As the immune reactions in human white blood cells of certain substances from insects to defend it when invaded by immune blood cells is increased. We experiment with changes in the total number of blood cells through the blood cells which increases and decreases, as well as to observe whether the immune response through any route is to evaluate what happens. Hemocyte population was analyzed in the last instar larvae of Spodoptera exigua. Granulocyte and plasmatocyte were predominant (>75%) types of hemocytes, whereas spherulocyte, prohemocyte, and oenocytoid hemocytes were observed in small densities (5~10%). Total hemocyte counts (THCs) were varied among different ages (day1-day5) of the last instar, in which day 3 larvae (L5D3) had the maximal density. Upon bacterial challenge to L5D3 larvae, THC was further enhanced within 2 h and then decreased to background level. This rapid THC increase in response to bacterial challenge was inhibited by injection with dexamethasone (1 ㎍ per larva). However, the addition of arachidonic acid reversed the inhibitory activity of dexamethasone and allowed the larvae to increase THC. This THC increase was mediated by cyclooxygenase products, but not by lipoxygenase products.

Teratocytes are originated from embryonic serosal membrane of some endoparasitoid wasps. Cotesia plutellae eggs release teratocytes in parasitoid host hemocoel at hatch in about 150 cells per egg. Teratocytes of C. plutellae were cultured in an insect culture medium for at least 14 days. Teratocytes cultured in vitro showed no increase in cell numbers but increased in cell size. Similarly,teratocytes in parasitized larvae did not increase cell numbers, but increased their cell size. Microinjection of invitro cultured teratocytes in to third instar larvae of nonparasitized Plutella xylostella showed a dose-dependently inhibitory effect on development and larval-pupal metamorphosis. In addition, teratocytes prolonged the immature developmental period and reduced the pupation rate, in which young aged host larvae were more sensitive to teratocytes treatment than old larvae. These results suggest that teratocytes play a crucial role in successful parasitization of C.plutellae by altering host developmental program.

An endoparasitoid wasp, Cotesia plutellae parasitized young larvae of diamondback moth, Plutella xylostella. Parasitized larvae exhibit sign ificant immunosuppression and fail to metamorphose to pupal stage. Especially, during last instar of parasitized P.xylostella, massive nutrients divert from host to wasp development. CpBV15α ,a host translation inhibitory factors encoded in C. Plutella bracovirus(CpBV), plays a crucial role in suppressing host usage of amino acids. Its promoter analysis shows that CpBV15α specifically inhibit host development in late larval period. To understand its inhibitory target, its specific expression was performed in non-parasitized P. xylostella by in vivo transient expression technique. Total plasma proteins were analyzed by 2D gel electrophoresis and determined target genes inhibited by CpBV15α. Immunoprecipation of cellular extract with CpBV15α antibody captured eIF2B. CpBV15α shares sequence homology with eIF5, especially at its eIF2B-binding region. Our results suggest that CpBV15α may sequester eIF2B, which result in malfunctioning of eIF2 cycling to form a translation initiation complex.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), is symbiotic to an endoparasitoid wasp, C.plutellae, which specifically parasitizes young larvae of the diamondback moth, Plutella xylostella. A recent study on CpBV replication by analysis of ovary transcriptome of C.Plutellae suggests several candidate coat protein genes. This study was conducted to confirm the coat protein genes by analyzing coat proteins of CpBV viral particles by a tandem mass MALDI-TOF. Immunoprecipitation of ovary protein extract with a polyclonal CpBV antibody captured three proteins named as p35, p60, and p70. More number of coat proteins were resolved in a protein extract directly from viral particles. All candidate coat proteins are analyzed in amino acid sequences by MALDI-TOF. A comprehensive analysis of viral proteomics and ovary transcriptome determined novel viral coat proteins from CpBV

Sound treatments have been considered as a non-chemical insect pest control technique. Different frequency and intensity sounds were applied to immune and adult stages to screen any stress sounds to alter physiological processes. At 95 dB, 5,000 Hz and 30,000 Hz were selected to be stress sounds in audible and inaudible sound ranges, respectively. Both stress sounds significantly inhibited larval and pupal development. In biochemical analyses, lipid and sugar levels in plasma signigicantly increased in response to the stress sound treatments. Moreover, a digestive phospholipase A2 enzyme activity in midgut was significantly reduced. In adult stage, ultrasound treatment significantly inhibited mating behavior, which resulted in a reduced fecundity. These stress sounds altered gene expressions of stress-related genes, such as heat-shock proteins and apolipophorin III. This study suggests that extreme sounds play a role in physiological stress factors in S. exigua by altering developmental and reproductive processes.

Parasitization by an endoparasitoid wasp, Cotesia plutellae, extends a larval period of Plutella xylostella and inhibits a larva-to-pupa metamorphosis. To determine antimetamorphic parasitic factor(s) in this host-parasitoid interaction, an effect of its symbiotic polydnavirus, Cotesia plutellae bracovirus (CpBV), was investigated by injecting purified virus particles to nonparasitized larvae of P. xylostella. Larvae injected with CpBV exhibited antimetamophosis in a viral dose-dependent manner. Also, the susceptibility to the viral injection was increased at young larval stages. Parasitized or virus-injected larvae shwed significant decrease in cell size of prothoracic gland and reduction in expression of ecdysone receptor (EcR) gene. However, they increased and maintained expression of insulin receptor (InR) gene. Twenty four CpBVsegments were individually injected to nonparasitized larvae. Only two segments (S22 and S27) had significant antimetamorphic effect. Subsequent RNA interference using double stranded RNA (dsRNA) was performed in each of encoded genes in each segment. Protein tyrosine phosphatase, ELP, and three hypothetical genes were determined to be antimetamorphic factors.

Understanding the spatial pattern of G. molesta and the temporal variation of their patterns are important to develop and maintain pest management programs in fruit orchards. The overwintering larvae of G. molesta pupate early in the spring and new adults begin a flight for several reasons such as mating, seeking resources (food or shelter) and oviposition. It was known that G. molesta presented “low movement activity” and male G. molesta flight behavior was closely related to the proximity of its host crops. Unmated males remain near the site of emergence in order to find and copulate with unmated females. The fruit-bearing status of orchards are important factors for G. molesta movement. To elucidate the spatial distribution and temporal variation of G. molesta within and among orchards, pheromone traps targeting male G. molesta were used because the trap represent a reliable and economic tool for monitoring adult G. molesta populations. The study was conducted in two apple orchards (one is isolated from other fruit orchards and another is surrounded by apple orchards), Andong and in seven plum orchards, Uiseong, 2010. Using spatial analysis by distance indices, the spatial pattern of G. molesta in each sampling date was presented. In the study of the spatial pattern within apple orchard, the index of aggregation (Ia) of isolated orchard were presented >1, indicating an aggregated distribution pattern, from monitoring results. The spatial association between successive monitoring using X (the index of spatial association) was negative during spring season and after then the value was changed to positive. In the experiment of the spatial pattern among orchards, the index of aggregation was >1 in most monitoring date and the index of spatial association was negative during early and late growing season. Factors influencing the spatial-temporal dynamics of G. molesta are discussed.

Three insect pests internally feed pome fruits in Korea. These include oriental fruit moth (Grapholita molesta), plum fruit moth (Grapholita dimorpha) and peach fruit moth (Carposina sasakii). Molecular markers discriminating these three species were developed using PCR-RFLP technique. Mitochondrial (mt) genomes were analyzed to locate polymorphic loci. Six mtDNA regions (CO-Ⅰ, CO-Ⅱ, CB, 16SrRNA-12SrRNA, ND3, ND4) of G. dimorpha were cloned and sequenced. These six sequences of G. dimorpha were aligned with those of C. sasakii and G. molesta to determine polymorphic restriction sites. Predicted PCR-RFLP markers were confirmed with known insect samples. With the validated PCR-RFLP markers, field male adults collected in traps baited with rubber sept lures impregnated with different ratios of major sex pheromone components of G. molesta were analyzed.

An entomopathogenic bacterium, Xenorhabdus sp., symbiotic to Steinernema monticolum was investigated in its insecticidal activity. The bacteria induced septicemia of two lepidopteran insects (Plutella xylostella, Spodoptera exigua), a coleopteran insect (Tribolium castaneum), and a hemipteran insect (Riptortus clavatus) when they were injected into hemocoel. The bacterial culture broth contained immunosuppressive factor(s) that inhibited hemocyte nodulation in response to heat-killed bacteria. The immunosuppressive activity appeared to be caused by inhibition of two main immune-associated enzymes, phospholipase A2 (PLA2) and phenoloxidase (PO). HPLC analysis of the bacterial culture broth contained several PLA2 inhibitors. The bacterial culture broth significantly enhanced Bt pathogenicity. There results support a novel insect pest control strategy using eicosanoid-biosynthesis inhibitors.

An entomopathogenic fungus Beauveria bassiana (Bb), is a potent pathogen against the beet armyworm, Spodoptera exigua. Phospholipase A2 (PLA2) activities were measured in both immune-associated tissues of hemocytes and fat body S. exigua. Upon the fungal PLA2s were significantly activated in both hemocytes and fat body. Considering inhibitory activity of BZA, we posed a hypothesis that BZA against PLA2 activity of hemocyte, resulting in shutdown of eicosanoid biosynthesis, subsequently inducing immunosupression, which leads to enhance Bb pathogenicity. This study directly analyzed the inhibitory activity of BZA on PLA2 extracted from different immune-related tissues. At low micromolar range, BZA significantly inhibited PLA2s of hemocytes, fat body, and plasma, in which most PLA2 activity was found in hemocytes. Interestingly, an immune signal receptor, Se-Toll, was related with PLA2 activation. RNA interference (RNAi) of Se-Toll significantly inhibited PLA2activity while nonspecific RNAi did not inhibit the PLA2 activity. The RNAi of Se-Toll also significantly suppressed hemocyte nodule response against Bb challenge. In addition, the fungal infection significantly induced activation of PLA2 activity, which would lead to production of immune mediating eicosanoids. This study addressed the synergistic effect of BZA on Bb pathegenicity by its inhibition of PLA2 activity, which was linked with Toll signal pathway.

Polydnaviruses (PDVs) are a group of insect double stranded DNA viruses and symbiotically associated with host endoparasitoid wasps. Their segmented genome is located in host chromosome(s) in a proviral form. Viral replication is initiated at the ovary during late pupal stages. Little is known about the factors involved in the viral replication. This study analyzed the ovarian transcripts of an endoparasitoid wasp, Cotesia plutellae, by 454 pyrosequencing and subsequent gene annotation. Out of 2,226 contigs and 12,457 singletons, 50 transcripts categorized in DNA replication, coat proteins, and viral origins were selected as putative viral replication factors. The selected genes were analyzed in their expressions according to host wasp development. Quantitative real-time RT-PCRs showed that some of the selected genes were expressed during the viral replication at late pupal stage. Using RNA interference, five putative genes were tested in their implication in the viral replication by analyzing viral DNA amplification, structure of ovarian calyx, and parasitism. RNA interference of contig#1004 (broad complex) or contig#174 (a viral DNA polymerase gene) significantly inhibited DNA amplification without any impairment of viral formation, and subsequently resulted in significant reduction in the wasp parasitism. This study reports that two wasp genes (or not encapsidated viral genes) are implicated in the viral DNA amplification and viral coat protein production during the polydnaviral replication.

Parasitization by Cotesia plutellae inhibits pupal metamorphosis of diamondback moth, Plutella xylostella. Two questions are raised : (1) which parasitic factor(s) is responsible for the antimetamorphosis and (2) how the parasitized larvae are altered in endocrine signals. This study addressed both questions. When C. plutellae bracovirus (CpBV), a parasitic factor of the wasp, alone was injected to nonparasitized P. xylostella larvae, it significantly inhibited pupal metamorphosis in a dosedependent manner. Corpora allata (CA) and prothoracic gland (PTG) were compared in both nonparasitized and parasitized P. xylostella. In both groups, size and shape of CA were not different. However, PTG was detected on prothoracic tracheal trunk in nonparasitized larvae, but not detected in parasitized. CpBV injection to nonparasitized larvae inhibited the growth of PTG. Transcriptional factor, broad complex, was partially cloned and expressed in nonparasitized P. xylotella. In parasitized or CpBV-injected larvae, broad complex gene was not expressed during late larval stage.

Insect blood cells (hemocytes) play a key role in defense against parasites and other pathogenic organisms that infect insects. Cellular immune responses exhibited by hemocytes are acute and effective to initially suppress pathogenic processes. Subsequently humoral immune responses executed by antimicrobial peptides completely cleared the pathogens with help of hemocytes. Two immune mediators, plasmatocyte-spreading peptide (PSP) and eicosanoid, are known to mediate cellular immune responses by activating hemocyte behavior. This study was focused on how these two immune mediators work together to express hemocyte spreading behavior. Both PSP and prostaglandins stimulate hemocyte spreading in dose-dependent manners in the beet armyworm, Spodoptera exigua. Interstingly, inhibition of eicosanoid biosynthesis inhibited PSP activity on mediating the hemocyte-spreading behavior. However, the addition of eicosanoid biosynthesis precursor, arachidonic acid, rescued the hemocytespreading activity. Inhibition of PSP or its receptor by each RNA interference are now under investigation to test whether PSP triggers eicosanoid signaling. These results suggest that there is a cross-talk between PSP and eicosanoid to express hemocyte-spreading behavior in response to bacterial challenge

A polydnavirus, Cotesia plutellae bracovirus (CpBV), is a symbiotic provirus to an endoparasitoid wasp, C. plutellae. When the wasp parasitizes its natural host, Plutella xylostella, larvae, CpBV viral particles are translocated to hemocoel of P. xylostella along with the wasp eggs. CpBV-ELP1 is encoded in a viral segment and expressed in the parasitized larvae during entire parasitization period. A recombinant baculovirus expressing CpBV-ELP1 was constructed and applied to a non-natural host, Spodoptera exigua, larvae. When the recombinant baculovirus was injected to hemocoel, CpBV-ELP1 was expressed in hemocytes as early as 2h postinjection and then later expressed in other tissues. When it was applied to diet, CpBV-ELP1 was expressed in midgut epithelium at 12 h and subsequently expressed in internal tissues. Both application methods of the recombinant baculovirus caused significantly higher mortality of S. exiguathan non-recombinant baculovirus. Interestingly, midgut epithelial cells expressing CpBV-ELP1 by infection of the recombinant baculovirus showed poor cell-cell interactions. Integrin, a cell surface molecule associated with cell-cell interaction, was cloned in S. exigua and was confirmed in its expression in the midgut epithelium. A hypothesis was raised that CpBV-ELP1 interrupts integrin function by direct binding or by blocking internal integrin signaling.

Previous studies indicated that Matsumuraeses phaseoli and M. falcana (Lepidoptera: Tortricidae) are separate species since a few differences were observed in genitalia morphology and female sex pheromone composition. A clear difference was detected in the DNA sequences of cytochrome oxidase I of the two species separately collected in different plants and regions. A hybridization test also showed that a post-zygotic reproductive isolation occurred between the species. In field monitoring, however, both species have been caught simultaneously and together in the separate sex pheromone traps installed for the two species around neighboring soybean and red bean fields. Molecular marker-assisted identification with several adults sampled from the trapped insects showed that only ca. 40% of M. phaseoli adults identified as the species by genitalia morphology was the M. phaseoli, while ca. 97% of M. falcana adults identified as the species was the M. falcana. The result indicated that the observation of genitalia did not make a decisive criterion for classification of the insects. Conclusively, it suggested that the sex pheromones of the two species should be studied more precisely although there is a possibility that the two species are hybridized in fields as in laboratory, and speciation is under process.

Based on Drosophila model, Toll and Imd signals have regared as central intracellular pathways in insect immune cells in response to various pathogens. Current insect genome studies have identified the corresponding orthologs in other insets. This study reports two immune signaling genes, Se-Toll-1 and Se-Relish-1, and suggests Toll and Imd pathways in the hemocytes of Spodoptera exigua. Partial Se-Toll-1 and Se-Relish-1 share high sequence homologies with known Toll and Relish genes of lepidopteran and dipteran species. Their expressions were detected from all developmental stages. In larval stage, there two genes were expressed in all tested tissues including hemocytes. Real time quantitative RT-PCR indicates that expression of both genes were highly up-regulated by bacterial and fungal infections. Various antimicrobial peptides (AMPs) were expressed in the hemocytes of S.exigua, in which their expressions appeared to be controlled by Se-Toll-1 and Se-Relish-1. However, Se-Toll-1 and Se-Relish-1 were proved to controlled different AMP genes from their RNA interference assays. These results suggest Toll and Imd signals in the hemocytes of S. exigua.

As an environment-friendly phytosanitary measure, CATTS (controlled atmosphere temperature treatment system) has been developed to kill several quarantine insect pests infesting subtropical agricultural commodities. This study tested any possibility to apply CATTS to apples to effectively eliminate the peach fruit moth, Carposina sasakii, which has been regarded as a quarantine insect from the imported countries. When the larvae of C. sasakii were directly exposed to 46℃ (an installed lethal temperature of CATTS), they showed a median lethal time at 14.66 min. Addition of high carbon dioxide to the temperature treatment enhanced the thermal limit susceptibility of C. sasakii to 46℃. The larvae internally infesting apples were tested using this CATTS device and showed 100% lethality after 60 min exposure to a treatment of 46℃ under 15% CO2 in the chamber. This study suggests a possibility that CATTS can be applied as a quarantine measure to kill the larvae of C. sasakii locating inside the apples. To understand the CATTS effect, a heat shock protein was cloned. Hsp90 was partially sequenced and showed its expression in response to heat treatment. CATTS was likely to suppress hsp90 expression.

Bacillus thuringiensis (Bt) is effective to control the diamondback moth, Plutella xylostella. However, its relative slow and unstable control efficacy limits its wide use by farmers. To facilitate pathogenic rate of Bt, a bacterial mixture technique has been developed in this study. Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata temperata (Ptt), possess high immunosuppressive activity against several lepidopteran insects. The mixture treatments using Bt + Xn or Bt + Ptt significantly enhanced Bt pathogenicity in median lethal concentration and time. Though live Xn and Ptt bacterial cells gave significant effect on the pathogenicity, their 48 h culture broth after removing the bacterialcells still possessed the synergistic effect on the Bt pathogenicity. The larvae fed with the bacterial culture broth suffered significant immunosuppression in response bacterial to infection

Antimicrobial peptides (AMPs) are a group of immune proteins that protect the host from microial infection. Gallerimycin is one of the AMPs most commonly found in Galleria mellonella and Spodoptera frugiperda. In this paper, we found Gallereimycin in Spodoptera exigua by expressed sequence tag library 0analysis. The gallerimycin of S. exigua gene is 332 bp long and the predicted open reading frame contains 75 amino acids with a signal peptide. After removing signal peptide, S. exigua gallrimycin was estimated to be 5.9 kDa and pI at 8.53. The gallerimycin of S. exigua shared maximum sequence homology with that of S. frugiperda. In naive S. exigua larvae, not much gene expression was detected, but strongly induced in fat body and hemocytes following immune challenge with entomopathogenic bacteria and fungus. A recombinant gallerimycin was prepared using a bacterial expression system and showed significant antibacterial and antifungal activities. RNA interference using double stranded RNA could knock down the expression of gallerimycin and significantly suppressed immune capacity.