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. Parasitization by an endoparasitoid wasp, Cotesia plutellae, delays the larval development and metamorphosis in Plutella xylostella. Nutritional deprivation by the wasp may induce these developmental alterations in growing host. This study focussed on the change of insulin signaling of the parasitized host. The parasitized larvae exhibit a significant suppression in insulin-like peptide (ILP) expression, which was induced only by the injection of the CpBV. Reduced ILP expression significantly increased the blood sugar level (trehalose) level in the parasitized host, which was mimicked by starvation. Foxo was expressed in the parasitized larvae, but localized mostly in the nucleus. Overexpression of ILP gene in the parasitized larvae induced translation of Foxo to cytoplasm and significantly decreased trehalose level in the plasma. Interestingly, the overexpression of ILP gene significantly prevented the successful parasitization and allowed the host metamorphosis.

A phase variation has been reported in an entomopathogenic bacterium, Xenorhabdus nematophila. Compared to a wild type primary form, a secondary form usually lose several physiological and biochemical characters. This study showed that the phase variation of X. nematophila caused a significant alteration in its immunosuppressive activity and subsequent entomopathogenicity. A secondary form of X. nematophila was detected in laboratory colonies and exhibited significant differences in dye absorption and entomopathogenicity. In addition, the secondary form was different in production of eicosanoid-biosynthesis inhibitors (EBIs) compared to the primary form of X. nematophila. Production of oxindole and p-hydroxypropionic acid was significantly reduced in the culture broth of the secondary form of X. nematophila. The reduced EBI production resulted in significant suppression in the inhibitory effects on a cellular nodule formation and phenoloxidase activity. Culture broth of the primary form of X. nematophila significantly more enhanced the pathogenicity of Bacillus thuringiensis (Bt) than the culture broth of the secondary form. Furthermore, this study developed a high efficient ‘Dual Bt-Plus’ to control both lepidopteran insect pests of Plutella xylostella and Spodoptera exigua by mixing two effective Bt strains along with the addition of potent bacterial metabolites or 100-fold concentrated X. nematophila culture broth.

An entomopathogenic bacterium, Xenorhabdus nematophila (Xn), is symbiotic to a nematode, Steinernema carpocapsae, and exhibits high pathogenicity to lepidoptera insects. Its metabolites released into the bacterial culture broth and also virulent in oral especially when they are treated with Bacillus thuringiensis (Bt). This study devised a high efficacy microbial insecticide by combining Xn culture broth and Bt. Bt kurstaki (Btk) exhibited relatively higher pathogenicity to Plutella xylostella than Spodoptera exigua larvae. By contrast, Bt aizawai (Bta) showed a reverse pathogenicity pattern. Phase Ⅰ type of Xn (XnK1) was isolated from S. carpocapsae Pochun and exhibited high pathogenicity than phase Ⅱ bacteria. Three bacterial mixtures of Bta+XnK1, Btk+XnK1, and Bta+Btk+XnK1 were prepared and analyzed in their target insects. Bta+XnK1 showed higher pathogenicity than those of Bta alone or Btk+XnK1 in P. xylostella. Btk+XnK1 showed higher pathogenicity than those of Btk alone or Bta+XnK1 in S. exigua. Bta+BtK+XnK1 showed high pathogenicity against both P. xylostella and S. exigua.

Cotesia plutellae has been known as a natural enemy against the Diamondback moth, Plutella xylostella via laying eggs into a larva. When the larva hatches from the egg, teratocytes also are released and expected to work as immune suppressor via secreting immune suppressive factors. In order to analyze the gene expression in teratocytes, total RNAs were isolated and genes expressed in the teratocyte were sequenced by Illumina HiSeq2000 RNASeq analysis. The information on RNA sequences was assembled by Trinity and contigs were annotated by Blast analysis. The levels of gene expression were calculated by FPKM. Approximately, 6.3 Gbs were obtained and 34,686 contigs were found and annotated. Forty two percent of contigs were homologous to previously reported genes and classified by gene ontologies: the highly abundant components are metabolic process, biological regulation and cellular process in biological function; binding, catalytic activity and transporter activity in molecular function; cell part, membrane part and organelle in cellular function, respectively. In addition, some teratocyte transcripts of C. plutellae are related to host regulation such as immunosuppression and nutrition: Ankyrin repeat proteins, Serpin, protease, lipase, chitinase and scavenger receptor.

Cadherin gene, which is a receptor of the Bacillus thuringiensis toxins, was predicted from 454 pyrosequencing transcripts from fifth instar larvae of the beet armyworm, Spodoptera exigua. The S. exigua cadherin gene (SeCad1) encodes 9 cadherin repeats and a tranmembrane domain. The SeCad1 gene was expressed in all developmental stage specifically in gut tissue by RT-PCR analysis. Expression of SeCad1 gene was suppressed by both injection and feeding of its specific dsRNASeCad1 in 5th instar larval stage. The suppression of SeCad1 expression did not significantly influence on pupal and adult development of S. exigua. However, the larval treated with dsRNASeCad1 (100 ng/larva) significantly reduced susceptibility to B. thuringiensis ssp. aizawai (3 × 106 CFU/larva). By contrast, the dsRNASeCad1-treated larvae did not show any change in susceptibility to B. thuringiensis ssp. krustaki (4 × 107 CFU/larva). These results suggest that SeCad1 is a specific receptor of Cry1A toxin from B. thuringiensis in S. exigua, but not Cry1C toxin.

The beet armyworm, Spodoptera exigua, is a freeze-susceptible species and overwinters without diapause in temperate zone. Depression of supercooling point (SCP) and rapid cold hardiness (RCH) allow S. exigua to survive at low temperatures. This study reports a polyol which is responsible for the cold hardiness of S. exigua. Pre-exposure of S. exigua larvae to 4°C for 6 h significantly enhanced survival under a freezing temperature (-10°C). This pre-exposure treatment also significantly depressed larval SCPs. Analysis of polyols indicated that glycerol titers significantly increase with increase of pre-exposure time. Glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) are involved in glycolysis pathway of insect. The S. exigua GK (SeGK1) and G3PDH (SeG3PDH1) genes were predicted from 454 pyrosequencing transcripts from fifth instar larvae of the beet armyworm, S. exigua. The SeGK1 and SeG3PDH1 genes both were expressed in all larval stage by RT-PCR analysis. Expression of SeGK1 and SeG3PDH1 genes were suppressed by its specific dsRNASeGK1 or dsRNASeG3PDH1 injection into hemocoel of 5th instar larva. Each 200 ng of dsRNASeGK1 or dsRNASeG3PDH1 injection also significantly decreased glycerol amount in hemolymph. Larval treated by either dsRNASeGK1 or dsRNASeG3PDH1 significantly lost the RCH under -10°C exposure. These results indicate that glycerol is a crucial RCH agent and its synthesis is regulated by SeGK1 and SeG3PDH1 genes in S. exigua.

An entomopathogenic bacterium, Xenorhabdus nematophila, secretes at least eight bacterial metabolites, which have suppressive effects on insect immunity. This study quantified their sequential production during bacterial growth and analyzed their individual immunosuppressive activities against an insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth in both insect host and culture medium, in which eight metabolites were secreted in different time points. At early growth phase (6 to 12 h), Ac-FGV, Cis-cPY, PHPP and indole metabolites were detected in the culture broth. During early growth phase, PHPP was highly potent to inhibit phenoloxidase activity as well as nodule formation. At late growth phase (24 to 48 h), BZA, HPA, PY were detected at 10 – 140 ppm in the culture broth, their metabolites were highly potent to inhibit phospholipase A2 and to induce cytotoxicity to hemocytes. These results suggest that X. nematophila sequentially produces the immune suppressive metabolites, which cooperatively inhibit different steps of insect immune responses.

Immune defense is indispensible for insect survival. However, uncontrolled and excessive immune responses would be highly detrimental and energy-consuming processes. An insect cytokine, plasmatocyte-spreading peptide (PSP), induces hemocyte-spreading behavior as well as activating phenoloxidase (PO) in the beet armyworm, Spodoptera exigua. A hemocyte transcriptome of S. exigua contains a partial sequence of a putative PSP-binding protein (SePSP-BP). SePSP-BP was expressed in all developmental stages especially in hemocytes and fat body. A quantitative RT-PCR showed that the bacterial infection significantly up-regulated the expression level of SePSP-BP. A double-stranded RNA specific to SePSP-BP (dsRNASePSP-BP) was injected and suppressed SePSP-BP expression even in response to bacterial challenge. The larvae treated with dsRNASePSP-BPsuffered high mortality to infection of nonpathogenic bacteria and prolonged high PO activity after the immune challenge. These results suggest that SePSP-BP may play a role in suppressing immune responses as a negative controller

Two Grapholita congeners, G. dimorpha and G. molesta, are internal fruit feeders and their young larvae cause serious damages to pome and stone fruits in Korea. They share similar morphological and biological characters not to be easily discriminated. We needed to develop molecular markers using diagnostic primers and PCR-RFLP with specific sequences in ND4 region. Two species have similar sex pheromone components (Z8-12:Ac and E8-12:Ac) although their composition ratios are different. In fields, G. molesta males were more captured in lures with higher Z8 component ratio than G. dimorpha males. Addition of Z8-12OH, minor sex pheromone component prevented G. dimorpha from capturing G. molesta males. In electroantennogram (EAG) bioassay, these two species males showed significant electric responses in their own sex pheromone ratios. An addition of Z8-12:OH to the major sex pheromone components significantly suppressed the EAG response of G. dimorpha, while it did not change that of G. molesta. A deep sequencing analysis of transcripts of both species pheromone glands identified sex pheromone biosynthesis genes including fatty acid synthase, desaturases, fatty acyl reductase (FAR), and aldehyde reductase. The presence of delta 10 desaturase in both species suggests that a double bond at C8 position in dodecenyl acetate is produced by desaturation at C10 position of tetradecenyl fatty acid and subsequent β-oxidation, which is then reduced at carboxylic acid by FAR to be acetylated by acetyl transferase. High sequence variation of FAR genes of G. molesta and G. dimorpha suggests their stereoisomer substrate preference, which may exert a driving force for this speciation with delta 10 desaturase.

최근 사과원에서 복숭아순나방붙이(Grapholita dimorpha)의 발생이 보고되었다. 복숭아순나방붙이는 이와 유사한 복숭아순나방(G.molesta)이 발생하는 또 다른 기주에서도 동시에 발생이 가능하다고 제기되었다. 본 연구는 국내 여러 지역의 배과원에서 복숭아순나방붙이의 발생이 있음을 보고한다. 복숭아순나방붙이의 종 동정은 형태적 특징과 분자마커를 이용하여 실시되었다. 이들 두 종의 공통된 성페로몬 주성분으로 상호 교차 포획이 이뤄질 수 있다. 복숭아순나방붙이 페로몬트랩에 복숭아순나방붙이와 복숭아순나방이 포획되고, 복숭아순나방 페로몬트랩에 복숭아순나방과 복숭아순나방붙이가 포획되었다. 복숭아순나방붙이 트랩에 이뤄진 교차 포획비율은 지역적으로 다른 배과원에서 상호 뚜렷한 차이가 나타났다. 더욱이 이 두 종의 발생 피크도 조사한 모든 야외 지역에서 시기적으로 뚜렷한 차이를 보였다. 이러한 결과는 배 과수원에서 두 종의 성페로몬 트랩으로 얻은 모니터링 자료는 각각 서로 다른 종의 포획이 혼재하며, 이는 해당종의 발생빈도와 발생밀도가 확대 해석될 수 있음을 본 연구 자료는 제시하고 있다.

콩과(Fabaceae) 작물 해충인 팥나방(Matsumuraeses phaseoli)과 어리팥나방(M. falcana) (나비목: 잎말이나방과)은 형태적으로 매우 유사하여 종 구별이 힘든 것으로 알려져 있다. 본 연구에서는 PCR-SSP(PCR with Sequence Specific Primers) 방법으로 두 종을 빠르고 정확하게 구별할 수 있는 판별법을 찾고자 두 종의 미토콘드리아 시토크롬 옥시다제 I(mtCOI) DNA 부분영역(439 bp)의 염기서열을 해독하였다. 그리고 다른 나방 종의 mtCOI 염기서열과 함께 나열하여 비교한 후 팥나방과 어리팥나방에서 종 특이적으로 차이가 나는 단일 뉴클레오티드를 프라이머의 3ʹ말단으로 하는 염기서열 특이 프라이머 조합을 만들었다. PCR 산물들을 전기영동 한 결과, 어리팥나방은 245 bp, 팥나방은 409 bp와 245 bp의 특이적 밴드 패턴을 보여 두 종을 구별할 수 있었다.

아이코사노이드는 곤충의 다양한 세포성 면역 반응을 중개한다. 본 연구는 면역반응에 따라 혈구세포 밀도 변화에 대한 아이코사노이드의 새로운 중개 기능을 밝히기 위해 수행되었다. 파밤나방(Spodoptera exigua) 5령충은 세균 감염에 따라 2 시간이 지나면 총혈구수의 현격한 증가를 보였다. 이 총혈구수 증가는 주로 부정형혈구와 소구형혈구 밀도의 증가로 해석되었다. 파밤나방 유충에 phospholipase A2 (PLA2) 억제자인 dexamethasone을 처리하면 세균 처리에 의한 총혈구수 변화가 일어나지 않았다. 하지만 dexamethasone을 처리한 유충에 PLA2의 촉매산물인 arachidonic acid를 첨가하면 총혈구수 증가가 회복되었다. 이러한 혈구 밀도 변화에 원인으로서 아이코사노이드 종류를 추적하기 위해 cyclooxygenase (COX)의 억제자인 naproxene을 처리한 결과 총혈구수 증가가 억제되고, lipoxygenase (LOX)의 억제자인 esculetin을 처리하면 총혈구수 증가가 유지되어 COX 산물이 세균 침입에 따른 총혈구수 증가에 관여하는 것으로 나타났다. COX의 생산물인 prostaglandin E2(PGE2)를 세균 없이 단독으로 처리할 때도 총혈구수의 뚜렷한 증가를 나타냈다. 이러한 결과는 파밤나방의 세포성 면역반응 과정에서 총혈구수 증가를 중개하는 아이코사노이드의 새로운 기능을 제시하고 있다.

Parasitism by an endoparasitoid wasp, Cotesia plutellae, results in significant immunosuppression of the diamondback moth, Plutella xylostella. Parasitized larvae significantly suffered higher susceptibility to a microbial biopesticide, Bacillus thuringiensis (Bt) than nonparasitized (NP) larvae. To find out an immunosuppressive agent causing the enhanced Bt efficacy, viral ankyrin (=vankyrin) genes encoded in C. plutellae bracovirus (CpBV) were analyzed by transient expression in NP larvae. CpBV segments containing different vankyrins were microinjected to NP larvae and expressed their encoded vankyrins. Expression of some vankyrins significantly inhibited immune response and enhanced Bt efficacy. This study suggests that expression of vankyrins suppress a cellular immune response and lose Bt tolerance of P. xylostella larvae.

An endoparasitoid wasp, Cotesia plutellae, parasitizes young larvae of the diamondback moth, Plutella xylostella, with its parasitic factors of polydnavirus, venom, ovarian proteins, and teratocytes (TC). TCs are originated from embryonic serosal membrane at hatch of C. plutellae eggs. TCs, after released in hemocoel of parasitized larvae, increased their average cell size from 20.6 μm to 77 μm during whole developmental period of the parasitoid larvae, but did not increase their cell number by maintaining about 150 cells per larvae. TCs of C. plutellae, are considered to be involved to extend the host larval development period and to arrest larval-pupal metamorphosis, were cultured in an insect cell culture medium for 21 days. Like TCs in parasitized larvae, in vitro cultured TCs showed increase in cell size, but did not show increase of cell number. Microinjection of in vitro cultured TCs significantly inhibited larva-to-pupa metamorphosis of nonparasitized P. xylostella, in which pupated host also showed extended larval period. Larvae injected with TCs exhibited alteration in expression of ecdysone receptor (EcR) and insulin receptor (InR) as well as in parasitized larvae. Teratocyte-secretory factors in culture medium showed this antimetamorphic effect on P. xylostella, while heat treated TC culture medium lost the effect. However, a successful parasitization of C. plutellae required both TCs and polydnavirus to alter host physiology.

An endoparasitoid wasp, Cotesia plutellae, contains a polydnavirus called C. plutellae bracovirus (CpBV) and induces various physiological alterations of parasitized host along with expressions of viral genes. Two host translation inhibitory factors (HTIFs) encoded in CpBV specifically inhibit host mRNAs at post-transcriptional level. They are expressed in late larval stage of Plutella xylostella parasitized by C. plutellae. To understand their late expression control, promoter region of an HTIF gene called CpBV15α was cloned by inverse PCR. The cloned HTIF upstream region (1,113 bp) possessed a putative JH response element (JHRE) and other promoter elements. The putative promoter region was rejoined with an open reading frame of enhanced green fluorescence protein (EGFP). When the recombinant vector construct was injected into early third instar larvae of nonparasitized P. xylostella, it was expressed in fourth larval instar at 72 h after injection, compared to relatively early expression in 24 h after injection of control construct containing a baculovirus immediate-early promoter. However, recombinant EGFP construct lost the late expression pattern when its promoter region was incomplete by truncating JHRE region. PYR application inhibited EGFP expression of the recombinant construct, but gave little influence on truncated constructs. Interestingly, when the complete promoter construct was injected to pupal stage, its late expression pattern was lost and showed early expression pattern. However, an addition of PYR to pupae, which had been injected with the complete promoter construct, inhibited the reporter gene expression. These results suggest that late expression of a HTIF (CpBV15α) is controlled by its promoter, which is sensitive to host JH titer.

Insect blood cells, hemocytes, inhibit spreading behavior upon bacterial challenge to perform cellular immune responses. Hemocyte spreading is accomplished by cytoskeleton rearrangement, which is activated by various immune mediators, such as biogenic monoamins, plasmatocyte-spreading peptide(psp), and eicosanoids. However, little is known how these, immune mediators. acitvate hemocyte spreading behavior. A small G protein, Rac1, gene was identified in hemocytes of Spodoptera exiqua. Its expressed in most developmental stages accept egg and expecially expresses in hemocytes and fat body of Larval stage. In response to bacterial challenge, its expression was segnificantly up-regulated. However, RNA inteference (RNAi) of Rac1 expression inhibited hemocyte spreading behavior. under RNAi condition of Rac1, octopamine and psp failed to activate hemocyte spreading behavior. Interestingly, as addition of prostaglandinE2 to the RNAiconditioned Larval rescued the mediation of octopamine and psp. These results indicate that Rac1 is required for mediation of octopamine and psp on hemocytespreading behavior and suggest that Rac1 may activate eicosanoid biosnthesis.

The oriental fruit moth (Grapholita molesta) and the plum fruit moth (G. dimorpha) are internal feeders of stone and pome fruits and highly similar in morphological characters and feeding behaviors. These two species share their two main sex pheromone components, Z8-dodecenyl acetate(Z8-12Ac) and E8-dodecenyl acetate(E8-12Ac) although pheromone compositions are different. But, two males of these species were cross-attracted to G. molesta and G. dimorpha pheromone trap, respectively. Their host plants are also very similar in Rosaceae including apples, plums, paches, etc. These sympatric and similar pheromone ratios and biological characters suggest their recent speciation divergence. To determine genetic origin of this speciation, were analysed transcriptomes associated in sex pheromone biosynthesis in these sibling species. Total RNAs were collected from pheromone glands and read by a short read deep sequencing technology using an lllumina HiSeq2000. Almost 3-4 Gb reads were de novo assembled and resulted in 76,361 contigs of G. dimorpha and 104,463 contigs of G. molesta. More than 70% of these contigs were annotated and classified by a typical GO analysis. Transcriptomes related with sex pheromone biosynthesis were selected and grouped into fatty acid synthase, fatty acid oxidation, desaturase, reductase, and isomerase. These analyses identified sex pheromone biosynthesis machineries, which showed significant differential expressions between two sibling species. Field monitoring assays indicated the minor components (Z8-12OH) resulted from fatty acid reductase were crucial in isolating two sibling species.

The oriental fruit moth (Grapholita molesta) and the plum fruit moth (G. dimorpha) are internal feeders of apples. Their sympatric and similar sex pheromone compositions suggest their recent divergence in speciation. This study aims to determine genetic factors in this speciation by comparing transcriptomes associated in sex pheromone biosynthesis in these sibling species. Total RNAs were collected two female abodominal tips and read by a short read deep sequencing technology using an lllumina HiSeq. Almost 3-4 Gb reads were de novo assembled and resulted in 76,361 contigs of G. dimorpha and 104,463 contigs of G. molesta. More than 70% of these contigs were annotated and classified by a typical GO analysis. Transcriptomes related with sex pheromone biosynthesis were selected and grouped into fatty acid synthase, fatty acid oxidation. These analyses identified sex pheromone biosynthesis machineries

Three tortricid pests, Grapholta dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura) are known as internal apple feeders in Korea. For identify young larvae which occurring serious damage in fruits, the molecular maker was developed from their mitochondrial DNA (mtDNA) sequences. To develop of PCR-RFLP marker, ND4 locus was digested with Swa Ⅰ. ND4-Swa Ⅰ digests showed two bands (396, 292 bp), one band (700 bp), and three bands (408, 178, and 103 bp) of G. dimorpha, G. molesta, and C. sasakii, respectively. Species-specific diagnostic PCR primers were developed in the ND4 locus and gave species-specific PCR products. Finally, these markers were applied to diagnose larvae infesting apples and showed species-specific fruit damage patterns, in which most feeders of G. dimorpha, G. molesta, and C. sasakii showed major feedings in apex, surface, and core of apple fruits, respectively.

An entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata subsp temperata (Ptt), suppresses insect immune responses and facilitates its symbiotic nematode development in target insect. Benzylideneacetone (BZA), PY, cPY, Ac-FGV, indole, 2-oxindole and 3-(4-hydroxyphenylpropionic) acid (PHPP) were compounds derived from the bacterial. Their immunosuppressive activities have been induced by inhibitory activity against eicosanoid biosynthesis and used to develop an additive to enhance control efficacy of other commercial microbial insecticides. This study investigated any cytotoxicity of their culture broth and bacterial metabolites on Spodoptera exigua hemocyte. When Xn or Ptt (<100 cells per larva) were injected to larval of S. exigua, the bacteria increased in density with incubation time, while the insent hemocyte numbers significantly and the resulting culture broths were sampled for analysis of their cytotoxicity against S. exigua hemocytes. In addition, the sequential culture broth samples were analyzed in active component chemicals using a reverse phase HPLC. Finally, seven bacterial metabolites were analyzed in relative cytotoxicity against S. exigua. These results suggest that BZA is a major cytotoxic compound.