HMGB1-Like Dorsal Switch Protein 1 Triggers a Damage Signal in Mosquito Gut to Activate Dual Oxidase via Eicosanoids.

Several mosquitoes transmit human pathogens by blood feeding, with the gut being the main entrance for the pathogens. Thus, the gut epithelium defends the pathogens by eliciting potent immune responses. However, it was unclear how the mosquito gut discriminates pathogens among various microflora in the lumen. This study proposed a hypothesis that a damage signal might be specifically induced by pathogens in the gut. The Asian tiger mosquito, Aedes albopictus, encodes dorsal switch protein 1 (Aa-DSP1) as a putative damage-associated molecular pattern (DAMP). Aa-DSP1 was localized in the nucleus of the midgut epithelium in naïve larvae. Upon infection by a pathogenic bacterium, Serratia marcescens, Aa-DSP1 was released to hemocoel and activated phospholipase A2 (PLA2). The activated PLA2 increased the level of prostaglandin E2 (PGE2) in the gut and subsequently increased Ca2+ signal to produce reactive oxygen species (ROS) via dual oxidase (Duox). Inhibition of Aa-DSP1 via RNA interference or specific inhibitor treatment failed to increase PGE2/Ca2+ signal upon the bacterial infection. Thus, the inhibitors specifically targeting eicosanoid biosynthesis significantly prevented the upregulation of ROS production in the gut and enhanced mosquito mortality after the bacterial infection. However, such inhibitory effects were rescued by adding PGE2. These suggest that Aa-DSP1 plays an important role in immune response of the mosquito gut as a DAMP during pathogen infection by triggering a signaling pathway, DSP1/PLA2/Ca2+/Duox.

Glycerol biosynthesis plays an essential role in mediating cold tolerance the red imported fire ant, Solenopsis invicta.

The red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and known as a global problematic invasive species. At low temperatures, several investigations have demonstrated an increase in glycerol as a primary rapid cold hardening (RCH) component and an increase in the supercooling point. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), have been identified as being involved in the glycerol production process. In this study, one GPDH and two GK sequences were extracted from RIFA transcriptome analysis (Si-GPDH, Si-GK1, and Si-GK2). All three genes were expressed in different body parts and different tissues of S. invicta that Si-GK2 showed a higher expression level than the others. According to gene expression levels by qRT-PCR analysis, the highest expression levels of three genes were observed in fat body tissues. After 1 h of exposure to low temperatures (5°C or lower), the mRNA levels of these genes significantly increased, according to expression analyses. RNA interference (RNAi) of Si-GPDH or Si-GK1 and Si-GK2 exhibited a significant downregulation at the mRNA level. The mortality rate of treated RIFA by double-stranded RNA (dsRNA) specific to GPDH and GK2 significantly increased at low temperatures. This study indicates that GPDH and GK2 as glycerol biosynthesis genes in RIFA have a high expression level to synthesize a high level of glycerol as an RCH factor and they play crucial roles in survival during the cold period.

Knockdown of Helicoverpa armigera protease genes affects its growth and mortality via RNA interference.

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a destructive pest which is famous for its resistance to a variety of insecticides. RNA interference is a posttranscriptional gene silencing mechanism that has become a popular tool to control insect pests, triggered by double-stranded RNAs (dsRNAs). The effect of ingestion and injection delivery methods of dsRNA related to some protease genes including Trypsin (Ha-TRY39 and Ha-TRY96), Chymotrypsin (Ha-CHY), and Cathepsin L (Ha-CAT) on growth and development of H. armigera was investigated in this study. All protease genes encoded full ORFs and were expressed in all H. armigera larvae stages and tissues. In both injection and feeding bioassays, Ha-RNAi CHY's performance outperformed that of other protease genes. CHY enzyme activity in the midgut of larvae was significantly reduced after treatment with ds-HaCHY. Oral administration of ds-CHY also resulted in significant mortality of H. armigera larvae. However, because of the high RNase activity in the midgut lumen of lepidoptera, a large amount of dsRNA was needed to effectively kill instars of H. armigera. To reduce dsRNA degradation, bacterial expression and dsRNA formulation were used. After oral administration, it was toxic to H. armigera larvae. Before oral administration, bacterial cells were sonicated to increase dsRNA release. The RNA interference efficiency of sonicated bacteria was significantly increased, resulting in higher larval mortality when administered orally. All of these findings point to Ha-CHY as a new candidate for developing an effective dsRNA-based pesticide for H. armigera control.

PGE2 upregulates gene expression of dual oxidase in a lepidopteran insect midgut via cAMP signalling pathway.

In insect midgut, prostaglandins (PGs) play a crucial role in defending bacterial and malarial pathogens. However, little is known about the PG signalling pathway in the midgut. A dual oxidase (Se-Duox) with presumed function of catalysing reactive oxygen species (ROS) production in the midgut was identified in beet armyworm, Spodoptera exigua. Se-Duox was expressed in all developmental stages, exhibiting relatively high expression levels in the midgut of late larval instars. Se-Duox expression was upregulated upon bacterial challenge. RNA interference (RNAi) of Se-Duox expression significantly suppressed ROS levels in the midgut lumen. The suppression of ROS levels increased insecticidal activity of Serratia marcescens after oral infection. Interestingly, treatment with a PLA2 inhibitor prevented the induction of Se-Duox expression in response to bacterial challenge. On the other hand, addition of its catalytic product rescued the induction of Se-Duox expression. Especially, PG synthesis inhibitor significantly suppressed Se-Duox expression, while the addition of PGE2 or PGD2 rescued the inhibition. Subsequent PG signals involved cAMP and downstream components because specific inhibitors of cAMP signal components such as adenylate cyclase (AC) and protein kinase A (PKA) significantly inhibited Se-Duox expression. Indeed, addition of a cAMP analogue stimulated Se-Duox expression in the midgut. Furthermore, individual RNAi specific to PGE2 receptor (a trimeric G-protein subunit), AC, PKA or cAMP-responsive element-binding protein resulted in suppression of Se-Duox expression. These results suggest that PGs can activate midgut immunity via cAMP signalling pathway by inducing Se-Duox expression along with increased ROS levels.

A prophylactic role of a secretory PLA2 of Spodoptera exigua against entomopathogens.

Phospholipase A2 (PLA2) hydrolyses phospholipids at sn-2 position to release free fatty acids and lysophospholipids. Secretory type of PLA2 (sPLA2) has been found in many different animals including insects. Insect sPLA2s have been divided into venomous and nonvenomous PLA2s. A non-venomous sPLA2 (Se-sPLA2) has been identified in beet armyworm, Spodoptera exigua. Its high enzyme activity is detected in hemolymph of naïve larvae. However, the physiological role of high sPLA2 activity in hemolymph remains unclear. To determine the physiological role of sPLA2 in hemolymph, a recombinant Se-sPLA2 (rSe-sPLA2) was expressed in a bacterial expression system and purified to test antimicrobial activity against various microbes. Purified rSe-sPLA2 exhibited typical enzyme kinetic properties, including becoming saturated at high substrate concentrations, exhibiting optimal activity at pH 7-9, and being inactivated at high temperatures. However, a reducing agent (dithiothreitol) or calcium chelator treatment inhibited the catalytic activity. A specific inhibitor to sPLA2 also inhibited the enzyme activity of rSe-sPLA2 while other type PLA2 inhibitors did not. Furthermore, eight bacterial metabolites of Xenorhabdus and Photorhabdus known to be inhibitory against insect PLA2 significantly inhibited the enzyme activity of rSe-sPLA2. High concentrations of rSe-sPLA2 (above 0.5 mM) showed significant cytotoxicity to hemocytes of S. exigua. At concentrations without showing cytotoxicity, rSe-sPLA2 possessed significant antimicrobial activities against entomopathogenic bacteria (Serratia marscens and Entercoccus mondtii) and fungi (Beauveria bassiana and Metarhyzium rileyi). Hemolymph obtained from larvae treated with RNA interference specific to Se-sPLA2 significantly lost such antimicrobial activities. However, the addition of rSe-sPLA2 to the hemolymph significantly rescued such antimicrobial activities. These results indicate that Se-sPLA2 possesses antimicrobial activity, suggesting that it might act as a prophylactic agent against microbial pathogens in the hemolymph of S. exigua.