Jiyeong Park

Rac1 mediates cytokine-stimulated hemocyte spreading via prostaglandin biosynthesis in the beet armyworm, Spodoptera exigua

Cell spreading is an integral component of insect hemocytic immune reactions to infections and invasions. Cell spreading is accomplished by cytoskeleton rearrangement, which is activated by three major immune mediators, biogenic monoamines, plasmatocyte-spreading peptide (PSP), and eicosanoids, particularly prostaglandin E2 (PGE2). However, little is known about how these immune mediators activate hemocyte spreading at the intra-cellular level. A small G protein, Rac1, acts in cytoskeleton arrangements in mammalian cells. Based on this information, we identified a Rac1 transcript (SeRac1) in hemocytes prepared from Spodoptera exigua. SeRac1 was expressed in most developmental stages and in the two main immunity-conferring tissues, hemocytes and fat body, in larvae. In response to bacterial challenge, its expression was up-regulated by >37-fold at 2h post-injection and returned to a basal level about 2h later. Silencing SeRac1 expression inhibited hemocyte spreading in response to three immune mediators, octopamine, 5-hydroxytryptamine, and PSP. Addition of PGE2 to SeRac1-silenced larvae rescued the influence of these three mediators on hemocyte spreading. These compounds also increased phospholipase A2 activity via SeRac1, which leads to prostaglandin biosynthesis. We infer that SeRac1 transduces OA, 5-HT, and PSP signaling via activating biosynthesis of prostaglandins and possibly other eicosanoids.

Roles of peroxinectin in PGE2-mediated cellular immunity in Spodoptera exigua.

Background Prostaglandins (PGs) mediate insect immune responses to infections and invasions. Although the presence of PGs has been confirmed in several insect species, their biosynthesis in insects remains a conundrum because orthologs of the mammalian cyclooxygenases (COXs) have not been found in the known insect genomes. PG-mediated immune reactions have been documented in the beet armyworm, Spodoptera exigua. The purpose of this research is to identify the source of PGs in S. exigua. Principal Findings Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (SePOX-A ∼ SePOX-J) were expressed in S. exigua. Expressions of SePOX-F and -H were induced by bacterial challenge and expressed in the hemocytes and the fat body. RNAi of each POX was performed by hemocoelic injection of their specific double-stranded RNAs. dsPOX-F or, separately, dsPOX-H, but not the other eight dsRNA constructs, specifically suppressed hemocyte-spreading behavior and nodule formation; these two reactions were also inhibited by aspirin, a COX inhibitor. PGE2, but not arachidonic acid, treatment rescued the immunosuppression. Sequence analysis indicated that both POX genes were clustered with peroxinectin (Pxt) and their cognate proteins shared some conserved domains corresponding to the Pxt of Drosophila melanogaster. Conclusions SePOX-F and -H are Pxt-like genes associated with PG biosynthesis in S. exigua.

Change in Hemocyte Populations of the Beet Armyworm, Spodoptera exigua, in Response to Bacterial Infection and Eicosanoid Mediation

Eicosanoid mediates various cellular immune responses in insects. This study aimed to discover its novel action on the modulation of hemocyte populations in response to an immune challenge. Upon bacterial challenge, the last instar larvae of the beet armyworm, Spodoptera exigua, increased their total hemocyte density in 2 h, and then decreased it to a basal hemocyte density level. This rapid increase in total hemocyte density was explained by an increase of plasmatocyte and spherulocyte densities. When larvae were treated with dexamethasone (a specific phospholipase () inhibitor), they did not show any increase in hemocyte density in response to bacterial challenge. However, the addition of arachidonic acid (a catalytic product of ) to larvae treated with dexamethasone recovered the up-regulation of hemocyte density in response to bacterial infection. Among eicosanoid, cyclooxygenase (COX), but not lipoxygenase (LOX), products seemed to mediate the increase of hemocyte density in response to bacterial infection because naproxene (a COX inhibitor) inhibited the hemocyte density increase, though esculetin (a LOX inhibitor) did not. Prostaglandin , a COX product, significantly increased the hemocyte density even without bacterial infection. These results suggest that eicosaniod mediates a rapid increase in total hemocyte density in response to immune challenge.

PGE2 MEDIATES OENOCYTOID CELL LYSIS VIA A SODIUM-POTASSIUM-CHLORIDE COTRANSPORTER

Prostaglandin E2 (PGE2 ) mediates immune responses of the beet armyworm, Spodoptera exigua, including oenocytoid cell lysis (a class of lepidopteran hemocytes: OCL) via its specific membrane receptor to release inactive prophenoloxidase (PPO) into hemolymph. PPO is activated into phenoloxidase in the plasma to play crucial roles in the immune responses of S. exigua. The mechanism of OCL has not been elucidated, however we posed the hypothesis that a rapid accumulation of sodium ions within the oenocytoids allows a massive influx of water by the ion gradient, which leads to the cell lysis. It remains unclear which sodium channel is responsible for the OCL in response to PGE2 . This study identified a specific sodium channel called sodium-potassium-chloride cotransporter 1 (Se-NKCC1) expressed in hemocytes of S. exigua and analyzed its function in the OCL in response to PGE2 . Se-NKCC1 encodes a basic membrane protein (pI value = 8.445) of 1,066 amino acid residues, which contains 12 putative transmembrane domains. Se-NKCC1 was expressed in all developmental stages and tissues. qPCR showed that bacterial challenge significantly induced its expression. A specific inhibitor of NKCC, bumetanide, prevented the OCL in a dose-dependent manner. When RNA interference (RNAi) using double-stranded RNA specific to Se-NKCC1 suppressed its expression, the OCL and PPO activation were significantly inhibited in response to PGE2 . The RNAi treatment also reduced nodule formation to bacterial challenge. These results suggest that Se-NKCC1 is associated with OCL by facilitating inward transport of ions in response to PGE2 .

Identification and Physiological Characters of Intestinal Bacteria of the Black Soldier Fly, Hermetia illucens

ABSTRACT: The black soldier fly, Hermetia illucens , larvae may depend on indigenous bacteria in the intestine to feed and digest diversefood sources. To prove this hypothesis, we isolated and identified the intestinal bacteria of the black soldier fly for their digestive andantimicrobial abilities. The last instar larvae had long digestive tracts, which were about seven times longer than its body length. An individual of H. illucens larvae possessed a total of 5.0 x 10 ⁶ bacteria in the whole intestine, of which more than 98% bacteria were locate d in the hindgut. Three different bacterial isolates cultured on nutrient agar (NA) medium were detected in the intestine and ide ntified asMorganella morganii, Providencia rettgeri and Bacillus halodurans by Biolog microbial identification system. Analysis of 16S rDNA sequences of the intestinal bacteria detected the additional bacteria of Proteus mirabilis , Providencia alcalifaciens , and Providencia sp. Thes eintestinal bacteria cultured on NA medium exhibited high resistance to 4 antibiotics and inhibited growth of other microbes whi ch aremainly plant pathogens. Also, these bacteria exhibited catalytic activities to degrade cellulose, lipid, proteins, and carbohyd rates. Thes eresults suggest that H. illucens larvae possess intestinal bacteria that may play crucial roles in their digestive physiology. Key words: Black soldier fly, Enterobacterium, Antimicrobial activity, Digestive enzyme, Symbiosis초 록: 다양한 먹이 조건에서 생활하는 아메리카동애등에(Hermetia illucens)는 장내 세균의 의존성을 가질 수 있다. 이 가설을 증명하기 위해 본 연구는 종령 유충의 소화관에 존재하는 세균을 분리, 동정하고 효소활성 및 항균 능력을 분석하였다. 종령 유충의 소화관은 몸 체장에 약 7 배의 길이를 나타냈다. 한 개체의 소화관 내 존재하는 세균 수는 5.0 x 10⁶ cfu로 98% 이상이 후장에 존재했다. 소화관에는 3 종류의 상이한 세균이 존재했고, 미생물 동정 장치는 이들이 각각 Morganella morganii, Providencia rettgeri 및 Bacillus halodurans로 동정하였다. 이들 소화관 세균을 16S rDNA 서열을 분석한 결과 이 외에 Proteus mirabilis , Providencia alcalifaciens , Providencia sp.를 검출하였다. 이들 장내세균은 항생제 내성을 보였고, 타 미생물의 성장을 억제하였다. 또한 섬유소, 지질, 단백질 및 탄수화물의 분해 능력을 보유하였다. 본 연구 결과들은 아메리카동애등에 소화관에 유용성이 높은 세균을 보유하고 있다고 제시하였다.검색어: 아메리카동애등에, 장내세균, 항균력, 소화효소, 공생*Corresponding author: hosanna@andong.ac.krReceived July 17 2013; Revised September 24 2013Accepted October 4 2013아메리카동애등에(Hermetia illucens )는 파리목 동애등에과에 속하는 곤충으로 북아메리카 원산지이나 전 세계로 퍼져나가 온대지역 전역에 분포하는 범존종으로 분류된다 (Sheppard et al., 2002). 유충은 잡식성으로 주로 인축의 음식물 폐기물에서 발생하여 환경 정화 곤충으로 주목을 받아 왔다 . 또한 이 유기 폐기물에서 다수의 유충이 함께 섭식하면서 고온 (50-60℃)을 발생하고, 항생물질을 분비하여 타 생명체의 접근을 억제한다(Landi, 1960; Erickson et al., 2004; Choi et al., 2012). 즉, 다양한 항생물질을 생산하는 아메리카동애등에는 의약용 물질 생산 측면에서도 산업적 이용 가능성을 보이고 있다 (Natori, 1995; Sherman et al., 2000). 다 자란 유충은 약 2 cm 크기가 되

Effect of Stress Sound on the Development of the Black Soldier Fly, Hermetia illucens

The black soldier fly (BSF), Hermetia illucens, is a generalist detritivore that is usually exploited for recycling environmental wastes, including food remnants. These insects can also be used to produce antibiotics for medical applications. For this purpose, flies need to be mass-reared using an automatic rearing system that generates various sounds. Insects use sound as a means of communication for mating or warning against predators. Thus, extrinsic sounds may interrupt the normal physiological processes of insects. In this study, we analyzed the physiological changes due to various sound treatments at and 8:16 h (L:D) photoperiod. At 95 dB, larvae were exposed to different sound frequencies (0-5,000 Hz). The treated larvae were adversely affected by the high-frequency sounds and showed significant reduction in pupation and adult emergence. Different sound intensities (0-105 dB) at 5,000 Hz caused significant changes in the pupation and survival of the adults at 85 dB or above. Under a stress sound condition (5,000 Hz, 95 dB), the BSF larvae exhibited aberrations in the expression of genes related to the immune system and digestion. These results indicate that high-frequency sound treatments at or above 85 dB act as a stress sound to BSF development.

Effect of Cellular Phospholipase A 2 Inhibition on Enhancement of Bt Insecticidal Activity

Some bacterial metabolites of Xenorhabdus nematophila (Xn) inhibit phospholipase A2 (PLA2) activity to shutdown eicosanoid biosynthesis in target insects. However, little has been known about the target insect PLA2 of these bacterial metabolites. Eight bacterial metabolites identified in Xn culture broth exhibited significant insecticidal activities against larvae of both lepidopteran species of Plutella xylostella and Spodoptera exigua. Moreover, these bacterial metabolites significantly enhanced insecticidal activities of Bacillus thuringiensis (Bt). To determine target PLA2, we cloned and over-expressed cellular PLA2 (SecPLA2) of S. exigua. Purified SecPLA2 catalyzed phospholipids derived from the fat body and released several polyunsaturated fatty acids. Most Xn metabolites significantly inhibited SecPLA2 activity, but were different in their inhibitory activities. There was a positive correlation between the inhibition of SecPLA2 and the enhancement of Bt insecticidal activity. These results indicate that SecPLA2 is a molecular target inhibited by Xn metabolite.