Microbe have been considered as potential control agents for pest, as alternative to chemical control methods. Among entomopathogens, fungi cause the mortality by penetrating the cuticle of pest and/or by metabolites such as toxin. Not only this direct control effect of fungi, but repellency of fungi also may be used to prevent the pest. Repellence effect of fungi is considered as inhibitory factor to control termite. A study was reported in Japan that termite was able to detect and remove the conidia of fungi on their surface. The termite can escape from fungal infection and protect their colony. There is few study that insect pest such as moth can detect and avoid the virulence fungi. Therefore, we has been conducting the detection and avoidance of beet armyworm to high pathogenic fungi, Paecilomyces fumosoroseus. Adult of the beet armyworm avoided oviposition at Chinese cabbage treated with P. fumosoroseus compare to control. This result may be used to prevent the infestation of moth in crop production.

Entomopathogenic nematodes (EPNs) of the genus Steinernema are pathogenic to the insects and well known as ideal models for understanding parasite-host interaction. EPNs harbor a number of bacterial symbionts in their gut belonging to the noble genus Xenorhabdus which are capable of killing insects by themselves or by combination with nematodes by suppressing insect immune defense. Here, we report host range of Steinernema monticolum and its symbiont Xenorhabdus hominickii. S. monticolum has a diverse host range including lepidopteran and coleopteran insects although they showed higher pathogenicity to the lepidopteran insects. Especially, X. hominickii suppressed insect immune responses. A target insect, Spodoptera exigua, exhibited both cellular and humoral immune responses by expressing antimicrobial peptides and forming nodules in response to heat-killed X. hominickii. However, live bacteria significantly suppressed the immune responses. An addition of arachidonic acid to the bacterial infection significantly rescued the immune responses, suggesting eicosanoid biosynthetic pathway as a pathogenic target of X. hominickii.

기후변화는 국내 해충상의 변화에 영향을 주는 주요 요인 가운데 하나로 알려지고 있다. 특히 지구 온난화 모델에 따라 남방계 곤충의 서식 지 확대가 예상되고 있다. 휴면 기작을 가지고 있지 않은 파밤나방(Spodoptera exigua)과 배추좀나방(Plutella xylostella)은 시설재배지를 중심으 로 국내에서 월동이 가능한 것으로 알려지고 있다. 그러나 두 해충은 계절적 발생 양상에서 뚜렷한 차이를 보여주었다. 배추좀나방은 이른 봄철과 가을기간에 발생하고 여름 기간 중에는 발생하지 않았다. 반면에 파밤나방은 늦은 봄철에 나타나기 시작해 가을까지 지속적으로 발생하였다. 본 연구는 이러한 두 남방계 곤충이 계절적 발생 차이를 보이는 것이 이들이 갖는 고온에 대한 감수성 차이에 기인한 것으로 가정하였다. 이 가설을 증명하기 두 곤충의 내열성을 비교 분석하였다. 동일한 열처리(42℃)에서 배추좀나방 유충은 40 분 노출에 100% 사망률을 보인 반면, 파밤나방 은 대부분의 유충이 80 분의 노출에서도 생존하였다. 이러한 내열성은 두 곤충 모두 이들의 발육시기에 따라 상이했다. 배추좀나방은 4령 유충과 성충이 가장 높은 내열성을 보인 반면, 파밤나방은 1령 유충에서 가장 높게 나타냈다. 두 곤충 모두는 37℃에서 30 분간 전 처리 후 고온에 노출시 키면 생존율이 뚜렷하게 증가했다. 이러한 내열성유기는 두 곤충 모두 혈림프의 글리세롤 함량 증가와 관련성을 보였다. 또한 파밤나방의 경우는 열충격단백질의 발현도 증가하였다. 따라서 이상의 결과는 여름기간 배추좀나방의 발생이 없는 것은 이 곤충의 고온에 대한 높은 감수성에 기인 된 것으로 보이고, 반면에 파밤나방은 비교적 높은 내열성을 보유하여 여름 기간에도 발생을 지속시킨 것으로 해석되었다.

곤충 생리현상의 가소성은 후생유전적 변화와 밀접하게 관련을 지을 수 있다. 이 가설을 증명하기 위해 광식성인 파밤나방(Spodoptera exigua)을 대상으로 상이한 먹이 조건에 따라 이 곤충의 발육과 DNA 메틸화에 영향을 주는 지 분석하였다. 동일한 코호트로 부터 얻은 갓 부화한 유충을 최종령에 이르기까지 세 가지 다른 먹이(대파, 배추, 인공사료)로 섭식 처리하였다. 이 결과 상이한 먹이 조건에 따라 유충발육속도, 용화 율 및 우화율에서 뚜렷한 차이를 보였다. 인공사료로 사육된 유충이 가장 빠른 유충발육속도와 높은 용화율 및 우화율을 나타냈다. 반면에 두 자 연 기주 가운데는 대파가 배추에 비해 파밤나방 발육에 양호하였다. 이러한 먹이에 따른 변이는 혈림프 단백질 및 혈당에서도 차이가 나타났다. 또한 발육과 연계되었을 것으로 추정되는 인슐린유사펩타이드(SeILP1) 유전자의 발현 정도도 먹이조건에 따라 상이했다. 단일항체를 이용하여 파밤나방 게놈 DNA의 시토신 메틸화를 분석한 결과 이 부위에 DNA 메틸화가 검출되었으며, 메틸화 정도는 먹이 조건에 따라 상이했다. 이 결 과들은 동일 집단의 파밤나방이 상이한 먹이 조건에 따라 발육차이를 나타내고 또한 시토신 메틸화에 변이를 보여 이 곤충의 생리적 가소성에 후 생유전적 인자가 작용하고 있는 것을 제시한다.

Trehalose is a major blood sugar in insects. It functions as carbohydrate nutrient source to all tissues with a relatively constant level and also serves as a cryoprotectant at high levels in some insects. Thus, its hemolymph levels need to be controlled according to physiological conditions. An insulin-like peptide, SeILP1, was identified and showed to mediate down-regulation of the hemolymph trehalose levels of Spodoptera exigua larvae. This study showed that hemolymph trehalose levels of S. exigua significantly increased upon immune-challenge and under starvation in a time-dependent manner. During up-regulation of the hemolymph trehalose, the gene expression of trehalose phosphate synthase significantly increased, but that of trehalase significantly decreased. Injection of biogenic monoamines, such as octopamine and serotonin, did not increase the hemolymph trehalose levels. Either treatment of arachidonic acid or eicosanoid biosynthesis inhibitor did not change the hemolymph trehalose levels. However, injection of adipokinetic hormone (AKH) of Manduca sexta significantly increased the trehalose levels. Interrogation of Spodobase identified an AKH-like gene of S. exigua. Its expression increased with starvation in the larvae. Its RNA interference significantly prevented the up-regulation of the hemolymph trehalose levels in S. exigua.

Entomopathogenic fungi have been studied to develop for biological control agents as an alternative to chemical control agents in insect pest management. Two Lepidopteran insects, Spodoptera exigua and Plutella xylostella, are serious insect pests infest various crops, but not effectively controlled by commercial chemical pesticides due to its high insecticide resistance. A fungal isolate was isolated from S. exigua larvae collected from green onion field in Andong, Korea. To identify the fungal isolate, 18srRNA sequence for internal transcribed spacer (ITS) and β-tubulin regions were sequenced. The ITS and β-tubulin sequence were highly matched to Beauveria bassiana and morphological characteristics also was fit to known B. bassiana. Finally, isolated fungus has identified as B. bassiana and named B. bassiana ANU1. The result of bioassay, median lethal concentrations were 2.7×103 and 0.9×103 conidia/ml and medial lethal times were 65.6 and 60.8 h to S. exigua and P. xylostella, respectively. B. bassiana ANU1 showed high pathogenicity to two insect pests from 20℃ to 30℃ at 50% relative humidity (RH) and more than 40% RH at 25℃ with 107 conidia/ml of concentration.

Like vertebrate insulins, insulin-like peptides (ILPs) play crucial roles in controlling immature growth, adult lifespan, and plasma sugar level in some insects. An ILP gene (SeILP1) was predicted from a transcription database of Spodoptera exigua. SeILP1 encodes 95 amino acid sequence, which shares sequence homologies (33~83%) with other insects ILPs. The predicted B and A chains possess six cysteine residences. SeILP1 was expressed in all developmental stages of S. exigua. However, its expression was detected in fat body, gut and epidermis, but not in hemocytes. Its expression increased with feeding activity. Plasma trehalose levels of fifth instar larvae maintained at relatively stable concentration of 2.31±0.62 mM. However, starvation induced a significant increase of plasma trehalose level by more than two fold in 48 h, at which SeILP1 expression kept at a low level. RNA interference of SeILP1 induced a significant increase of plasma trehalose level. Interestingly, a bovine insulin decreased plasma trehalose level in a dose-dependent manner. These results indicate mat SeILP1 plays a role in suppressing plasma trehalose level in S. exigua.

Double-stranded RNA(dsRNA) had been used to specitically suppress target gene expression at post-tanscription level. Injection of dsRNA to hemocoel is the most efficient to knockdown target mRNA. However, some insects have shown to be susceptible to feeding dsRNA. Spodoptera exigua was susceptible to dsRNA at oral treatment. Especially dsRNA specific to β-integrin was potent to survival of S.exigua larvae. This study advanced our dsRNA application technology by generating recombinant E.coli expressing dsRNA specific the β-integrin. A recombinant vector L4440 was constructed with a partial β-integrin gene under T7 RNA polymerase promoter. The recombinant vector was used to transform HT115 competent cells of E.coli. The transformed E.coli expressed the dsRNA. The production of dsRNA was proportional to the bacterial number. By feeding the recombinant E.coli, S.exigua underwent significant mortality. By adding E.coli expressing Cry1Ca Bt toxin to E.coli expressing dsRNA, S.exigua exhibited highly enhanced mortality. This study suggests a possibility to use a recombinant E.coli expressing dsRNA to control S.exigua.

Phospholipase A2 (PLA2) catalyzes an ester hydrolysis at sn-2 position of phospholipids. Various PLA2 genes are classified into at least 15 groups. However, on the basis of physiological functions, PLA2 genes are classified into calcium dependent cellular PLA2 (cPLA2), calcium independent cellular PLA2 (iPLA2) and secretary PLA2 (sPLA2). In insects, several sPLA2 genes are known to be associated with venom or immune functions. However, no known cellular PLA2 genes are identified. This study reports an iPLA2 (SeiPLA2) encoded in Spodoptera exigua. SeiPLA2 has an open reading frame of 2448 bp encoding a sequence of 816 amino acid residues. Its predicted protein is 89.55 KDa and 6.15 pI. SeiPLA2 is expressed in egg, larva, pupa and adult stages. In larval stage, SeiPLA2 is expressed in hemocytes, fat body, epidermis, gut, malpighian tubules and salivary gland. To understand its physiological function, its RNA interference is under investigation.

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.

The antifeedant activity of 20 plant essential oils, constituents from clove stem oil and related compounds were tested against the third instar larvae from both moth Spodoptera litura and Spodoptera exigua by used leaf dipping bioassay. Among the oils tested, clove stem (94%), thyme oil red (85%), and savory oil (80%) were showed high antifeedant activity against both S. litura and S. exigua third instar larvae. Thyme oil white (91%), geranium (90%), and cinnamon bark oil (85%) were shown high antifeedant activity against only S. exigua when compare other oils. The other plant essential oils were showed moderate (40-50%) or low (>20%) antifeedant activity against third instar larvae of both S. litura and S. exigua. The most active clove stem oil constituents were indentified by GC-MS. The major constituents eugenol (95%), farnesene (81%) and structurally related compounds isoeugenol (96%), nerolidol (80%) were showed significant antifeedant activity against both S. litura and S. exigua. Global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment justify further studies on the essential oils and their constituents describes as potential insecticides for the control of moth population with antifeedant activity.

파밤나방(Spodoptera exigua)의 발육을 일으키는 최저온도를 결정하고, 이 상태의 생리적 특성을 서로 다른 기능군(대사, 신경, 면역 및 스트레스) 유전자의 발현 양상을 이해하기 위해 본 연구를 수행하였다. 알부터 번데기까지 파밤나방의 발육영점온도는 5.5~11.6℃로 다양하였다. 유충은 알과 번데기에 비해 비교적 낮은 온도에서 발육이 가능하였다. 5령충의 경우 생리적 발육영점온도가 추정치(10.3℃)와 다르게 이보다 높은 15℃에서 관찰되었다. 정량적 RT-PCR로 분석된 유전자의 발현양상은 유충 영기가 진행됨에 따라 모든 기능군의 대부분 유전자의 발현량이증가하였고, 또한 5령 시기에서도 처리온도가 증가함에 따라 이들 유전자의 발현량도 증가하였다. 비록 동일한 갓 탈피한 5령이라 하더라도 이전에 노출된 외부 온도에 따라 발현량이 상이하였다. 5령충의 생리적 발육영점온도인 15℃에서 대부분의 유전자 발현량은 저하되었다. 그러나 높은 온도에서와 마찬가지로 발육기간이 증가함에 따라 이들 유전자의 발현량이 증가하였다. 이상의 결과는 발육영점온도에서 파밤나방의 발육 관련 유전자의 발현이 전체적으로 수준은 낮지만 지속적으로 진행되고 있다는 것을 의미한다.

Beet armyworm, Spodoptera exigua are difficult to control using chemical insecticides because of the development of insecticide resistance. For eco-friendly beet armyworm managements, various control agents are required. Entomopathogenic fungus is one of promise control agents as an alternative to chemical control agent. We isolated entomopathogenic fungi from soil samples of suwon by insect-bait method using Galleria mellonella and conducted bioassay to larva of beet armyworm. As a result of bioassay isolate FG274, FG340, FG344 had high virulence as 100% against second instar larva of S. exigua. To identify the fungus isolates, their’s morphological characteristic was observed and ITS of 18srRNA was sequenced. ITS sequence of FT274, 340, 344 were highly matched (100%) to that of Beauveria bassiana, Paecilomyces fumosoroseus, Metarhizium anisopliae. To investigate the optimal concentration, three isolates were sprayed at three different concentration(1×106 ,107 and 108 conidia/㎖) in laboratory conditions. 나타내었다.

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%.

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.

Melanism is one of the most marked phenotypic variations that naturally occur in a wide range of organisms. In this study, we established a homozygous melanism mutant strain with black pupae spontaneously occurring within a wild-type population of Spodoptera exigua. The S.exigua pupal melanic strain showed several viability advantages. The melanism is associated with faster development, heavier pupa weight and higher fecundity after eating seven different host plants. Female adults of the two strains both tend to attract xenogeneic male adults for mating, and the fecundity of the melanic strain is significantly higher than the wild-type strain. However, the melanism is associated with slower mean flight seed, shorter mean flight duration and distance. The melanic strain adults have weaker flight capacity in different ages. The viability advantages above will contribute in more generations per year, increasing population and more serious damage. Meanwhile, based on the mating competition results, the melanic strain will be able to interfere with the reproduction of wide-type strain and replace it. However, decreased flight capacity will influence the long-distance migration ability of the melanic strain and limit its range of damage

Beet armyworm, Spodoptera exigua are difficult to control using chemical insecticides because of the development of insecticide resistance. For eco-friendly beet armyworm managements, various control agents are required. Entomopathogenic fungus is one of promise control agents as an alternative to chemcal control agent. We conducted bioassays with entomopathogenic fungi to select high virulence isolate to larva of beet armyworm. The bioassay was used 150 entomopathogenic fungal isolates which were isolated from soil samples of nine provinces by insect-bait method using Galleria mellonella and Tenebrio molitor. . Three isolates, Metarhizium spp. FT83, FT89 and FT90 had high virulence as 84.6%, 100% and 100%, respectively, against S. exigua. The medial lethal time(LT50) of conidia of three isolates was 5.01, 2.99 and 2.92days respectively.

Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces parasporal crystal proteins known as endotoxins or Cry proteins. The Cry protoxins are then cleaved by insect midgut proteinases to form active Bt toxins. The activated Cry protein then binds to specific receptors at the midgut epithelium. Cadherin-like and aminopeptidase N (APN) proteins are involved in Bt toxin binding by interacting sequentially with different toxin structures. Aminopeptidase N (APNs) from several insect species have been shown to be putative receptors for these toxins. We have characterized four different midgut APNs(APN1, APN2, APN3, APN4) cDNAs from S. exigua. Forward primers and reverse primers for confirmation of four different midgut APNs were designed based on their sequences cloned from the cDNA libraries. Quantitative RT-PCR procedures includes 42℃ for 20min (cDNA synthesis), 99℃ for 5min, and 35 cycles (94℃ for 1min, and 60℃ for 50 s) for collection. Four aminopeptidase N isoforms were confirmed with qRT-PCR. Sequence analysis was performed by BlastX search the National Center for Biotechnology Information(NCBI) nucleotide. Furthermore, double-stranded RNAs(dsRNAs) were synthesized. DsRNAs were determined for bioassay.

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.