Cytotoxic effects of ß-asarone on Sf9 insect cells.

ß-Asarone is the predominant component of the essential oil of rhizomes of Acorus calamus Linn ( Sweet flag). Although rhizome extracts from this plant have long been used for insect pest control, their cytotoxic effects on insect cells are not well understood. In this study, we evaluated the potency of ß-asarone as a natural insecticide by using a Spodoptera frugiperda cell line (Sf9). To assess the cytotoxic effects of ß-asarone on Sf9 cells, we observed morphologic changes in treated cells and performed a cell proliferation assay and a DNA fragmentation assay. After 24 and 48 h of treatment with ß-asarone, the proliferation of the Sf9 cells was inhibited in a dose-dependent manner, with IC50 values of 0.558 mg/ml at 24 h and 0.253 mg/ml at 48 h. Morphologic changes in ß-asarone-treated cells were typical of apoptosis and included loss of adhesion, cell shrinkage, and small apoptotic bodies. The DNA laddering present in ß-asarone-treated SF9 cells and annexin V assay confirmed that this compound can induce apoptosis in insect cells. Together, these findings suggest that apoptosis induction may be one mechanism through which ß-asarone inhibits the proliferation of insect cells and thus exerts insecticidal effects.

Effects of two transglutaminases on innate immune responses in the oriental armyworm, Mythimna separata.

Transglutaminase (TGase) is a key enzyme that mediates hemolymph coagulation and is thought to contribute to the elimination of pathogenic microorganisms in invertebrates. The objective of this study was to elucidate the involvement of TGase in insect immune responses via functional analysis of this enzyme in the oriental armyworm, Mythimna separata, using recombinant proteins and RNA interference technique. We identified two TGase genes, mystgase1 and mystgase2, in Mythimna separata and found that both genes are expressed in all surveyed tissues in M. separata larvae. Significant changes were induced in hemocytes following Escherichia coli injection. Injection of Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Escherichia coli and Serratia marcescens) into larvae triggered a time-specific induction of both mystgase1 and mystgase2 in hemocytes. Recombinant MysTGase1 and MysTGase2 proteins bound to both E. coli and M. luteus, localizing within bacterial clusters and resulting in agglutination in a Ca2+-dependent manner. The hemocytes of larvae injected with recombinant MysTGase1 or MysTGase2 exhibited enhanced phagocytic ability against E. coli, improved in vivo bacterial clearance, and increased resistance to S. marcescens, decreasing larval mortality rate. Conversely, RNA interference targeting mystgase1 or mystgase2 significantly reduced hemocyte phagocytic capability, decreased bacterial clearance, and increased susceptibility to S. marcescens infection, thereby increasing larval mortality rate. The findings of this study are anticipated to expand our understanding of the function of TGases within insect immune responses and may contribute to developing new pest control strategies.

Cloak Scavenges the Reactive Oxygen Species around the Larvae of Drino inconspicuoides (Diptera: Tachinidae).

Drino inconspicuoides (Diptera: Tachinidae) is an endoparasitoid that develops inside the lepidopteran host. When the larva of D. inconspicuoides penetrates into the host, Mythimna separata (Lepidoptera: Noctuidae), the larva creates a cap-like structure, called the funnel, by using host hemocytes, forming a respiratory attachment to permit efficient respiration. A newly described cloudy and cottony structure, called the "cloak", is formed outside the funnel within 24 h of parasitism. The cloak contains the host fat body and hemocytes. In this study, we aimed to clarify the function of the cloak, which has to date remained unknown. We hypothesized that the funnel generates reactive oxygen species (ROS) through melanization, and that the cloak detoxifies them. We confirmed that the black pigments of the funnel were caused by melanization, which inevitably generates ROS that are potentially harmful to the D. inconspicuoides larva inside the funnel. The cloak showed high activities of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, and catalase. These results suggest that the cloak scavenged the ROS from the melanized funnel through the diversion of antioxidant enzymes in the fat body, thereby protecting the D. inconspicuoides larva from oxidative damage.

Inoculation with black soldier fly larvae alters the microbiome and volatile organic compound profile of decomposing food waste.

The black soldier fly (BSF; Hermetia illucens) is used in sustainable processing of many types of organic waste. However, organic waste being decomposed by BSF produces strong odors, hindering more widespread application. The odor components and how they are produced have yet to be characterized. We found that digestion of food waste by BSF significantly alters the microbial flora, based on metagenomic analyses, and the odor components generated, as shown by thermal desorption gas chromatography mass spectrometry analysis. Inoculation with BSF significantly decreased production of volatile organic sulfur compounds (dimethyl disulfide and dimethyl trisulfide), which are known to be released during methionine and cysteine metabolism by Lactobacillus and Enterococcus bacteria. BSF inoculation significantly changed the abundance of Lactobacillus and Enterococcus and decreased microbial diversity overall. These findings may help in optimizing use of BSF for deodorization of composting food waste.

An Effective Chemical Permeabilization of Silkworm Embryos.

The lipid layer surrounding the vitelline membrane of insect eggs has a critical role in the waterproofing and desiccation resistance of embryos. However, this lipid layer also prevents the flux of chemicals into the embryos, such as cryoprotectants, which are required for successful cryopreservation. The permeabilization studies of silkworm embryos remain insufficient. Therefore, in this study, we developed a permeabilization method to remove the lipid layer in the silkworm, Bombyx mori, and examined factors affecting the viability of dechorionated embryos, including the types and exposure times of chemicals and embryonic stages. Among the chemicals used, hexane and heptane were effective for permeabilization, whereas Triton X-100 and Tween-80 were less effective. Regarding the embryonic stages, there were significant differences between 160 and 166 h after egg laying (AEL) at 25 °C. Consequently, we found that the treatment of 160 AEL embryos with hexane for 30 s was the best condition for the permeability and viability of embryos, in which over 62% of the permeabilized embryos grew up to the second larval instar and their moths could lay fertilized eggs. Our method can be used for various purposes, including permeability investigations using other chemicals and embryonic cryopreservation.

Reference Genome Sequences of the Oriental Armyworm, Mythimna separata (Lepidoptera: Noctuidae).

Lepidopteran insects are an important group of animals, including those used as biochemical and physiological model species in the insect and silk industries as well as others that are major agricultural pests. Therefore, the genome sequences of several lepidopteran insects have been reported. The oriental armyworm, Mythimna separata, is an agricultural pest commonly used to study insect immune reactions and interactions with parasitoid wasps as hosts. To improve our understanding of these research topics, reference genome sequences were constructed in the present study. Using long-read and short-read sequence data, de novo assembly and polishing were performed and haplotigs were purged. Subsequently, gene predictions and functional annotations were performed. To search for orthologs of the Toll and Immune Deficiency (IMD) pathways and for C-type lectins, annotation data analysis, BLASTp, and Hummer scans were performed. The M. separata genome is 682 Mbp; its contig N50 was 2.7 Mbp, with 21,970 genes and 24,452 coding sites predicted. All orthologs of the core components of the Toll and IMD pathways and 105 C-type lectins were identified. These results suggest that the genome data were of sufficient quality for use as reference genome data and could contribute to promoting M. separata and lepidopteran research at the molecular and genome levels.

Effect of chemical dechorionation on silkworm embryo viability.

The chorion covering/protecting insect egg, which has some effective functions such as providing mechanical strength, protecting eggs from external environments, and keeping moisture adjustment, is one of the principal barriers to manipulation, cryopreservation, and study of insect embryos. Here we evaluated the silkworm embryo viability after dechorionation using chemical reagents. We have developed an easy and effective method for chemical dechorionation that enables embryos to develop in culture, so that the larvae could normally grow. Eggs attached to a nylon net were treated with potassium hydroxide (KOH) and sodium hypochlorite (NaClO) to remove the chorion, washed with the Grace's insect medium, and then cultured using a dry-moist method which we created. The most effective treatment with regard to embryonic development, hatching, and production of second instar larvae was 30% KOH for 7 min and 2% NaClO for 5 min at 27 °C. Embryos at later embryonic stages were more tolerant to chemical dechorionation and over 75% of embryos treated at 168 h-old (Stage 25, appearance of taenidium) survived to the second larval instar, moreover, the larvae derived from the dechorionated embryos have developed into the moths which can lay the fertilized eggs. Our method would contribute to the establishment of cryopreservation using embryos and analysis of silkworm embryogenesis and might also be applicable to other insect species.

CpG methylation in the hexamerin 110 gene in the European honeybee, Apis mellifera.

The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), has a full set of machinery for functional CpG methylation of its genome. A recent study demonstrated that DNA methylation in the honeybee is involved in caste differentiation. In this study, the expression and methylation of the hexamerin 110 gene (Hex110), which encodes a storage protein, was analyzed. High levels of the Hex110 transcript were expressed in both worker and queen larvae. Low levels of this transcript were also detected in adult fat bodies, and the expression level was higher in the queen than in the worker. Bisulfite sequencing revealed that the Hex110 gene is overall methylated at a low level, with a limited number of CpG sites methylated at relatively high levels. These highly methylated sites were exclusively located in the exon regions. The average methylation rate of the Hex110 gene was higher in the adult stage than in the larval stage. Furthermore, several CpG sites were differentially methylated between the worker and queen larvae. These observations suggest that the methylation of the Hex110 gene is regulated at the developmental stage and in a caste-dependent manner.

Complete WO phage sequences reveal their dynamic evolutionary trajectories and putative functional elements required for integration into the Wolbachia genome.

Wolbachia endosymbionts are ubiquitously found in diverse insects including many medical and hygienic pests, causing a variety of reproductive phenotypes, such as cytoplasmic incompatibility, and thereby efficiently spreading in host insect populations. Recently, Wolbachia-mediated approaches to pest control and management have been proposed, but the application of these approaches has been hindered by the lack of genetic transformation techniques for symbiotic bacteria. Here, we report the genome and structure of active bacteriophages from a Wolbachia endosymbiont. From the Wolbachia strain wCauB infecting the moth Ephestia kuehniella two closely related WO prophages, WOcauB2 of 43,016 bp with 47 open reading frames (ORFs) and WOcauB3 of 45,078 bp with 46 ORFs, were characterized. In each of the prophage genomes, an integrase gene and an attachment site core sequence were identified, which are putatively involved in integration and excision of the mobile genetic elements. The 3' region of the prophages encoded genes with sequence motifs related to bacterial virulence and protein-protein interactions, which might represent effector molecules that affect cellular processes and functions of their host bacterium and/or insect. Database searches and phylogenetic analyses revealed that the prophage genes have experienced dynamic evolutionary trajectories. Genes similar to the prophage genes were found across divergent bacterial phyla, highlighting the active and mobile nature of the genetic elements. We suggest that the active WO prophage genomes and their constituent sequence elements would provide a clue to development of a genetic transformation vector for Wolbachia endosymbionts.

Functional characterization of a cactus homolog from the silkworm Bombyx mori.

A cDNA encoding an IkappaB family protein was identified and the full nucleotide sequence was determined in the silkworm Bombyx mori. The IkappaB gene, designated BmCactus, was constitutively expressed mainly in the fat body and hemocytes. Transfection experiments on a B. mori cell line, NIAS-Bm-aff3, with expression vectors containing BmCactus, BmRelA, BmRelB, or the active portion of BmRelish1 showed that activation of the CecB1 gene promoter by either BmRelA or BmRelB, but not the active portion of BmRelish1, was strongly inhibited by BmCactus. In addition, activation of CecB1 gene by autoclaved E. coli in the cultured cells was observed regardless of the presence or absence of BmCactus. A glutathione S-transferase pull-down assay and analysis using a yeast two-hybrid system demonstrated that BmCactus interacted with the BmRel Rel homology domain, but not with the BmRelish Rel homology domain. These results suggest that BmCactus is involved in the Toll signal transduction pathway in B. mori.

High-Performance Organic Energy-Harvesting Devices and Modules for Self-Sustainable Power Generation under Ambient Indoor Lighting Environments.

Organic photovoltaics (OPVs) that perform more efficiently under artificial indoor lighting conditions than they do under sunlight are attracting growing interest as they can potentially serve as ambient energy harvesters for powering low-power electronics and portable devices for the Internet of Things. Herein, solution-processed small-molecule OPVs are demonstrated to exhibit high power conversion efficiencies exceeding 16% under white LED illumination, delivering high output power densities of up to 12.4 and 65.3 µW cm-2 at 200 and 1000 lx, respectively. Increasing the open-circuit voltage ( Voc) of OPVs is a critical factor for achieving higher indoor photovoltaic performance. Toward real applications, this small-molecule OPV system is adopted to fabricate six series-connected modules with an active area of ∼10 cm2 that are capable of generating a high output power surpassing 100 µW and a high Voc of over 4.2 V even under dimly lit indoor conditions of 200 lx. These results indicate that OPVs are promising as indoor electric power sources for self-sustainable electronic devices.

Identification and functional analysis of Relish homologs in the silkworm, Bombyx mori.

Two cDNAs designated BmRelish1 and 2, that encode Relish homologs, were cloned from the silkworm, Bombyx mori. BmRelish1 had an IkappaB-like domain with 5 ankyrin repeats in addition to Rel homology domain (RHD), nuclear localization signal (NLS), and acidic and hydrophobic amino acids (AHAA) rich regions. On the other hand, BmRelish2 lacked the AHAA and ankyrin repeats (ANK). Knockdown of the BmRelish gene in transgenic silkworms resulted in failure of the activation of antimicrobial peptide genes by Escherichia coli, suggesting that BmRelish plays an important role in antimicrobial peptide gene expression. Functional analysis of BmRelish1 and 2 in mbn-2 cells showed that both Relish homologs do not activate promoters of B. mori antimicrobial peptide genes encoding cecropin B1, attacin, lebocin 3 and lebocin 4. However, a gene construct BmRelish1-d2 lacking the ANK strongly activated promoters of these genes. Another gene construct lacking AHAA and ANK failed to activate these genes, suggesting that BmRelish becomes active by removal of the ANK and that the AHAA-rich region is a transactivation domain. BmRelish2 was shown to repress activation of Cecropin B1 gene expression by BmRelish1-d2, suggesting that BmRelish2 plays a role as a dominant negative factor against the BmRelish1 active form. Necessity of kappaB sites of Cecropin B1, Attacin and Lebocin 4 genes for the full activation of these genes by BmRelish1-d2 was confirmed. The requirement of the mandatory kappaB sites for Lebocin 4 gene expression was different between BmRelish1 active form and BmRelA, suggesting differential roles for kappaB sites in antimicrobial peptide gene activation by different transcription factors. The binding of the RHD portion of BmRelish1 fusion protein to the kappaB sites of Cecropin B1 and Attacin genes was also confirmed.

High-Crystallinity π-Conjugated Small Molecules Based on Thienylene-Vinylene-Thienylene: Critical Role of Self-Organization in Photovoltaic, Charge-Transport, and Morphological Properties.

Narrow-band-gap small molecules with π-extended backbones are promising donor materials for solution-processed bulk-heterojunction (BHJ) organic solar cells (OSCs). Herein, a series of acceptor-donor-acceptor (A-D-A) photovoltaic small molecules incorporating thienylene-vinylene-thienylene (TVT) as a central D unit and alkyl-substituted rhodanine or 2-(1,1-dicyanomethylene)rhodanine as terminal A units are designed and synthesized. Their physical properties including photoabsorption, electronic energy levels, hole mobility, and morphological characteristics are systematically investigated. Using solvent vapor annealing (SVA), the morphologies of the BHJ photoactive layers composed of these small-molecule donors and a [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM) acceptor can be properly modulated. As a result of increased crystallinity of the donors and desired phase segregation between the donors and PC71BM upon rapid SVA treatment, the photovoltaic performances of the resultant OSC devices undergo drastic enhancement. The results reported here indicate that high-efficiency small-molecule OSCs can be achieved through rational design of the TVT-based molecular framework and optimization of the nanoscale phase-segregated morphology via proper SVA treatment.

Oligothiophene-Indandione-Linked Narrow-Band Gap Molecules: Impact of π-Conjugated Chain Length on Photovoltaic Performance.

Solution-processed organic solar cells (OSCs) based on narrow-band gap small molecules hold great promise as next-generation energy-converting devices. In this paper, we focus on a family of A-π-D-π-A-type small molecules, namely, BDT- nT-ID ( n = 1-4) oligomers, consisting of benzo[1,2- b:4,5- b']dithiophene (BDT) as the central electron-donating (D) core, 1,3-indandione (ID) as the terminal electron-accepting (A) units, and two regioregular oligo(3-hexylthiophene)s ( nT) with different numbers of thiophene rings as the π-bridging units, and elucidate their structure-property-function relationships. The effects of the length of the π-bridging nT units on the optical absorption, thermal behavior, morphology, hole mobility, and OSC performance were systematically investigated. All oligomers exhibited broad and intense visible photoabsorption in the 400-700 nm range. The photovoltaic performances of bulk heterojunction OSCs based on BDT- nT-IDs as donors and a fullerene derivative as an acceptor were studied. Among these oligomers, BDT-2T-ID, incorporating bithiophene as the π-bridging units, showed better photovoltaic performance with a maximum power conversion efficiency as high as 6.9% under AM 1.5G illumination without using solvent additives or postdeposition treatments. These favorable properties originated from the well-developed interpenetrating network morphology of BDT-2T-ID, with larger domain sizes in the photoactive layer. Even though all oligomers have the same A-D-A main backbone, structural modulation of the π-bridging nT length was found to impact their self-organization and nanostructure formation in the solid state, as well as the corresponding OSC device performance.

Gene expression and molecular characterization of a novel C-type lectin, encapsulation promoting lectin (EPL), in the rice armyworm, Mythimna separata.

Insect cellular immune reactions differ depending on the target species. Phagocytosis is activated to scavenge microorganisms such as bacteria and fungi. On the other hand, larger invaders such as parasitoid wasps are eliminated by activation of encapsulation. In this study, we hypothesized that novel determinants regulate cellular immunities independent of surface molecular pattern recognition involving pattern recognition receptors (PRRs). Immune-related genes differentially expressed depending on the treated material size were screened in larval hemocytes of the rice armyworm, Mythimna separata. Consequently, we identified a novel C-type lectin gene up-regulated by injection of large beads but not small beads of identical material. Examination of in vitro effect of the recombinant protein on the immune reactions clarified that the protein activated encapsulation reaction, while it suppressed phagocytosis. These results suggest that this novel C-type lectin designated "encapsulation promoting lectin (EPL)" regulates cellular immunity by a novel immune target size-recognition mechanism.

A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori.

Two cDNAs encoding novel Rel proteins were cloned from the silkworm, Bombyx mori. These cDNA clones (BmRelA and BmRelB) showed identical nucleotide sequences except for the 5'-region. BmRelB cDNA derived probably from an alternatively spliced mRNA lacked 241 bp nucleotides at the 5'-region of the BmRelA cDNA, resulting in a loss of the first 52 amino acids. Expression of antibacterial peptide genes was strongly inhibited upon infection with Micrococcus luteus in transgenic silkworms in which BmRel gene expression was knocked down, suggesting that these two Rel proteins are involved in activation of antibacterial peptide genes. Co-transfection experiments indicated that BmRelB activated the Attacin gene strongly and other genes to a lesser extent, whereas BmRelA activated Lebocin 4 gene strongly and Attacin and Lebocin 3 genes very weakly. The Rel homology domain of BmRelA and BmRelB was shown to bind specifically to kappaB sites of antibacterial peptide genes. Proline-rich domains of the BmRels were necessary for activation of antibacterial peptide genes. These results illustrate that a minor structural change in Rel proteins can provoke a dramatic differential activation of antibacterial peptide genes, suggesting a novel regulatory mechanism for insect antibacterial peptide gene expression.

Characterization of a homologue of the Rel/NF-kappaB transcription factor from a beetle, Allomyrina dichotoma.

A cDNA encoding a Rel/NF-kappaB homologue was cloned from a beetle, Allomyrina dichotoma, by reverse transcriptase-polymerase chain reactions (RT-PCR) taking advantage of the conserved Rel homology domain (RHD) to synthesize primers. The Rel/NF-kappaB homologue was designated A. dichotoma (A.d.) Rel A. The amino acid sequence of the A.d. Rel A RHD was compared with those of insect RHDs. The result showed that it has 70% identity with Tribolium castaneum Dorsal, 66% with Drosophila melanogaster Dorsal, 61% with Anopheles gambiae Gambif1, and 55% with D. melanogaster Dif. A putative phosphorylation site in the RHD, RRPS, and two putative nuclear localization signals were conserved in A.d. Rel A. A recombinant fusion protein containing the A.d. Rel A RHD was confirmed to bind specifically to the NF-kappaB site of a gene encoding A.d. coleoptericin A, an antibacterial peptide from A. dichotoma. The activity of A.d. Rel A in modulating a gene construct of the A.d. coleoptericin A promoter-luciferase reporter by expressing the A.d. coleoptericin A cDNA in a Bombyx mori cell line was analyzed. The result showed that A.d. Rel A strongly activates the A.d. coleoptericin A gene construct, whereas A.d. Rel A failed to activate the gene construct containing the mutated NF-kappaB site, suggesting the importance of the interaction between the NF-kappaB site and A.d. Rel A in the signal transduction for gene expression of antibacterial peptides in A. dichotoma.

BmEts upregulates promoter activity of lebocin in Bombyx mori.

The Ets family protein BmEts is assumed to be implicated in determination of diapause in the embryogenesis of Bombyx mori. In this study, we found that expression of BmEts was increased in the fat body and other tissues of the 5th instar larvae in response to Escherichia coli injection. Cotransfection experiments using a silkworm cell line revealed that overexpression of BmEts significantly elevated the activity of lebocin promoter but not of cecropin B1, cecropin D, attacin, and moricin promoters. Activation of the lebocin promoter by BmEts was dependent on at least two κB elements and the most proximal GGAA/T motif located on the 5'-upstream region. BmEts further synergistically enhanced E. coli or BmRelish1-d2 (active form)-stimulated lebocin promoter activation. Two κB elements were also found to be involved in promoter activation by BmRelish1-d2 and in synergistic promoter activation by BmEts and BmRelish1-d2 in the silkworm cells. Specific binding of recombinant BmEts to the proximal κB element and the most proximal GGAA/T motif and interaction between BmEts and BmRelish1 were also observed. To our knowledge, this is the first report of an Ets family protein directly regulating immune-related genes in invertebrates.

Expression of antimicrobial peptide genes encoding Enbocin and Gloverin isoforms in the silkworm, Bombyx mori.

Antimicrobial peptides, Enbocin and Gloverin isoforms from the silkworm Bombyx mori, were analyzed for expression of these peptide genes. Tissue-specific expression of Enbocin and Bmgloverin isoform genes was observed mainly in the fat body upon injection of Escherichia coli. Peptidoglycan and lipopolysaccharide triggered expression of these genes in vivo. On the other hand, lipid A activated Bmgloverin isoform genes but not Enbocin isoform genes. These results illustrate the fact that expression of Enbocin and Bmgloverin isoform genes is inducible by bacteria and that the effects of bacterial cell wall components on the activation of these peptide genes are not necessarily the same. In addition, selective activation of the Enbocin2, Bmgloverin2, and Bmgloverin4 genes by BmRelB rather than BmRelA was observed, providing additional evidence for the occurrence of selective activation of antimicrobial peptide genes by a Rel protein. These results suggest complex regulatory mechanisms in insect antimicrobial peptide genes by bacterial cell wall components.

A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus.

A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.