After firstly identified sex pheromone components of Bombxy mori, those of many insect pests were synthesized by organic chemistry methodology. These synthesized components were used for monitoring, mass trapping, and mating disruption during five decades. For identification of pheromone biosynthesis mechanisms and control to many pests bring to serious damages also were proceeded. The transcriptome analysis from pheromone glands by Next Generation Sequence (NGS) showed many genes and pathway involved on sex pheromone biosynthesis.. The two main genes involved on production of acetate and alcohol, and aldehyde from fatty acid, fatty acid desaturase and fatty acid reductase (FAR) were identified and functional characterized via gene introduction to Brewer’s yeast Saccharomyces cerevisiae. This S. cerevisiae now used as a mediator as well as cell factory for sex pheromone producing. Recently, One group was published that the plant factory for producing via genetically modified plant (tobacco, Nicotiana benthamiana) as a step of semisynthetic preparation. These trials will be suggest that firstly, the possibility of yeast as a molecular toolbox to produce pheromone components and secondly, a novel and cost-effective way of producing moderate to large quantities of pheromones with high purity and a minimum of hazardous waste.

The genus Diadegma is a well known parasitoid group and some are known to have symbiotic virus, PDV. A novel IV was discovered from the calyx of D. fenestrale female. This virus was named as D. fenestrale Ichnovirus (DfIV). The encapsidated DfIV genome contains 65 circular DNA segments with an aggregate size of 247,191 bp. Based on BLAST analysis, a total of 120 ORFs were predicted as follows: rep; 48, cys-motif; 11, vinnexin; 10, vankyrin; 9, PRRP; 3 and other unassigned genes (39). These viral genes were expressed in lepidopteran hosts (Phthorimaea operculella and Plutella xylostella) after parasitization which means DfIV genome segments were integrated into lepidopteran hosts. This study was focused on this result. Based on gene expression profile, candidate promoter and integration motifs were selected and then, fused with eGFP as a reporter gene. Modified DfIV genome segment was ligated to a commercial containing f1 ori and Ampr gene to propagate in E. coli. We have named this fusion vector as pIN. The construction methodology of pIN and its application would be further discussed in detail.

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus. Our previous study indicated that overexpression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. This study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to analyze its inhibitory activity against cysteine protease and physiological role in the parasitism of an endoparsitoid wasp, Cotesia plutellae. The open reading frame (ORF) of CpBV-CST1 encodes a polypeptide of 138 amino acids (15 kDa). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was overexpressed by 0.5 mM IPTG for 4 h. In biological activity assay, partially purified GST-fused rCpBV-CST1 showed inhibitory activity against papain. It also inhibited larval development of P. xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 plays a role in retardation of larval development of P. xylostella during parasitism.

Insulin in vertebrates plays a crucial role in maintaining homeostasis of blood sugar level. Insulin-like peptide (ILP) has been identified in insects, such as Drosophila melanogaster and Aedes aegypti. Plasma sugars and polyols of the diamondback moth, Plutella xylostella were separated by a Bio-LC. Among seven peaks, trehalose was the most predominant blood sugar and maintained at approximately 3.5 mM in the larval plasma. However, the feeding activity affected the plasma trehalose level, in which starvation significantly up-regulated the trehalose level. Analysis of ILP expression upon feeding indicated that feeding stimulated the gene expression of ILP. Interestingly, an injection of a vertebrate insulin significantly suppressed the hypertrehalosemia induced by starvation. These results suggest that ILP is a endocrine signal to down-regulate the plasma trehalose level in P. xylostella.

Prostaglandins (PGs) mediate insect immune responses. However, their biosynthesis in insects is little understood due to lack of cyclooxygenase (COX) ortholog. This study aimed to identify PG-biosynthetic factor(s) in Spodoptera exigua, which has been a well-known insect in possessing immune responses mediated via PGs. Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (POX-A ∼POX-J) were expressed in S. exigua. Especially, expressions of POX-F and POX-H were inducible to bacterial challenge and expressed in hemocytes and fat body. Individual RNA interference (RNAi) of each of ten POXs was performed by hemocoelic injection of their specific double-strnaded RNAs (dsRNAs). Only RNAi of POX-F or POX-H specifically suppressed hemocyte-spreading behavior and nodule formation. Addition of PGE2 significantly nescued the immunosuppression in either dsRNA treatment of POX-F or POX-H. Structural analysis indicated that both POX-F and POX-H have conserved domain and residues corresponding to peroxinectin of Drosophila melanogaster, which mimics COX-like activity. These results suggest that POX-E and POX-H are involved in PG biosynthesis in S. exigua.

Polydnavirus are well known to interfere with the host endocrine system, causing immune suppression and other physiological disorders. The Cotesia rubculla polydnavirus gene products, CrV1, are known to be a potent immunosuppressive agent. CrV1 protein express within 12 h after viral infection at oviposition during deposition of parasitoid eggs and are mainly secreted in to host hemocyte, where it functions like phagocytosis and cell spreading. This study identified its homolog in CpBV and analyzed its molecular characteristics motif called “coiled-coil. A point mutation of Alanine to Proline of CpBV-CrV1 could lose the coiled-coil motif from in silico assay. The coiled type CpBV-CrV1 could inhibit host cellular immunity, however, interestingly the mutant CpBV-CrV1 lacking in coiled-coil motif completely lost the immunosuppressive activity. This study suggests that the coiled-coil motif is functional to inhibit host cellular immune responses. RNA interference against CrV1 significantly loses the inhibitory activity and thus further supporting the immunosuppressive activity of CrV1. In this study we also have analyzed the localization of CrV1 by transient transfection in HiFive Cell lines by in situ hybridization.

Teratocytes (TCs) are the cells derived from the embryonic serosal membrane of some parasitic hymenopteran insects. As a parasitic factor, TCs are multifunctional in host regulation by inducing nutritional, immune, and developmental alterations. However, little is understood about their genetic constituents. This study reveals a comprehensive view of the genes expressed by TCs through a transcriptome analysis based on RNAseq technology. More than 6.29 Gb sequences were used to assemble 34,686 contigs (>200 bp) and annotated into different functional categories. The TC transcriptome profile was clearly distinct from those of hemocytes and the fat body. The TC transcriptome contained components of insulin signaling and biosynthesis of juvenile hormone and 20-hydroxyecdysone. TCs also expressed various groups of digestive enzymes, supporting its nutritional role for the growing parasitoid larvae in parasitism. Furthermore, this transcriptome analysis annotated two kinds of immunosuppressive serine protease inhibitors (serpins) and Rho GTPase-activating proteins (RhoGAPs). To determine the biological functions of these factors, we devised ex vivo RNA interference (RNAi) by conducting knockdown of gene expression in in vitro cultured TCs followed by injection of the treated TCs to test insects. Ex vivo RNAi revealed that some serpins and RhoGAPs expressed in TCs inhibited host cellular immunity. This study reports a transcriptome of the unique TC animal cell, and its immunosuppressive genetic factors using ex vivo RNAi technology.

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2,517 bp) of a integrin subunit β1 (HaITGβ1) was cloned from the oriental tobacco budworm, Helicoverpa assulta. Phylogenetic analysis showed that HaITGβ1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (61%) to β1 of other Noctuidae such as Spodoptera exigua and S. litura. Structural analysis of the HaITGβ1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that HaITGβ1 was expressed in all developmental stages and all tested tissues of H. assulta. Injection of double-stranded HaITGβ1 RNA (dsHaITGβ1) into third instar of H. assulta suppressed HaITGβ1 expression and resulted in significant delay from last larval stage to pupal stage. The dsHaITGβ1 injection significantly impaired nodule formation of H. assulta in response to bacterial challenge and hemocyte adherence. These results suggest that HaITGβ1 plays crucial roles in cellular immune responses as well as development in H. assulta.

Insects have a highly efficient innate immunity consisting of cellular and humoral responses. Upon microbial pathogen infection, pattern recognition receptors specifically recognize pathogen types and trigger the programmed immune responses via immune mediators, such as cytokine, biogenic monoamines, and eicosanoids. In addition to these canonical immune mediators, two insect developmental hormones of juvenile hormone (JH) and 20-hydroxyecdysone (20E) also modulate immune responses. JH suppresses a cellular immune response, while 20E antagonizes the JH action. In Tribolium castaneum, JH mediates cellular immune responses via non-nuclear receptor(s). When Met (the JH nuclear receptor) expression was silenced by its specific double-stranded RNA, T. castaneum underwent precocious metamorphosis. Under these RNA interference (RNAi) conditions, JH still mediates the cellular immune responses. The addition of calphostin C (a PKC inhibitor) or ouabain (a Na + -K + ATPase inhibitor) significantly suppressed the JH mediation on the cellular immune responses, suggesting the presence of a novel JH receptor(s) at nonnuclear regions. Eicosanoids are a group of oxygenated C20 polyunsaturated fatty acids. They mediate various insect physiological processes including reproduction, excretion, and immunity. Arachidonic acid (AA) is a main precursor of eicosanoid biosynthesis. Phospholipase A2 (PLA2) catalyzes AA release from phospholipids. Subsequent oxygenases of cyclooxygenase (COX) or lipoxygenase (LOX) transform AA to prostaglandins (PGs) or leukotrienes (LTs). In a model insect, Spodoptera exigua, both PGs and LTs mediate both cellular and humoral immune responses. In addition to the physiological significance of the secretory PLA2 (sPLA2), a recent genome analysis identified a novel cellular PLA2 (cPLA2) in S. exigua, which mediates the immune responses. Especially, eicosanoids mediate prophenoloxidase (PPO) activation by stimulating release of PPO from oenocytoid hemocytes. A G protein-coupled receptor is identified in oenocytoids and highly specific to PGE2. Subsequent calcium and PKC pathways mediate PG signal to activate a specific ion channel, Na + -K + -Cl - -cotransporter (NKCC), which generates an osmotic shock to induce cell lysis of oenocytoids to release internal PPO. The released PPO would be activated by a cascade of serine proteases and involved in various immune responses. Eicosanoids also mediate antimicrobial peptide gene expression in response to the bacterial infection. Lack of eicosanid biosynthesis results in a significant immunosuppression. This kind of immunosuppression is exploited by an entomopathogenic bacterium, Xenorhabdus nematophila, to proliferate in the infected larvae without adverse attack to the bacteria by host immune defense. Eight PLA2 inhibitors have been chemically identified and used to develop novel biopesticides. Even though several chemical analyses using various insect systems indicate PG and LT eicosanoids, COX and LOX genes are not identified. Furthermore, little information is available in PG receptors in insects. Thus, signal transduction of eicosanoids in insects remains unresolved. Eicosanoid biosynthesis and signal transduction need to be explored to understand the significant roles of eicosanoids in insect physiological processes.

Postharvest insect pest control is highly demanding in agricultural industry including domestic consumer markets and exporting products for a quarantine purpose. Especially, the organic or environmentally friendly agricultural products do not fit to the traditional chemical postharvest treatments using methyl bromide (MeBr) or phosphine (PH3). As an alternative, a physical treatment called CATTS (controlled atmosphere and temperature treatment) has been developed to control various insect and mite pests on ornamental products. The oriental fruit moth, Grapholita molesta, infects the apple or pear fruits and is limited in importing and exporting the infected products. To apply CATTS on this insect pests, the most heat-tolerant stage was determined. Among the immature stages locating on the fruits, the fifth instar larvae were the most tolerant to 44℃ for 20 min. A ramping step of CATTS is to increase chamber temperature from 25℃ to 46℃ under 15% CO2 and less than 1% O2. The ramping rate was positively correlated with the CATTS efficiency. After the ramping step, the duration of CATTS was positively correlated with CATTS efficiency. However, fruit damage by CATTS was negatively correlated with the ramping rate was positively correlated with the CATTS duration. in addition, the CATTS efficiency was highly dependent on the fruit internal temperature at 44℃. From all these parameters, we developed a standard protocol yielding 100% control efficiency of CATTS.

Putative cadherin genes, which are a receptor of the Bacillus thuringinesis toxins, were predicted from a whole genome sequencing data from the diamondback moth, Plutella xylostella. After the sequence and expression analysis, a Bt receptor cadherin gene was selected. The P. xylostella cadherin gene (PxCad1, GenBank Accession no. GU901158.1) encodes 11 cadherin repeats and a transmembrane domain. The PxCad1 gene was expressed in all developmental stage specifically in gut tissue by RT-PCR analysis. Expression of PxCad1 gene was suppressed by feeding of its specific dsRNA PxCad1 in 4th instar larval stage. The suppression of PxCad1 expression did not significantly feeding of its specific dsRNA PxCad1 in 4th instar larval stage. The suppression of PxCad1 expression did not significantly influence on pupal and adult development of P. xylostella. However, the larval treated with dsRNA PeCad1 (150 ng/larva) significantly reduced susceptibility to B. thuringiensis Cry1Ac (4.83 μg/ml). By contrast, the dsRNA PxCad1 -treated larvae did not show any change in susceptibility to B. thuringiensis Cry1Ca (0.24 μg/ml). These results suggest that PxCad1 is a specific receptor of Cry1Ac toxin from B. thuringiensis in P. xylostella.

Juvenile hormone is the most important hormone inside the insect, but its circulating titer should be under tight control. Then enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs) play critical roles in insect metamorphosis and reproduction. Within a set of 11 JHEs predicted in the diamondback moth (DBM) genome, Plutlla xylostella (Linn.) we identified one gene that contained the main functional motifs of insect JHEs. Its expression and transcript levels during egg, different instar of larvae, prepuae, pupae and adult stages were measured. Also, its expression in the epidermis, midgut and fatbody of the 4 th instar larvae was compared. The changes in enzyme activity in the 4th instar larvae were determined.

A nuclear receptor, Met, mediates juvenile hormone (JH) action to control gene expressions associated with metamorphosis in many insects. In this study, we showed that RNA interference (RNAi) of the Met or Kruffel homolog 1 (Kr-h1) induced the precocious metamorphosis of Tribolium castaneum larvae. JH significantly inhibited cellular immune response of T. castaneum hemocyte by suppressing hemocyte-spreading behaviour and nodule formation in response to bacterial injection. However, either RNAi of Met or Kr-h1 expression did not prevent the JH-inhibitory effect on hemocyte behaviors. However, several inhibitors specific to JH membrane action significantly inhibit the JH action hemocytes. These results suggest that JH responsiveness of hemocyte is not mediated by the nuclear receptor.

A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV), which is symbiotic to an endoparasitoid wasp, C. plutellae. Compared to general histone H4, the viral H4 possesses an extra N-terminal tail containing 38 amino acid residues, which has been presumed to control host gene expression in an epigenetic mode. This study addressed the mutational analysis of extra N-terminal amino acid residues of a viral histone H4 and their epigenetic control efficacy. Mutational analysis was performed by serially deleting each of the nine amino acid residues from N-terminal tail of a viral histone H4. Transient expression of each truncated mutants (K1M-K19) in diamondback moth, Plutella xylostella, was performed by microinjection of a recombinant expression vector and confirmed by RT-PCR. Under transient expression, we analysed the effect of these mutations on target gene, transferin. Interestingly, we found that truncated mutants (K1M-K15) did not inhibit the expression of target gene but mutations thereafter (K6M-K9M) significantly alter its expression. As expected these truncated mutants (K1M-K5M) also inhibit hemocyte nodule formation and development of Plutella xylostella. This suggest that lysine residue (K6) in the N-terminal tail is very crucial for the epigenetic control efficacy of viral histone H4.

A viral histone H4 (=CpBV-H4) is encoded in a polydnavirus, Cotesia plutellae bracovirus, and symbiotically associated with an endoparasitoid wasp, C. plutellae. It has an extended N-terminal tail consisting of 38 amino acid residues, compared to the host H4 and this extended N-terminal tail has been postulated to play a crucial role in an epigenetic control of gene expression. The (SSH) suppression subtractive hybridization analysis was analyzed in transcriptome by short-read sequencing technology. The SSH analysis provided several target and nontarget genes of a viral histone H4. In this study, we analyzed the effect CpBV-H4 on the expression of two target genes serpins and histone lysine N-methyl transferase. Transient expression of CpBV-H4 by microinjecting recombinant expression vector to non parasitized larvae of Plutella xylostella showed that it was expressed up to 70 h. Under this transient expression condition, we analyzed the effect of CpBV-H4 on the expression of target genes by RT-PCR at different time points. Interestingly, the CpBV-H4 significantly inhibited the expression of target genes after 44 h, while the truncated CpBV-H4 deleting the N-terminal tail did not show the inhibitory activity.

The oriental fruit moth (Grapholita molesta) and the plum fruit moth (G. dimorpha) share the same major sex pheromone components, Z8-dodecenyl acetate (Z8-12Ac) and E8-dodecenyl acetate (E8-12Ac) with different ratio. However, these two congener male species were cross-attracted to the counter sex pheromone traps. For development of the specific monitoring lures, the minor sex pheromone components were added to the major components. G. molesta females emit two minor components of Z8-12OH and 12OH and G. dimorpha females emit four minor of 12Ac, 14Ac, Z8-14Ac, and E8-14Ac. For a specific monitoring lure of G. molesta, only Z8-12Ac major component attracted only G. molesta males, but did not any G. dimorpha. For a specific monitoring lure of G. dimorpha, the addition of Z8-14Ac to the major component (Z8-12Ac:E8-12Ac = 85:15) attracted G. dimorpha males with less than 5% G. molesta males. Other with components (12Ac, 14Ac, and E8-14Ac) was not effective in both trapping efficiency and selectivity.

The genus Diadegma is a well known parasitoid group and some are known to have symbiotic virus, PDV. A novel IV was discovered from the calyx of D. fenestrale female. D. fenestrale has more than two hosts, including PTM and DBM. The oviposition and survival rate results showed that D. fenestrale preferred PTM to DBM as hosts. Nevertheless, the developmental period and morphology of D. fenestrale were not significantly different between PTM and DBM. To identify these phenomena, DfIV genome expression patterens were compared between PTM and DBM under various conditions. DfIV genes were more widely expressed in PTM than in DBM after parasitized by D. fenestrale, particularly at the initial point. In addition, large numbers of DfIV genes were expressed only in PTM and they showed differential expression patterns between two lepidopteran hosts. This DfIV genome expression plasticity showed a dependency on the lepidopteran host species and parasitization time, suggesting that it may contribute to the parasitoid survival rate increase. This may be one of the key elements that determine the symbiotic relationship between PDV and parasitoid.

Immune mediators play crucial roles in amplifying the emergency signals with massive amounts of de novo synthesized mediators and relaying the specific recognition signals to the immune-associated target tissues. Eicosanoids are the representative immune mediators and synthesized from a polyunsaturated fatty acid (PUFA), arachidonic acid. Compared to mammalian systems, insects have relatively low levels of arachidonic acid in the biological membranes. This has raised a fundamental issue that eicosanoids may be not significant in insect system. Our previous chemical analysis suggests that the hemocytes of Spodoptera exigua have less than 5% arachidonic acid. We postulated that S. exigua may store arachidonic acid in other tissues, such as fat body. This analysed fatty acid compositions of two immune-associated tissues using a gas chromatography (GC) eguipped with FID detector or GC-MS. Our analysis of PUFA in the immune tissues suggests that insects maintain a low level of PUFA including arachidonic acid due to its evolutionary origin from the paleozoic era at which the oxygen level was 35%, compared to the present era 21%.

Cold tolerance of the palm thrips, Thrips palmi Karny, was investigated to predict its survival in field during winter. Supercooling temperatures of T. palmi ranged from -26.4 to -18.4°C. However, exposure to subzero temperatures (from -5°C to -15°C) gave significant mortality to all developmental stages of T. palmi. Thus, T. palmi was determined to be a freeze-susceptible and suffered with cold injury. A brief pre-exposure to a low temperature (4°C) for 7 h significantly increase the cold tolerance of all stages of T. palmi with respect to survival at -10°C and supercooling capacity. A pre-exposure of T. palmi at 4°C significantly increased the survival rate on all developmental stages at -10°C. The rapid cold hardiness (RCH) was dependent on the duration of the pre-exposure period at 4°C in adult stage. Cryoprotectant analysis using an HPLC showed that the pre-exposure treatment increased the adult to synthesize glycerol, trehalose, mannitol, and mannose, at which trehalose represented the highest content. This study suggests that all stages of T. palmi are able to become cold-hardy by RCH, in which several polyols may play crucial roles as cryoprotectant.

Phospholipase A2 (PLA2) catalyze the committed step for eicosanoid biosynthesis and releases arachidonic acid (AA), which is oxygenated into eicosanoids that mediate immune responses in insects. Thus, any inhibition of PLA2 activity would lead to a significant immuno suppression due to lack of eicosanoids. Among more than 15 families of PLA2s, group Ⅳ cytosolic PLA2 (cPLA2) has been mainly associated with the production of eicosanoids associated with immune responses. However, no cPLA2 has been reported in all invertebrates including insects. AcPLA2 candidate gene (SecPLA2) has been identified from a hemocyte transcriptome of the beet armyworm, Spodoptera exigua. RNA interference of SecPLA2 expression significantly reduced cellular immune responses of hemocytes. When the SecPLA2 was expressed and purified, the recombinant SecPLA2 catalyzed a substrate, phosphoatidyl choline, atsn-2 position. Its catalytic activity was sensitive to pH, temperature, and calciumlevel. Furthermore, there combinant SecPLA2 was specifically sensitive to a cPLA2-specificinhibitor, methyl arachidonyl fluorophosphonate.