termini (LLKELWTKMKGAGKAVLGKIKGLL) was found, corresponding to a previously described peptide from ant venom and here denominated as Bm-ponericin-L1. The antibacterial activity of Bm-ponericin-L1 was determined against ESKAPE pathogens. Scanning electron microscopy confirmed the membrane disruption potential of Bm-ponericin-L1. Moreover, this peptide also showed promising antibiofilm activity. Finally, cell viability and hemolytic assays revealed that Bm-ponericin-L1 is non-toxic toward primary fibroblasts cell lines and red blood cells, respectively. This study opens up new perspectives toward an alternative approach to overcoming multiple-antibiotic-resistance by means of AMPs through invertebrates' infection with human pathogenic bacteria.

Bio-molecule functionalized rapid one-pot green synthesis of silver nanoparticles and their efficacy toward the multidrug resistant (MDR) gut bacteria of silkworms (Bombyx mori).

The present study aimed to synthesise bio-molecule functionalized silver nanoparticles (AgNPs) using leaf extract from mulberry variety S-1635 (Morus alba L.) and to explore its antibacterial efficacy against multidrug resistant (MDR) gut bacteria isolated from natural infection observed from silkworm larvae in rearing conditions. AgNPs formation was established by surface plasmon resonance at 480 nm. The crystallinity of the synthesised AgNPs was checked by HR-TEM and XRD analysis. SEM and TEM characterisation further exhibited the spherical, monodispersed, well scattered nature of the AgNPs with an average particle size of 11.8 nm ± 2.8. The presence of (111), (200), (220) and (311) planes in Bragg's reflections confirmed the face-cantered-cubic crystalline silver. EDX analysis confirmed the presence of elemental silver. FT-IR spectra revealed functional groups were responsible for the reduction of silver ions. The zeta potential value of -17.3 mV and -25.6 mV was recorded in MH and DMEM/F-12 media, respectively. The LC-QTOF/MS and HRMS spectra disclosed the presence of bioactive compounds like flavonoid, gallic acid, and stigmasterol, which are probably involved in the reduction and functionalization of AgNPs. The antibacterial efficacy of bio-molecule functionalized AgNPs and the naked AgNPs was tested on Gram-positive and Gram-negative bacteria isolated from silkworms and characterized by using 16S rDNA and gyrB genes. The cytotoxicity of AgNPs was tested on WRL-68, HEK-293, ACHN, and HUH-7 cell lines using MTT assay. This study provides an insight into the application of bio-molecule functionalized AgNPs for combating various silkworm pathogens which severely affect the agro-rural economy of developing countries.

Effect of feed supplementation with biosynthesized silver nanoparticles using leaf extract of Morus indica L. V1 on Bombyx mori L. (Lepidoptera: Bombycidae).

Herein, we report the synthesis of silver nanoparticles (AgNPs) by a green route using the aqueous leaf extract of Morus indica L. V1. The synthesized AgNPs exhibited maximum UV-Vis absorbance at 460 nm due to surface plasmon resonance. The average diameter (~54 nm) of AgNPs was measured from HR-TEM analysis. EDX spectra also supported the formation of AgNPs, and negative zeta potential value (-14 mV) suggested its stability. Moreover, a shift in the carbonyl stretching (from 1639 cm-1 to 1630 cm-1) was noted in the FT-IR spectra of leaf extract after AgNPs synthesis which confirm the role of natural products present in leaves for the conversion of silver ions to AgNPs. The four bright circular rings (111), (200), (220) and (311) observed in the selected area electron diffraction pattern are the characteristic reflections of face centered cubic crystalline silver. LC-MS/MS study revealed the presence of phytochemicals in the leaf extract which is responsible for the reduction of silver ions. MTT assay was performed to investigate the cytotoxicity of AgNPs against two human cell lines, namely HepG2 and WRL-68. The antibacterial study revealed that MIC value of the synthesized AgNPs was 80 µg/ml against Escherichia coli K12 and Staphylococcus aureus (MTCC 96). Finally, the synthesized AgNPs at 10 µg/ml dosages showed beneficial effects on the survivability, body weights of the Bombyx mori L. larvae, pupae, cocoons and shells weights via enhancing the feed efficacy.