Prostaglandin E2(PGE2) is an autocrine and paracrine signal in insects and other animals. Its signal pathways in target cells are well understood in mammalian system but not in insects. Here, we assessed PGE2 signaling in hemocytes of Spodoptera exigua through knocking-down of signal component genes by RNA interference (RNAi) and knocking-out (KO) of PGE2 receptor using CRISPR-Cas9. From S. exigua transcriptome, we selected hemocyte signaling components and analyzed their functions in cellular immune responses through RNAi. KO mutant against PGE2 receptor exhibited severely hampered larval development and adult fecundity.

본 연구는 둥글장수풍뎅이(Pentodon quadridens bidentulus (Famaire, 1887)) 유충의 혈림프에 존재하는 혈구세포들의 종류와 병원균 침 입시 세포 면역을 담당하는 세포를 특정하고 분석하였다. 둥글장수풍뎅이(P. quadridens bidentulus)유충 혈강내에는 과립혈구세포, 부정형혈구 세포, 편도혈구세포, 소구형혈구세포, 원시혈구세포, 지방혈구세포 총 여섯 종류의 혈구세포들이 존재하였다. 여섯 종류의 혈구세포 중 과립혈구 세포가 병원균 침입 시 형태적 변이와 함께 병원균들에 대한 식균작용을 수행하는 면역세포로 관찰 되었다. 특히, 생체 내에서 병원균 침입시 12 시간 안에 활발히 식균작용을 수행하였고 24시간 이후로는 면역 활성화가 줄어들면서 정상상태로 전환되는 것을 알 수 있었다. 세포질 혈구세포 의 식균작용도 간혹 관찰 되었으나 나머지 세포들은 면역과 연관된 활성화가 없는 것으로 나타났다.

A novel oxidant fumigation (NOF) has been considered as alternative fumigant to replace methyl bromide that is a serious ozone depleter. Its high oxidative activity has been used as a bleaching or sanitary agent. Though some reports an insecticidal activity of NOF, its insecticidal action is yet to be understood. This study was conducted with an observation of an insecticidal activity of NOF against Plodia interpunctella, which is a stored grain insect pest. Cytotoxicity test was performed by using MTT assay, NOF gave a significant cytotoxicity on both Sf9 cells and HiFive insect cell lines. Sf9 cells were higher susceptible (IC50 = 43.2+ 3.5 ppm) to chloride dioxide than HiFive cells (IC50 = 174.6 + 5.9 ppm). To understand its cytotoxic effect on P. interpunctella, the larval hemocytes were incubated in vitro with different doses of NOF for 40 min at room temperature. In a dose-dependent manner, NOF gave a significant toxicity to the hemocytes. When NOF was injected to larvae of P. interpunctella, it significantly reduced total hemocyte counts compared to control. These results indicate that NOF has cytotoxic effect against hemocytes of P. interpunctella. This hemolytic activity of NOF can be regarded as a lethal factor to the stored grain insect pest.

Scavenger receptors (SRs) are transmembrane cell surface molecules recognized in apophotic cells, bacteria and lipopolysaccharide. With no physiological information on SRs in insects except SR-CI of Drosophila melanogaster, a putative SR gene was cloned and characterized in Spodoptera exigua. A partial S. exigua SR gene was obtained from hemocyte transcripts and exhibited high homology with type C. Its expression was confirmed in all developmental stages. Among different tissues, S. exigua SR was expressed highly in hemocytes. To confirm change in SR expression by infection, Escherichia coli was injected to fifth instar and RNA was extracted after 10 hours. SR expression in hemocytes of E. coli injected larva was not significantly different from the control but SR expression in fat body of E. coli injected larva was higher than the control. It is expected that SRs of S. exigua are related with immune responses against bacteria such as E. coli. To address its function, S. exigua SR expression was suppressed by double-stranded RNA (dsRNA).

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.

본 연구는 꼬리명주나비(Sericinus montela Grey)의 유충을 재료로 하여 투과전자현미쟁과 주사전자현미경을 이용하여 혈구의 미세구조를 관찰하였다. 꼬리명주나비 종령유충의 혈구는 구조와 특징에 따라 5종류로 구분되었다. 원시혈구(Prohemocyte)는 세포내에 하나의 큰 핵과 미분화된 세포내 소기관을 가지고 있었다. 부정형혈구(Plasmatocyte)는 섬유다발을 가진 방추행 또는 난형의 비교적 큰 세포로, 특히 방추형의 세포는 길게 발달한 핵을 갖고 있었다. 과립혈구(Granular cell)는 세포질내에 다양한 과립과 잘 발달한 소기관을 함유하고 있었으며 원형질 돌기를 많이 가지고 있었다. 소구형혈구(Spherule cell)는 세포질내에 미세한 물질을 함유한 소구(spherule)를 가자고 있으며 성숙한 세포는 소구내의 물질을 혈림프내로 방출시켰다. 편도혈구(Oenocytoid)는 세포질내 전자밀도가 높은 물결로 채워져 있으며 소기관은 잘 발달되어 있지 않았다.