Two distinct amphipathic peptide antibiotics with systemic efficacy.

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.

Characterization of the active fragments of Spodoptera litura Lebocin-1.

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.

Identification and characterization of the glycoside hydrolase family 18 genes from the entomopathogenic fungus Isaria cicadae genome.

Fungal chitinases play essential roles in chitin degradation, cell wall remodeling, chitin recycling, nutrition acquisition, autolysis, and virulence. In this study, 18 genes of the glycoside hydrolase 18 (GH18) family were identified in the Isaria cicadae genome. Seventeen of the genes belonged to chitinases and one was an endo-ß-N-acetylglucosaminidase (ENGase). According to phylogenetic analysis, the 17 chitinases were designated as subgroups A (7 chitinases), B (7), and C (3). The exon-intron organizations of these genes were analyzed. The conserved regions DxxDxDxE and S/AxGG and the domains CBM1, CBM18, and CBM50 were detected in I. cicadae chitinases and ENGase. The results of analysis of expression patterns showed that genes ICchiA1, ICchiA6, ICchiB1, and ICchiB4 had high transcript levels in the different growth conditions or developmental stages. Subgroup A chitinase genes had higher transcript levels than the genes of all other chitinases. Subgroup B chitinase genes (except ICchiB7) presented higher transcript levels in chitin medium compared with other conditions. ICchiC2 and ICchiC3 were mainly transcribed in autolysis medium and in blastospores, respectively. Moreover, ICchiB1 presented higher transcript levels than genes of other chitinases. This work provides an overview of the GH18 chitinases and ENGase in I. cicadae and provides a context for the chitinolytic potential, functions, and biological controls of these enzymes of entomopathogenic fungi.

Molecular characterization and expression patterns of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) heat shock protein genes and their response to host stress.

As a polyphagous insect, little is known at the molecular level about the effects of different host plants on physiological changes in Phenacoccus solenopsis. In this study, four heat shock protein (Hsp) genes (PsHsp60, PsHsp70, PsHsp90, and PsHsp20.7) were identified from the transcriptome of P. solenopsis. Analysis of Hsp expression levels revealed significant differences in Hsp gene expression levels in P. solenopsis fed on different host plants. In host conversion tests, the expression levels of PsHsp90 and PsHsp60 were upregulated after transfer of second instar nymphs from tomato to cotton. The expression levels of PsHsp70 and PsHsp20.7 were, respectively, significantly upregulated at 9 and 48 hr after transfer from tomato to Hibiscus. The results of this study aid molecular characterization and understanding of the expression patterns of Hsp genes during different developmental stages and host transfer of P. solenopsis.

Investigation of the antimicrobial activity and biocompatibility of magnesium alloy coated with HA and antimicrobial peptide.

Implant-associated infection is one of the biggest problems in orthopedic surgery. Antimicrobial peptides (AMPs) are well-known components of the innate immunity and less susceptible to the development of pathogen resistance compared to conventional antibiotics. Magnesium alloys as potential biodegradable bone implants have been received much attention in biomaterials field. This study investigated the deposition of calcium phosphate (CaP) coatings and loading of AMPs on the magnesium alloy surface by a biomimetic method. Scanning electron microscope (SEM) results presented that a microporous and plate-like CaP coating was processed on the magnesium alloy surface. X-ray diffractometry (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis showed the main component of coating was hydroxyapatite (HA). Degradation assay in vitro showed that the HA coating deposited onto the magnesium alloy was corroded more slowly than the bare one. The amount of AMP loaded in the HA coating was 11.16±1.99 µg/cm2. The AMP loaded onto HA coatings had slow release for 7 days. The AMP-loaded coating showed antimicrobial activity against Staphylococcus aureus. Its bacterial inhibition rate exceeded 50% after 4 days and the antibacterial effect was sustained for 7 days. The coated magnesium alloys loaded with AMP could improve rat bone marrow mesenchymal stem cells (rBMMSCs) proliferation. Furthermore, it could also promote alkaline phosphatase (ALP) activity of rBMMSCs. Both radiographic evaluation and histopathology analysis demonstrated that implantation of the coated magnesium alloy into the rabbit femoral condyle had promoted bone repair and showed anti-inflammatory effect. The results showed that the AMP loaded onto HA coatings on the magnesium alloy surface could be considered an ideal orthopedic implant against S. aureus infection.

Antibacterial mechanism of the novel antimicrobial peptide Jelleine-Ic and its efficacy in controlling Pseudomonas syringae pv. actinidiae in kiwifruit.

BACKGROUND: Bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) poses a severe threat to kiwifruit production. Because of the insufficient efficacy and environmental safety of available treatments, novel antibacterial agents should be urgently developed. Antimicrobial peptides (AMPs) can be used as antimicrobials for disease control. In this study, we designed a novel AMP, Jelleine-Ic, and evaluated its antibacterial activity and mechanism of action against Psa. RESULTS: Jelleine-Ic with a half-maximal effective concentration of 1.67 µg/mL exhibited stronger antibacterial activity than did parent Jelleine-I. Jelleine-Ic targeted the Psa membrane, increased membrane permeabilization and dissipated membrane potential, resulting in calcium leakage. Electron microscopy revealed that Jelleine-Ic disrupted cell morphology and caused intracellular alterations. Moreover, this AMP penetrated the cell membrane, bound to DNA, and reduced the expression of genes related to DNA replication and repair. Jelleine-Ic also reduced esterase activity and induced intracellular reactive oxygen species generation. This peptide inhibited the development of Psa canker. The control efficiency of Jelleine-Ic against Psa in the leaf discs and leaves of kiwifruit was 81.83% and 70.53%, respectively, which was superior to that of the commercial agricultural streptomycin. Furthermore, Jelleine-Ic upregulated the expression of kiwifruit defense genes (PR-10 and WRKY70a). CONCLUSION: Jelleine-Ic effectively controls Psa in vitro and in vivo, and may be developed as a bactericide for plant disease control. © 2023 Society of Chemical Industry.

In vitro and in vivo antimicrobial activity of antimicrobial peptide Jelleine-I against foodborne pathogen Listeria monocytogenes.

As a human foodborne pathogen, Listeria monocytogenes can cause severe human listeriosis and develop resistance to antibiotics. Antimicrobial peptides (AMPs) are produced from all kingdoms of life and regarded as promising alternatives to conventional antibiotics. Jelleine-I is an AMP identified from honeybees royal jelly. In this study, we explored the activity and action mechanism of Jelleine-I against L. monocytogenes. We found its minimum inhibitory concentration to be 12.5 µg/mL. Membrane permeability analysis revealed that Jelleine-I increased L. monocytogenes cell membrane permeability, causing calcium leakage. Scanning, transmission electron microscopy and fluorescence microscopy revealed that Jelleine-I destroyed membrane integrity, disrupted intracellular structures and interacted with the bacterial DNA. DNA binding analysis demonstrated that Jelleine-I bound to bacterial genomic DNA. Results of reverse transcription-quantitative PCR revealed that Jelleine-I affected bacterial DNA replication gene expression levels. Moreover, Jelleine-I induced cellular reactive oxygen species (ROS) production from fluorescence intensity analysis, and inhibited bacterial biofilm formation. Results of immunomodulation in Galleria mellonella revealed that Jelleine-I increased host hemocyte counts, upregulated host AMP gene (Gloverin and Cecropin D) expression, and inhibited proinfammatory cytokine (tumor necrosis factor α and interleukin 6) production induced by bacterial infection. It efficiently killed bacteria and increased the survival rate of infected insects to 70 %. Furthermore, Jelleine-I increased the G1 to S phase transition in mammalian cells from cells cycle analysis, and cytotoxicity assay results indicated that it promoted cell proliferation without hemolysis or cytotoxicity. Collectively, Jelleine-I possesses antimicrobial, immunomodulatory and cell proliferative activities, and is a promising candidate for preventing L. monocytogenes emergence and dissemination.

Elongation factor 1ß' gene from Spodoptera exigua: characterization and function identification through RNA interference.

Elongation factor (EF) is a key regulation factor for translation in many organisms, including plants, bacteria, fungi, animals and insects. To investigate the nature and function of elongation factor 1ß' from Spodoptera exigua (SeEF-1ß'), its cDNA was cloned. This contained an open reading frame of 672 nucleotides encoding a protein of 223 amino acids with a predicted molecular weight of 24.04 kDa and pI of 4.53. Northern blotting revealed that SeEF-1ß' mRNA is expressed in brain, epidermis, fat body, midgut, Malpighian tubules, ovary and tracheae. RT-PCR revealed that SeEF-1ß' mRNA is expressed at different levels in fat body and whole body during different developmental stages. In RNAi experiments, the survival rate of insects injected with SeEF-1ß' dsRNA was 58.7% at 36 h after injection, which was significantly lower than three control groups. Other elongation factors and transcription factors were also influenced when EF-1ß' was suppressed. The results demonstrate that SeEF-1ß' is a key gene in transcription in S. exigua.

In vitro and in vivo antifungal activity of two peptides with the same composition and different distribution.

Candida albicans can cause local or systemic diseases when host immune status is disrupted. Drug resistance to C. albicans highlights the necessity of novel antifungal drugs. Antimicrobial peptides exhibit potential as antifungal drugs. PAF26 was found to exhibit favorable activity against plant pathogenic fungi. However, it showed low antifungal activity against C. albicans. Here, P255 and P256 with the same composition and different distribution were derived from PAF26. P256 exhibited higher antifungal activity against C. albicans than did P255 and PAF26. P256 and P255 exhibited synergism when combined with amphotericin B (AMB). Both peptides reduced cell wall integrity, rapidly increased membrane permeability, disrupted cell morphology and intracellular alterations. The peptides affected the expression of fungal DNA replication and repair, cell wall synthesis and ergosterol synthesis genes. They increased cellular reactive oxygen species production and bound with fungal genomic DNA. Antibiofilm activities were observed when peptide alone or combined with AMB. Finally, these peptides protected 70% of Galleria mellonella from infection-caused death. Insects treated with peptides exhibited fewer infection foci compared with the untreatment. These results demonstrate the therapeutic potential of the peptides, particularly P256 with clear amphipathicity, in the development of therapies for C. albicans infections.

Silencing ß-1,3-glucan binding protein enhances the susceptibility of Plutella xylostella to entomopathogenic fungus Isaria cicadae.

BACKGROUND: The diamondback moth, Plutella xylostella is a notorious pest of brassicaceae crops globally and has developed serious resistance to insecticide. Insects primarily rely on their innate immunity to defense against various pathogens. In this study, we investigated the immunological functions of a ß-1,3-glucan binding protein from P. xylostella (PxßGBP) and evaluated its potential for biocontrolling P. xylostella. RESULTS: The open reading frame of PxßGBP is 1422 bp encoding 473 amino acids residues. PxßGBP contained a CBM39 domain, a PAC domain and a GH16 domain and shared evolutionary conservation with other lepidoptera ßGRPs. PxßGBP was strongly expressed in the third instar larvae and fat body. PxßGBP transcript levels increased significantly after the challenge with microbes, including Isaria cicadae, Escherichia coli and Staphylococcus aureus. PxßGBP was identified in P. xylostella larvae challenged by I cicadae, but not in the naïve insects. Recombinant PxßGBP can directly bind fungal and bacterial cells, and also agglutinate the cells of I cicadae, S. aureus and E coli in a zinc-dependent manner. Knockdown of PxßGBP via RNA interference significantly down-regulated the expression of antimicrobial peptide gene gloverin, and enhanced the susceptibility of P. xylostella to I. cicadae infection, leading to high mortality. CONCLUSION: These results indicated that PxßGBP plays an important role in the immune response of P. xylostella against I. cicadae infection, and could serve as a potential novel target for pest control. © 2022 Society of Chemical Industry.

Structure and Activity of a Selective Antibiofilm Peptide SK-24 Derived from the NMR Structure of Human Cathelicidin LL-37.

The deployment of the innate immune system in humans is essential to protect us from infection. Human cathelicidin LL-37 is a linear host defense peptide with both antimicrobial and immune modulatory properties. Despite years of studies of numerous peptides, SK-24, corresponding to the long hydrophobic domain (residues 9-32) in the anionic lipid-bound NMR structure of LL-37, has not been investigated. This study reports the structure and activity of SK-24. Interestingly, SK-24 is entirely helical (~100%) in phosphate buffer (PBS), more than LL-37 (84%), GI-20 (75%), and GF-17 (33%), while RI-10 and 17BIPHE2 are essentially randomly coiled (helix%: 7-10%). These results imply an important role for the additional N-terminal amino acids (likely E16) of SK-24 in stabilizing the helical conformation in PBS. It is proposed herein that SK-24 contains the minimal sequence for effective oligomerization of LL-37. Superior to LL-37 and RI-10, SK-24 shows an antimicrobial activity spectrum comparable to the major antimicrobial peptides GF-17 and GI-20 by targeting bacterial membranes and forming a helical conformation. Like the engineered peptide 17BIPHE2, SK-24 has a stronger antibiofilm activity than LL-37, GI-20, and GF-17. Nevertheless, SK-24 is least hemolytic at 200 µM compared with LL-37 and its other peptides investigated herein. Combined, these results enabled us to appreciate the elegance of the long amphipathic helix SK-24 nature deploys within LL-37 for human antimicrobial defense. SK-24 may be a useful template of therapeutic potential.

Spotlight on the Selected New Antimicrobial Innate Immune Peptides Discovered During 2015-2019.

BACKGROUND: Antibiotic resistance is a global issue and new anti-microbials are required. INTRODUCTION: Anti-microbial peptides are important players of host innate immune systems that prevent infections. Due to their ability to eliminate drug-resistant pathogens, AMPs are promising candidates for developing the next generation of anti-microbials. METHODS: The anti-microbial peptide database provides a useful tool for searching, predicting, and designing new AMPs. In the period from 2015-2019, ~500 new natural peptides have been registered. RESULTS: This article highlights a selected set of new AMP members with interesting properties. Teixobactin is a cell wall inhibiting peptide antibiotic, while darobactin inhibits a chaperone and translocator for outer membrane proteins. Remarkably, cOB1, a sex pheromone from commensal enterococci, restricts the growth of multidrug-resistant Enterococcus faecalis in the gut at a picomolar concentration. A novel proline-rich AMP has been found in the plant Brassica napus. A shrimp peptide MjPen- II comprises three different sequence domains: serine-rich, proline-rich, and cysteine-rich regions. Surprisingly, an amphibian peptide urumin specifically inhibits H1 hemagglutinin-bearing influenza A virus. Defensins are abundant and typically consist of three pairs of intramolecular disulfide bonds. However, rat rattusin dimerizes via forming five pairs of intermolecular disulfide bonds. While human LL-37 can be induced by vitamin D, vitamin A induces the expression of resistin-like molecule alpha (RELMα) in mice. The isolation and characterization of an alternative human cathelicidin peptide, TLN-58, substantiates the concept of one gene multiple peptides. The involvement of a fly AMP nemuri in sleep induction may promote the research on the relationship between sleep and infection control. CONCLUSION: The functional roles of AMPs continue to grow and the general term "innate immune peptides" becomes useful. These discoveries widen our view on the anti-microbial peptides and may open new opportunities for developing novel peptide therapeutics for different applications.

Suppression of Transferrin Expression Enhances the Susceptibility of Plutella xylostella to Isaria cicadae.

Transferrins (Trfs) are multifunctional proteins with key functions in iron transport. In the present study, a Trf (PxTrf) from Plutella xylostella was identified and characterized. The PxTrf consisted of a 2046-bp open reading frame, which encoded a 681 amino acid protein with a molecular weight of 73.43 kDa and had an isoelectric point of 7.18. Only a single iron domain was predicted in the N-lobe of PxTrf. Although PxTrf was expressed ubiquitously, the highest levels of expression were observed in the fourth instar larvae. PxTrf transcript level was highest in fat bodies among various tissues. The PxTrf transcript levels increased significantly after the stimulation of pathogens. A decrease in PxTrf expression via RNA interference enhanced the susceptibility of P. xylostella to the Isaria cicadae fungus and inhibited hemocyte nodulation in response to the fungal challenge. In addition, a considerable increase in the pupation rate was observed in larvae treated with double-stranded PxTrf (dsPxTrf). Overall, according to the results, PxTrf may participate in P. xylostella immunity against fungal infection and insect development.

Resistome of Staphylococcus aureus in Response to Human Cathelicidin LL-37 and Its Engineered Antimicrobial Peptides.

Staphylococcus aureus is notoriously known for its rapid development of resistance to conventional antibiotics. S. aureus can alter its membrane composition to reduce the killing effect of antibiotics and antimicrobial peptides (AMPs). To obtain a more complete picture, this study identified the resistance genes of S. aureus in response to human cathelicidin LL-37 peptides by screening the Nebraska Transposon Mutant Library. In total, 24 resistant genes were identified. Among them, six mutants, including the one with the known membrane-modifying gene (mprF) disabled, became more membrane permeable to the LL-37 engineered peptide 17BIPHE2 than the wild type. Mass spectrometry analysis detected minimal lysyl-phosphatidylglycerol (lysylPG) from the mprF mutant of S. aureus JE2, confirming loss-of-function of this gene. Moreover, multiple mutants showed reduced surface adhesion and biofilm formation. In addition, four S. aureus mutants were unable to infect wax moth Galleria mellonella. There appears to be a connection between the ability of bacterial attachment/biofilm formation and infection. These results underscore the multiple functional roles of the identified peptide-response genes in bacterial growth, infection, and biofilm formation. Therefore, S. aureus utilizes a set of resistant genes to weave a complex molecular network to handle the danger posed by cationic LL-37. It appears that different genes are involved depending on the nature of antimicrobials. These resistant genes may offer a novel avenue to designing more potent antibiotics that target the Achilles heels of S. aureus USA300, a community-associated pathogen of great threat.

Membrane activity of two short Trp-rich amphipathic peptides.

Short linear antimicrobial peptides are attractive templates for developing new antibiotics. Here, it is described a study of the interaction between two short Trp-rich peptides, horine and verine-L, and model membranes. Isothermal titration calorimetry studies showed that the affinity of these peptides towards large unilamellar vesicles (LUV) having a lipid composition mimicking the lipid composition of S. aureus membranes is ca. 30-fold higher than that towards E. coli mimetics. The former interaction is driven by enthalpy and entropy, while the latter case is driven by entropy, suggesting differences in the forces that play a role in the binding to the two types of model membranes. Upon membrane binding the peptides acquired different conformations according to circular dichroism (CD) studies; however, in both cases CD studies indicated stacked W-residues. Peptide-induced membrane permeabilization, lipid flip-flop, molecular packing at the membrane-water interface, and lateral lipid segregation were observed in all cases. However, the extent of these peptide-induced changes on membrane properties was always higher in S. aureus than E. coli mimetics. Both peptides seem to act via a similar mechanism of membrane permeabilization of S. aureus membrane mimetics, while their mechanisms seem to differ in the case of E. coli. This may be the result of differences in both the peptides´ structure and the membrane lipid composition between both types of bacteria.

Bactrocerin-1: a novel inducible antimicrobial peptide from pupae of oriental fruit fly Bactrocera dorsalis Hendel.

A novel antimicrobial peptide, Bactrocerin-1, was purified and characterized from an immunized dipteran insect, Bactrocera dorsalis. Bactrocerin-1 has 20 amino acid residues with a mass of 2,325.95 Da. The amino acid sequence of Bactrocerin-1 showed very high similarity to the active fragment (46V-65S-NH(2)) of Coleoptericin A. The composition of amino acid residues revealed that Bactrocerin-1 is a hydrophobic, positively charged, and Lys/Ile/Gly-rich peptide. Minimal growth inhibition concentration (MIC) measurements for synthesized Bactrocerin-1 showed a very broad spectrum of anti-microbial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. Bactrocerin-1 did not show hemolytic activity toward mouse red blood cells even at a concentration of 50 microM. Analysis of the Helical-wheel projection and the CD spectrum suggested that Bactrocerin-1 contains the amphipathic alpha-helix.

Molecular and functional characterization of a c-type lysozyme from the Asian corn borer, Ostrinia furnacalis.

Some lepidopteran lysozymes have been reported to display activity against Gram-positive and Gram-negative bacteria, in contrast to most lysozymes that are active only against Gram-positive bacteria. OstrinLysC, a c-type lysozyme, was purified from the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae), and shows activity against Gram-positive and Gram-negative bacteria. The NH2-terminal amino acid sequence was determined by Edman degradation and used in a homology cloning strategy. The gene coding for OstrinLysC contains three exons and two introns. The expression profile of the OstrinlysC gene was examined by quantitative real-time PCR. Following injection of the larvae with bacteria, the OstrinlysC gene is strongly up-regulated in immune tissues. Transcripts were also detected in gut tissue. After feeding the larvae with bacteria, OstrinlysC transcripts increased in immune tissues. A very low level of transcript abundance was also detected in gut tissue. These results suggested that the OstrinlysC gene is involved in immune responses. The three dimensional structure of OstrinLysC was predicted. Based on comparison of the 3-D structure of OstrinLysC with that of silkworm lysozyme and chicken lysozyme, we hypothesize that the positive charge-rich surface and the short loop-2, which is close to the cluster of hydrophobic residues, may play important roles in the interaction with the outer membrane of Gram-negative bacterial cell walls.

Expression Patterns, Molecular Characterization, and Response to Host Stress of CYP Genes from Phenacoccus solenopsis (Hemiptera: Pseudococcidae).

The quarantine insect pest Phenacoccus solenopsis (Hemiptera: Pseudococcidae) has a broad host range and is distributed worldwide. Each year, P. solenopsis causes significant crop losses. The detoxification of various xenobiotic compounds involves the cytochrome P450 monooxygenase (CYP) superfamily of enzymes. However, the functions of CYPs in P. solenopsis are poorly understood. In the present study, P. solenopsis was reared from the egg to the adult stage on three host plants: Tomato, cotton, and hibiscus. Thirty-seven P. solenopsis CYP genes were identified and their phylogenetic relationships were analyzed. Eleven CYP genes (PsCYP4NT1, PsCYP4G219, PsCYP6PZ1, PsCYP6PZ5, PsCYP301B1, PsCYP302A1, PsCYP305A22, PsCYP315A1, PsCYP353F1, PsCYP3634A1, and PsCYP3635A2) were selected for quantitative real-time PCR analysis. The results demonstrated marked differences in CYP expression levels in P. solenopsis grown on different host plants. The results will aid the molecular characterization of CYPs and will increase our understanding of CYP expression patterns in P. solenopsis during development and growth on different hosts.

Housefly larvae hydrolysate: orthogonal optimization of hydrolysis, antioxidant activity, amino acid composition and functional properties.

BACKGROUND: Antioxidant, one of the most important food additives, is widely used in food industry. At present, antioxidant is mostly produced by chemical synthesis, which would accumulate to be pathogenic. Therefore, a great interest has been developed to identify and use natural antioxidants. It was showed that there are a lot of antioxidative peptides in protein hydrolysates, possessing strong capacity of inhibiting peroxidation of macro-biomolecular and scavenging free redicals in vivo. Enzymatic hydrolysis used for preparation of antioxidative peptides is a new hot-spot in the field of natural antioxidants. It reacts under mild conditions, with accurate site-specific degradation, good repeatability and few damages to biological activity of protein. Substrates for enzymatic hydrolysis are usually plants and aqua-animals. Insects are also gaining attention because of their rich protein and resource. Antioxidative peptides are potential to be exploited as new natural antioxidant and functional food. There is a huge potential market in medical and cosmetic field as well. RESULT: Protein hydrolysate with antioxidant activity was prepared from housefly larvae, by a two-step hydrolysis. Through orthogonal optimization of the hydrolysis conditions, the degree of hydrolysis was determined to be approximately 60%. Fractionated hydrolysate at 25 mg/mL, 2.5 mg/mL and 1 mg/mL exhibited approximately 50%, 60% and 50% of scavenging capacity on superoxide radicals, 1, 1-Diphenyl-2-picrylhydrazyl radicals and hydroxyl radicals, respectively. Hydrolysate did not exhibit substantial ion chelation. Using a linoneic peroxidation system, the inhibition activity of hydrolysate at 20 mg/mL was close to that of 20 µg/mL tertiary butylhydroquinone, suggesting a potential application of hydrolysate in the oil industry as an efficient antioxidant. The lyophilized hydrolysate presented almost 100% solubility at pH 3-pH 9, and maintained nearly 100% activity at pH 5-pH 8 at 0°C- 4°C and room temperature during the first 6 months of storage. Essential amino acids in the hydrolysate accounted for 43% of the total amino acids. CONCLUSIONS: The results suggesting that hydrolysate could be added to food oils as an efficient antioxidant. It might be useful for food additives, diet nutrients and pharmaceutical agents.

Effect of extracted housefly pupae peptide mixture on chilled pork preservation.

The peptide mixture from housefly pupae has broad spectrum antimicrobial activity but has not previously been reported as a food preservative. In this study, the preservation effects of a housefly pupae peptide mixture, nisin, and sodium dehydroacetate (DHA-S) on the number of mesophilic aerobic bacteria (MAB), total volatile basic nitrogen (TVB-N), and pH value of chilled pork were compared. All results showed that a good preservation effect was observed among 3 treatments with the peptide mixture of housefly pupae, nisin, and DHA-S and that there was no significant difference among them. These results indicate that housefly peptide mixture has a great potential as a food preservative. The results of scanning electron microscope and transmission electron microscopy suggest that the primary mechanism of housefly pupae peptide mixture may be bacterial cytoplasmic membrane lysis and pores induced in the membranes. Practical Applications: Peptide mixture extracted from housefly pupae using low-cost and simple method has broad spectrum antimicrobial activity. According to the effect on chilled pork preservation, extracted housefly peptide mixture has a great potential as a food preservative.