Degumming and characterization of Bombyx mori and non-mulberry silks from Saturniidae silkworms.

In this study, cocoons and degummed silk samples of Bombyx mori and twenty Saturniidae species of the genera Actias, Attacus, Argema, Antheraea, Caligula, Callosamia, Cricula, Epiphora, Hyalophora, Loepa, Samia and Saturnia are studied to gain an insight into their morphology, chemical composition and physical structure. For this purpose, silk samples are characterized by optical microscopy and FTIR spectroscopy in attenuated total reflection mode (ATR-FTIR spectroscopy). Furthermore, degummed silk samples are analyzed for their amino acid (AA) composition by GC-FID. In the course of method development, various degumming methods are tested using alkalis, citric acid, enzymes and detergents. A mixture of 0.1% sodium carbonate and 2.5% ethylenediamine proves to be an effective agent for degumming Saturniidae and B. mori cocoons. After hydrolysis of the fibroin filaments with 6 N hydrochloric acid and derivatization with propyl chloroformate, fifteen AAs are identified and qualified. This method shows a satisfactory overall analytical performance with an average recovery rate of 95% at the medium concentration level. The chemical composition of the different silks was considered comparatively. Within a genus, the analyses usually show a high degree of similarity in AA composition and the resulting structural indices, whereas differences are found between genera.

Design and Analysis of a Mutant form of the Ice-Binding Protein from Choristoneura fumiferana.

Ice-binding proteins are expressed in the cells of some cold adapted organisms, helping them to survive at extremely low temperatures. One of the problems in studying such proteins is the difficulty of their isolation and purification. For example, eight cysteine residues in the cfAF (antifreeze protein from the eastern spruce budworm Choristoneura fumiferana) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. To overcome this issue, in this work, we designed a mutant form of the ice-binding protein cfAFP, which is much easier to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in the presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and does not decrease the water freezing temperature.

Novel antimicrobial anionic cecropins from the spruce budworm feature a poly-L-aspartic acid C-terminus.

Cecropins form a family of amphipathic α-helical cationic peptides with broad-spectrum antibacterial properties and potent anticancer activity. The emergence of bacteria and cancer cells showing resistance to cationic antimicrobial peptides (CAMPs) has fostered a search for new, more selective and more effective alternatives to CAMPs. With this goal in mind, we looked for cecropin homologs in the genome and transcriptome of the spruce budworm, Choristoneura fumiferana. Not only did we find paralogs of the conventional cationic cecropins (Cfcec+ ), our screening also led to the identification of previously uncharacterized anionic cecropins (Cfcec- ), featuring a poly-l-aspartic acid C-terminus. Comparative peptide analysis indicated that the C-terminal helix of Cfcec- is amphipathic, unlike that of Cfcec+ , which is hydrophobic. Interestingly, molecular dynamics simulations pointed to the lower conformational flexibility of Cfcec- peptides, relative to that of Cfcec+ . Phylogenetic analysis suggests that the evolution of distinct Cfcec+ and Cfcec- peptides may have resulted from an ancient duplication event within the Lepidoptera. Finally, we found that both anionic and cationic cecropins contain a BH3-like motif (G-[KQR]-[HKQNR]-[IV]-[KQR]) that could interact with Bcl-2, a protein involved in apoptosis; this observation is congruent with previous reports indicating that cecropins induce apoptosis. Altogether, our observations suggest that cecropins may provide templates for the development of new anticancer drugs. We also estimated the antibacterial activity of Cfcec-2 and a ∆Cfce-2 peptide as AMPs by testing directly their ability in inhibiting bacterial growth in a disk diffusion assay and their potential for development of novel therapeutics.

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.

Key Amino Acid Residues Influencing Binding Affinities of Pheromone-Binding Protein from Athetis lepigone to Two Sex Pheromones.

Athetis lepigone is a polyphagous pest found around the world that feeds on maize, wheat, and various other important crops. Although it exhibits a degree of resistance to various chemical insecticides, an effective pest-control method has not yet been developed. The sex pheromone communication system plays an essential role in the mating and reproduction of moths, in which pheromone-binding proteins (PBPs) are crucial genes. In this study, we cloned and purified the protein AlepPBP1 using an E. coli expression system and found it had a higher binding affinity to two sex pheromones of A. lepigone, namely, Z7-12:Ac and Z9-14:Ac (with Ki 0.77 ± 0.10 and 1.10 ± 0.20 µM, respectively), than to other plant volatiles. The binding-mode analysis of protein conformation with equilibrium stabilization was obtained using molecular dynamics (MD) simulation and indicated that hydrophobic interactions involving several nonpolar residues were the main driving force for the binding affinity of AlepPBP1 with sex pheromones. Computational alanine scanning (CAS) was performed to further identify key amino acid residues and validate their binding contributions. Each key residue, including Phe36, Trp37, Val52, and Phe118, was subsequently mutated into alanine using site-directed mutagenesis. Binding assays showed that the efficient binding abilities to Z7-12:Ac (F36A, W37A, and F118A) and Z9-14:Ac (F36A, W37A, V52A, and F118A) were almost lost in the mutated proteins. Our results demonstrated that these key amino acid residues are crucial for determining the binding ability of AlepPBP1 to sex pheromones. These findings provide a basis for the use of AlepPBP1 in the studies as a specific target for the development of novel behavioral antagonists with marked inhibition or mating-disruption abilities using computer-aided drug design (CADD).

Antifungal Activity of Anionic Defense Peptides: Insight into the Action of Galleria mellonella Anionic Peptide 2.

Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.

Purification and Characterization of a Novel Rhamnose/Fucose-Specific Lectin from the Hemolymph of Oak Tasar (Antheraea proylei J.) Silkworm.

BACKGROUND: Lectins are proteins or glycoproteins of non-immune origin which bind specifically but reversibly to carbohydrates or glycoconjugates. They play a crucial role in various biological processes including host defense mechanism, inflammation and metastasis. Therefore, there is an expanding scientific emphasis on purification and characterization of novel lectins possessing different useful biological properties. OBJECTIVE: The present investigation is concerned with purification and characterization of a novel lectin from the hemolymph of oak tasar (Antheraea proylei J.) silkworm. METHODS: The lectin was purified from the hemolymph by a procedure involving successive steps of hemocyte-free hemolymph preparation, ammonium sulfate (0-40%) fractionation and affinity chromatography on a column of Sephadex G-50 covalently coupled with L-rhamnose. It was then characterized by various physico-chemical methods including SDS-PAGE, gel filtration, hemagglutination assay, hemagglutination inhibition assay and tandem mass spectrometry (LCMS/ MS) coupled with Mascot sequence matching software (Matrix Science). RESULTS: The lectin was purified to electrophoretic homogeneity from the silkworm hemolymph and was found to be a monomeric protein with a native molecular weight of 39.5 kDa. It was specifically inhibited by L-rhamnose and D-fucose, the former being sixteen times more inhibitory than the latter. The hemagglutinating activity was further characterized by independency of metal ion, optimum at pH 7-7.5 and thermal stability with t1/2 of 60°C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not purified and characterized earlier. CONCLUSION: A novel rhamnose/fucose-specific lectin was purified to electrophoretic homogeneity from the hemolymph of oak tasar (Antheraea proylei J.) silkworm. The lectin was found to be a monomeric protein with a native molecular weight of 39.5 kDa. Its activity was found to be independent of metal ion, optimum at pH 7-7.5 and characterized by thermal stability with t1/2 of 60°C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not characterized earlier.

Cationic Antheraea pernyi Silk Fibroin-Modified Adenovirus-Mediated ING4 and IL-24 Dual Gene Coexpression Vector Suppresses the Growth of Hepatoma Carcinoma Cells.

INTRODUCTION: Cancer gene therapy requires both effective tumor suppressor genes and safe vectors that express target genes efficiently. Inhibitor of growth 4 (ING4) inhibits tumor growth via multiple pathways. Interleukin-24 (IL-24) also has tumor-suppressive activity against a broad spectrum of human cancers. Adenovirus (Ad) vectors exhibit high infection efficiency, but potential toxicity related to high doses of adenovirus has led to careful reconsideration of their use in human clinical trials. Antheraea pernyi silk fibroin (ASF) is a cytocompatible and biodegradable natural polymer, and it possesses Arg-Gly-Asp sequences exhibiting a high binding affinity and selectivity for αvß3 and αvß5 integrin receptors, which are overexpressed in tumor vessels and most tumor cells. METHODS: In this study, an Arg-Gly-Asp peptide-modified Ad vector coexpressing ING4 and IL-24 was constructed by homologous recombination of the dual gene coexpression transfer plasmid and RGD-modified pAdEasy-1 adenoviral backbone plasmid. The cationic ASF (CASF) was prepared by modifying ASF with low-molecular-weight PEI. The negatively charged Ad vector was modified with CASF to form a CASF/Ad complex. RESULTS: Human hepatoma carcinoma SMMC-7721 cells and normal hepatic L-02 cells were infected with the CASF/Ad complex, which showed significantly higher infection efficiency than the naked Ad. The CASF/Ad complex could effectively mediate the expression of the target gene ING4 in SMMC-7721 cells and the secretion of the target gene IL-24 from SMMC-7721 cells, thus inducing apoptosis of hepatoma carcinoma SMMC-7721 cells. The viability of SMMC-7721 and L-02 cells infected with the CASF/Ad complex was further assessed, and it was found that the growth of SMMC-7721 cells was significantly inhibited but that the growth and proliferation of L-02 cells were not affected. CONCLUSION: The CASF/Ad complex constructed in this study, showing improved infection efficiency and enhanced suppressive effects on human hepatoma carcinoma SMMC-7721 cells, has the potential to reduce the dose of adenovirus and still maintain high infection efficiency and tumor inhibition.

Pheromone Perception: Mechanism of the Reversible Coil-Helix Transition in Antheraea polyphemus Pheromone-Binding Protein 1.

Pheromone-binding protein (PBP) in male moth antennae transports pheromone to the olfactory receptor neuron by undergoing a pH-dependent conformational switch, from PBPB at higher pH to PBPA at lower pH, associated with ligand binding and release, respectively. The characteristic feature of the dramatic protein switch is the pH-dependent reversible coil-helix transition of the C-terminus. In the PBPB conformation at pH >6.0, the C-terminus is exposed to the solvent as a coil while the ligand occupies the hydrophobic pocket. However, in the PBPA conformation at acidic pH, the C-terminus switches to a helix and releases the ligand by outcompeting it for the hydrophobic pocket. In Antheraea polyphemus PBP1 (ApolPBP1), the C-terminus (P129-V142) is composed predominantly of hydrophobic residues except for three strategically located acidic residues: Asp132, Glu137, and Glu141. Here, we report for the first time on the consequences of the mutation of one or more acidic residues in the pH-driven reversible coil-helix transition of the ApolPBP1 C-terminus through biophysical characterization. Mutation of any single acidic residue in the C-terminus to its neutral counterpart destabilizes the helix formation at lower pH; these mutants exist as a mixture of both conformations. However, mutation of the two terminal acidic residues together knocks out the protein switch and adversely affects both ligand binding and release functions. Thus, these mutant proteins remain in the open (PBPB) conformation at all pH levels.

The Role of Tyramine ß-Hydroxylase in Density Dependent Immunityof Oriental Armyworm (Mythmina separata) Larva.

High population density alters insect prophylactic immunity, with density-dependent prophylaxis (DDP) being reported in many polyphonic insects. However, the molecular mechanism for DDP remains unclear. In current study, the role of tyramine ß-hydroxylase (Tßh) in the immune response of M. separata larvae that were subject to different rearing densities conditions was investigated. The tyramine ß-hydroxylase activity of larvae from high density treatments (10 and 30 larvae per jar) was significantly higher than that of the larvae from low density treatments (one, two, and five larvae/jar). A tyramine ß-hydroxylase (designated MsTßh) containing a 1779 bp open reading frame was identified. Multiple sequence alignment and phylogenetic analysis indicated that MsTßh was orthologous to the Tßh that was found in other lepidopterans. Elevated MsTßh expression was observed in larvae under high density (10 larvae per jar). Silencing MsTßh expression by the injection of dsRNA in larvae from the high density treatment produced a 25.1% reduction in octopamine levels, while at the same time, there was a significant decrease in phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival against Beauveria infection 56.6%, 88.5%, 82.0%, and 55.8%, respectively, when compared to control larvae. Our findings provide the first insights into how MsTßh mediates the octopamine level, which in turn modulates the immune response of larvae under different population densities.

Evaluation of Cyclosaplin Efficacy Using a Silk Based 3D Tumor Model.

Development of novel anti-cancer peptides requires a rapid screening process which can be accelerated by using appropriate in vitro tumor models. Breast carcinoma tissue is a three-dimensional (3D) microenvironment, which contains a hypoxic center surrounded by dense proliferative tissue. Biochemical clues provided by such a 3D cell mass cannot be recapitulated in conventional 2D culture systems. In this experiment, we evaluate the efficacy of the sandalwood peptide, cyclosaplin, on an established in vitro 3D silk breast cancer model using the invasive MDA-MB-231 cell line. The anti-proliferative effect of the peptide on the 3D silk tumor model is monitored by alamarBlue assay, with conventional 2D culture as control. The proliferation rate, glucose consumed, lactate dehydrogenase (LDH), and matrix metalloproteinase 9 (MMP-9) activity of human breast cancer cells are higher in 3D constructs compared to 2D. A higher concentration of drug is required to achieve 50% cell death in 3D culture than in 2D culture. The cyclosaplin treated MDA-MB-231 cells showed a significant decrease in MMP-9 activity in 3D constructs. Microscopic analysis revealed the formation of cell clusters evenly distributed in the scaffolds. The drug treated cells were less in number, smaller and showed unusual morphology. Overall, these findings indicate the role of cyclosaplin as a promising anti-cancer therapeutic.

Immunomodulatory injectable silk hydrogels maintaining functional islets and promoting anti-inflammatory M2 macrophage polarization.

Islet transplantation is considered the most promising treatment for type 1 diabetes. However, the clinical success is limited by islet dysfunction in long-term culture. In this study, we have utilized the rapid self-gelation and injectability offered by blending of mulberry silk (Bombyx mori) with non-mulberry (Antheraea assama) silk, resulting in a biomimetic hydrogel. Unlike the previously reported silk gelation techniques, the differences in amino acid sequences of the two silk varieties result in accelerated gelation without requiring any external stimulus. Gelation study and rheological assessment depicts tuneable gelation as a function of protein concentration and blending ratio with minimum gelation time. In vitro biological results reveal that the blended hydrogels provide an ideal 3D matrix for primary rat islets. Also, A. assama fibroin with inherent Arg-Gly-Asp (RGD) shows significant influence on islet viability, insulin secretion and endothelial cell maintenance. Furthermore, utility of these hydrogels demonstrate sustained release of Interleukin-4 (IL-4) and Dexamethasone with effective M2 macrophage polarization while preserving islet physiology. The immuno-informed hydrogel demonstrates local modulation of inflammatory responses in vivo. Altogether, the results exhibit promising attributes of injectable silk hydrogel and the utility of non-mulberry silk fibroin as an alternative biomaterial for islet encapsulation.

Identification of Key Residues Associated with the Interaction between Plutella xylostella Sigma-Class Glutathione S-Transferase and the Inhibitor S-Hexyl Glutathione.

Glutathione S-transferases (GSTs) are important detoxification enzymes involved in the development of metabolic resistance in Plutella xylostella. Uncovering the interactions between representative PxGSTs and the inhibitor S-hexyl glutathione (GTX), helps in the development of effective PxGST inhibitors for resistance management. As the PxGST most severely inhibited by GTX, PxGSTσ (sigma-class PxGST) adopts the canonical fold of insect GSTs. The formation of the PxGSTσ-GTX complex is mainly driven by H-bond and hydrophobic interactions derived from the side chains of favorable residues. Of the residues composing the active site of PxGSTσ, Lys43 and Arg99 are two hot spots, first reported in the binding of GSH derivatives to GSTs. Such differences indicate the metabolism discrimination of different insect GSTs. Unfavorable interactions between the PxGSTσ active site and GTX are depicted as well. The research guides the discovery and optimization of PxGSTσ inhibitors.

Molecular Characterization and Expression of Vitellogenin and Vitellogenin Receptor of Thitarodes pui (Lepidoptera: Hepialidae), an Insect on the Tibetan Plateau.

Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in the vitellogenesis of insects. In this study, we cloned and characterized the two corresponding genes (TpVg and TpVgR) in an economically important insect, Thitarodes pui (Lepidoptera: Hepialidae), from the Tibetan plateau. The full length of TpVg is 5566 bp with a 5373 bp open reading frame (ORF) encoding 1,790 amino acids. Sequence alignment revealed that TpVg has three conserved domains: a Vitellogenin_N domain, a DUF1943 domain, and a von Willebrand factor type D domain (VWD). The full length of TpVgR is 5732 bp, with a 5397 bp ORF encoding 1798 amino acids. BLASTP showed that TpVgR belongs to the low-density lipoprotein receptor (LDLR) gene superfamily. Structural analysis revealed that TpVgR has a group of four structural domains: a ligand-binding domain (LBD), an epidermal growth factor (EGF)-precursor homology domain, a transmembrane (TM) domain, and a cytoplasmic domain. In addition, TpVgR has four cysteine-rich LDL repeats in the first ligand-binding site and seven in the second. Quantitative real-time polymerase chain reaction analysis revealed that the expression levels of TpVg and TpVgR are much higher in later pupa than in either the larval or adult stage, implying that the synthesis and uptake of Vg in T. pui occurs in the later pupal stage. These results will help us to understand the molecular mechanism of the reproductive capacity and will provide new insight into the mass rearing and utilization of T. pui.

Effect of antimicrobial peptides from Galleria mellonella on molecular models of Leishmania membrane. Thermotropic and fluorescence anisotropy study.

Antimicrobial peptides are molecules of natural origin, produced by organisms such as insects, which have focused attention as potential antiparasitic agents. They can cause the death of parasites such Leishmania by interacting with their membrane. In this study, additional information was obtained on how the anionic peptide 2 and cecropin D-like peptide derived from Galleria mellonella interact with liposomes that mimic the composition of the Leishmania membrane. In order to do this, lipid bilayers consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, and dimyristoylphosphatidylglycerol were constructed. The effect of the peptides on these membranes was evaluated using calorimetry analysis and fluorescence spectroscopy. The results obtained using differential scanning calorimetry indicated a concentration-dependent effect on membranes composed of phosphatidylserine and phosphatidylglycerol, showing a preference of both peptides for anionic lipids. The binding of the peptides drastically reduced the transition enthalpy in the phosphatidylserine and phosphatidylglycerol liposomes. The results suggest that the mode of action of anionic peptide 2 and cecropin D-like peptide is different, with a higher effect of cecropin D-like on the anionic lipids, which led to changes in the main transition temperature and a complete solubilization of the vesicles. Interactions between peptides and phosphatidylcholine, which is the most abundant lipid on the surface of Leishmania cells, were evaluated using isothermal titration calorimetry and the anisotropy of fluorescence of DPH. The peptides had a slight effect on the gel phase of the phosphatidylcholine, with changes in the anisotropy correlated with that observed by DSC. The results showed a selectivity of these peptides toward some lipids, which will direct the study of the development of new drugs.

Proteomic analysis of labial saliva of the generalist cabbage looper (Trichoplusia ni) and its role in interactions with host plants.

Insect saliva is one of the first secretions to come in contact with plants during feeding. The composition and role of caterpillar saliva has not been as thoroughly studied as that of sucking insects. This study focuses on characterizing the proteome of the cabbage looper (Trichoplusia ni) saliva using iTRAQ labeling and LC-MS/MS. We also measured how the saliva proteome changed when larvae were reared on different diets - cabbage, tomato, and an artificial pinto bean diet. We identified 254 proteins in the saliva out of which 63 were differentially expressed. A large percentage (56%) of the proteins identified function in protein metabolism, followed by proteins involved in vesicle transport (6%) and oxidoreductase activity (5%), among other categories. Several proteins identified are antioxidants or reactive oxygen species (ROS) scavengers. Among these ROS scavengers, we identified a catalase and further analyzed its gene expression and enzymatic activity. We also applied commercial, purified catalase on tomato and measured the activity of defensive proteins - trypsin proteinase inhibitor, polyphenol oxidase and peroxidase. Catalase gene expression was significantly higher in the salivary glands of larvae fed on tomato. Also, catalase suppressed the induction of tomato trypsin proteinase inhibitor levels, but not the induction of polyphenol oxidase or peroxidase. These results add to our understanding of proteomic plasticity in saliva and its role in herbivore offense against plant defenses.

Mating-Induced Differential Peptidomics of Neuropeptides and Protein Hormones in Agrotis ipsilon Moths.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used next-generation RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion, and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed postmating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

A small heat shock protein 21 (sHSP21) mediates immune responses in Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) are conserved among insects and play an important role in the regulation of many biological processes, including temperature stress, abiotic stress, immune responses, metamorphosis, and embryo development. Antheraea pernyi is an economically valuable silk-producing moth and source of insect food containing high-quality protein. The aim of this study was to quantify expression of the ApsHSP21 gene in response to pathogen-associated molecular patterns (PAMPs) and nucleopolyhedrovirus (NPV) challenge. The deduced ApsHSP21 protein sequence consists of 186 residues with a calculated molecular mass of 21.0 kDa and an isoelectronic point (pI) of 6.63. The protein contains a conserved α-crystallin domain (ACD), and includes two casein kinase II phosphorylation sites, a protein kinase C phosphorylation site, two tyrosine kinase phosphorylation sites, and various polypeptide binding sites. Phylogenetic analysis revealed that ApsHSP21 is closely related to homologs from other insects. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that expression of ApsHSP21 was significantly up-regulated at different timepoints following simulated pathogen challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan, and NPV. The results suggest sHSP21 is involved in innate immune responses in A. pernyi.

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.

Role of Antheraea pernyi serpin 12 in prophenoloxidase activation and immune responses.

Serine protease inhibitors play a key role in the immune system of invertebrates by controlling proteolytic cascades. Besides its importance, the knowledge on immune functions of serpins in most of insects is fragmentary. In the present study, we identified serpin-12 from Antheraea pernyi encoding a predicted 402 amino acid residue protein (Apserpin-12). We expressed the recombinant protein in Escherichia coli and the purified protein was used for the synthesis of rabbit anti-Apserpin-12 polyclonal antibodies and functional studies. Quantitative real-time ploymerase chain reaction (qRT-PCR) analysis revealed that the knock-down of Apserpin-12 enhanced the prophenoloxidase (PPO) cascade stimulated by Micrococcus luteus in hemolymph, whereas addition of recombinant Apserpin-12 protein along with same elicitor led to down-regulate PPO activation. Following different microbial challenge (E. coli, Beauveria bassiana, M. Luteus, and nuclear polyhedrosis virus), the expression of Apserpin-12 mRNA was induced significantly. Furthermore, the Apserpin-12 double-stranded RNA administration elicited the expression of antimicrobial peptides, while the treatment with recombinant protein suppressed their expression. Tissue profile of Apserpin-12 indicated that it is expressed in all examined tissues, that is, hemolymph, malpighian tubules, midgut, silk gland, integument, and fat body with variation in their transcript levels. We concluded that Apserpin-12 may regulate PPO activation and inhibit the production of antimicrobial peptides in A. pernyi, suggesting important role in its immune system.