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.

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.

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.

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.

Two entomopathogenic bacteria, Xenorhadus nematophila and Photorhabdus temperata temperata, are known to suppress immune responses of target insects by inhibiting eicosanoid biosynthesis. This study analyzed these bacterial metabolites in their effects on hemocyte-spreading behavior of the beet armyworm, Spodoptera exigua. Both bacterial culture broth significantly inhibited the hemocyte-spreading behavior, at which the culture broth derived from the stationary growth phase had the most potent effect. Three identified eicosanoid synthesis inhibitors (benzylideneacetone, PY and Ac-FGV) impaired the hemocyte-spreading behavior of S. xigua, at which benzylideneacetone was the most potent. These three compounds share a common chemical structure: a pentenebenzene ring. Alternation of this common structure resulted in significant loss of their inhibitory activity to the hemocyte-spreading behavior.

Innate immunity responses are triggered by the immune challenge and therefore involve signaling processes. The cellular response is initiated by hemocytes and mainly involves phagocytosis and encapsulation of intruders by these cells. To address whether Hc-STAT is activated upon bacterial challenge, we examined the subcellular location of STAT protein in hemocyte by immunostaining. A new insect member of the STAT family of transcription factors (Hc-STAT) has been cloned from the lepidopteran, Hyphantria cunea. The domain involved in DNA interaction and the SH2 domain are well conserved. The gene is transcribed at a low level during all stages of development, and the protein is present in hemocytes, fat body, midgut, epidermis, and Malphigian tuble (Mt). Especially, hemocytes and Mt showed transcriptional activation of Hc-STAT upon Gram (-) bacteria and fungal challenge. Gram (-) bacteria and fungal challenge specifically results in nuclear translocation of Hc-STAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vitro treatment with pervanadate translocates Hc-STAT to the nucleus in hemocyte cells. Here we report the first evidence for the involvement hemocyte JAK/STAT pathway upon microbial infection in lepidopteran insect.

Monoamines have been known to mediate cellular immune responses such as hemocyte nodulation and phagocytosis in some insects. Eicosanoids also mediate the cellular immune reactions responsible for clearing bacterial infection from hemolymph circulation in various insects. In this study, we investigated a functional relationship of both mediators in expressing hemocyte nodulation and phagocytosis of fifth instar larvae of the beet armyworm, Spodoptera exigua. Addition of octopamine or serotonin along with bacteria enhanced hemocyte nodulation and phagocytosis of S. exigua larvae. Phentolamine (octopamine inhibitor) and ketanserin (serotonin inhibitor) suppressed hemocyte nodulation and phagocytosis of S. exigua in response to a bacterial challenge. Each inhibitory effect of both monoamine inhibitors was recovered by addition of either octopamine or serotonin, suggesting their independent signaling pathways. Both inhibitor effects were also recovered by addition of arachidonic acid (a precursor of eicosanoid biosynthesis). Furthermore, the stimulatory effects of monoamines on the cellular immune responses were prevented by addition of dexamethasone (a specific phospholipase A2). However, the inhibitory effect of dexamethasone was not recovered by excess amounts of monoamines. These results suggest that monoamines mediate hemocyte nodulation and phagocytosis through a downstream eicosanoid signal pathway in S. exigua.