Dorsal switch protein 1 as a damage signal in insect gut immunity to activate dual oxidase via an eicosanoid, PGE2.

Various microbiota including beneficial symbionts reside in the insect gut. Infections of pathogens cause dysregulation of the microflora and threaten insect survival. Reactive oxygen species (ROS) have been used in the gut immune responses, in which its production is tightly regulated by controlling dual oxidase (Duox) activity via Ca2+ signal to protect beneficial microflora and gut epithelium due to its high cytotoxicity. However, it was not clear how the insects discriminate the pathogens from the various microbes in the gut lumen to trigger ROS production. An entomopathogenic nematode (Steinernema feltiae) infection elevated ROS level in the gut lumen of a lepidopteran insect, Spodoptera exigua. Dorsal switch protein 1 (DSP1) localized in the nucleus in the midgut epithelium was released into plasma upon the nematode infection and activated phospholipase A2 (PLA2). The activated PLA2 led to an increase of PGE2 level in the midgut epithelium, in which rising Ca2+ signal up-regulated ROS production. Inhibiting DSP1 release by its specific RNA interference (RNAi) or specific inhibitor, 3-ethoxy-4-methoxyphenol, treatment failed to increase the intracellular Ca2+ level and subsequently prevented ROS production upon the nematode infection. A specific PLA2 inhibitor treatment also prevented the up-regulation of Ca2+ and subsequent ROS production upon the nematode infection. However, the addition of PGE2 to the inhibitor treatment rescued the gut immunity. DSP1 release was not observed at infection with non-pathogenic pathogens but detected in plasma with pathogenic infections that would lead to damage to the gut epithelium. These results indicate that DSP1 acts as a damage-associated molecular pattern in gut immunity through DSP1/PLA2/Ca2+/Duox.

HMG-Like DSP1 Mediates Immune Responses of the Western Flower Thrips (Frankliniella occidentalis) Against Beauveria bassiana, a Fungal Pathogen.

Western flower thrips, Frankliella occidentalis, is a serious pest by directly infesting host crops. It can also give indirect damage to host crops by transmitting a plant virus called tomato spotted wilt virus. A fungal pathogen, Beauveria bassiana, can infect thrips. It has been used as a biopesticide. However, little is known on the defense of thrips against this fungal pathogen. This study assessed the defense of thrips against the fungal infection with respect to immunity by analyzing immune-associated genes of F. occidentalis in both larvae and adults. Immunity-associated genes of western flower thrips were selected from three immunity steps: nonself recognition, mediation, and immune responses. For the pathogen recognition step, dorsal switch protein 1 (DSP1) was chosen. For the immune mediation step, phospholipase A2 (PLA2) and prostaglandin E2 synthase were also selected. For the step of immune responses, two phenoloxidases (PO) genes and four proPO-activating peptidase genes involved in melanization against pathogens were chosen. Dual oxidase gene involved in the production of reactive oxygen species and four antimicrobial peptide genes for executing humoral immune responses were selected. All immunity-associated genes were inducible to the fungal infection. Their expression levels were induced higher in adults than in larvae by the fungal infections. However, inhibitor treatments specific to DSP1 or PLA2 significantly suppressed the inducible expression of these immune-associated genes, leading to significant enhancement of fungal pathogenicity. These results suggest that immunity is essential for thrips to defend against B. bassiana, in which DSP1 and eicosanoids play a crucial role in eliciting immune responses.

Thelytokous Reproduction of Onion Thrips, Thrips tabaci Lindeman 1889, Infesting Welsh Onion and Genetic Variation among Their Subpopulations.

Parthenogenesis is not uncommon in thrips. This asexual reproduction produces males (arrhenotokous) or female (thelytokous). Only females are found in the onion thrips (Thrips tabaci Lindeman 1889) infesting Welsh onion (Allium fistulosum) in several areas of Korea. To determine the reproduction mode of T. tabaci, thrips infesting Welsh onion were collected from different localities in Korea. Cytochrome oxidase I (COI) sequences were then assessed. Results showed that all test local populations had signature motif specific to a thelytokous type. These COI sequences were clustered with other thelytokous populations separated from arrhenotokous T. tabaci populations. In a laboratory test, individual rearing produced female progeny without any males. These results support that Korean onion thrips infesting Welsh onion have the thelytokous type of parthenogenesis. Local thrips populations exhibited significant variations in susceptibility to chemical and biological insecticides. Random amplified polymorphic DNA (RAPD) analysis indicated genetic variations of local populations. However, the genetic distance estimated from RAPD was independent of the actual distance among different local populations. These results suggest that genetic variations of T. tabaci are arisen from population subdivision due to asexual thelytokous reproductive mode.

Toll signal pathway activating eicosanoid biosynthesis shares its conserved upstream recognition components in a lepidopteran Spodoptera exigua upon infection by Metarhizium rileyi, an entomopathogenic fungus.

Eicosanoids play crucial roles in mediating immune responses in insects. Upon a fungal infection, Toll signal pathway can mediate immune responses of Spodoptera exigua, a lepidopteran insect, by activating eicosanoid biosynthesis. However, upstream signal components of the Toll signal pathway activating eicosanoid biosynthesis remain unclear. This study predicted pattern recognition receptors (PRRs) and serine proteases (SPs) as upstream components of the Toll pathway with reference to known signal components of Manduca sexta, another lepidopteran insect. S. exigua infected with Metarhizium rileyi, an entomopathogenic fungus, activated phospholipase A2 (PLA2) and phenoloxidase (PO) enzymes along with marked increases of expression levels of genes encoding three specific antimicrobial peptides, cecropin, gallerimycin, and hemolin. Among ten Toll receptors encoded in the genome of S. exigua, seven Toll genes were associated with immune responses against fungal infection by M. rileyi through individual RNA interference (RNAi) screening. In addition, two Spätzles (ligands of Toll receptor) were required for Toll signaling against the fungal infection. All predicted upstream components of the Toll pathway were inducible by the fungal infection. Individual RNAi screening showed that three PRRs (ßGRP-1, ßGRP-2, and GNBP3) and five SPs (ModSP, HP21, HP5, HP6, and HP8) were required for immune responses of S. exigua mediated by Toll signal pathway against the fungal infection. However, two PO-activating proteases (PAP1 and PAP3) were not required for PLA2 activation, although they were required for PO activation. These results suggest that PRRs and SPs conserved as upstream components in Toll signal pathway play crucial roles in triggering eicosanoid biosynthesis of S. exigua to mediate various immune responses against fungal infection.

CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt.

Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.

Antiviral Treatment Reveals a Cooperative Pathogenicity of Baculovirus and Iflavirus in Spodoptera exigua, a Lepidopteran Insect.

NPVThe beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Doublestranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.

Thromboxane Mobilizes Insect Blood Cells to Infection Foci.

Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2via a Ca2+ signal through a PG receptor.

Variations of Indole Metabolites and NRPS-PKS Loci in Two Different Virulent Strains of Xenorhabdus hominickii.

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Tolerance of the mealworm beetle, Tenebrio molitor, to an entomopathogenic nematode, Steinernema feltiae, at two infection foci, the intestine and the hemocoel.

An entomopathogenic nematode, Steinernema feltiae K1, exhibits pathogenicity in various insect hosts, however, its virulence among the target insect species varies. Specifically, a coleopteran insect, Tenebrio molitor, is less susceptible to S. feltiae than are lepidopteran insects. We analyzed the low virulence of S. feltiae against T. molitor sequentially, in entering the gut lumen and penetrating the hemocoel, and in hemocoelic immune defenses by comparing the responses to those of a lepidopteran insect, Spodoptera exigua. Infective juveniles (IJs) of S. feltiae exhibited higher virulence and produced more progeny IJs in S. exigua than in T. molitor. The difference in IJ behavior was observed in the IJ invasion rate (IJs in gut lumen/IJs treated) after treatment, in which a lower rate was observed in T. molitor (20.4%) than in S. exigua (55.5%). Also, a lower hemocoelic penetration rate of IJs (IJs in hemocoel/IJs in gut) was observed in T. molitor (54%) than in S. exigua (74%) 24 h after feeding treatment. To investigate the immune defense in the hemocoel, insect hemolymph samples were incubated with IJs. The encapsulation behavior and phenoloxidase activity was higher in T. molitor hemolymph than in S. exigua hemolymph, which resulted in a significantly higher nematicidal activity in S. exigua. The humoral immune responses against S. feltiae were also different between the two species. The expression of two antimicrobial peptides, cecropin and attacin 1, was much higher in T. molitor. Furthermore, eicosanoid biosynthetic activity against S. feltiae was different in the two host species; sPLA2 activity was highly inducible in T. molitor but not in S. exigua. These results suggest that variability of the immune defense in the target insects, as well as in the invasion and penetration rates of IJs to the hemocoel, plays a crucial role in determining the insecticidal virulence of S. feltiae.

sPLA2 behaves like a prophylactic agent and mediates cellular and humoral immune responses in Plutella xylostella.

Most immune effectors are inducible to microbial pathogen infection while some are already present to act as prophylactic immunity against as yet unseen infection. This study identified secretory phospholipase A2 (sPLA2 ) as a prophylactic factor in diamondback moth, Plutella xylostella. Western blotting using a polyclonal antibody raised against other lepidopteran sPLA2 reacted specifically with ∼25 kDa protein, which was present at approximately 0.4 mM in the plasma of naïve larvae. Interrogation of P. xylostella transcriptomes revealed an open-reading frame for sPLA2 (Px-sPLA2 ), exhibiting high homology with other Group III sPLA2 s. Px-sPLA2 was expressed in all developmental stages. In the larval stage, bacterial challenge induced its expression in hemocytes and fat body but not in gut or epidermis. RNA interference (RNAi) suppressed Px-sPLA2 messenger RNA level and sPLA2 activity in plasma. An inhibition zone assay showed that Px-sPLA2 exhibited antibacterial activities against different species, because specific RNAi knockdown impaired the activity. The RNAi treatment also suppressed the cellular immune response assessed by hemocyte nodule formation and humoral immune response assessed by antimicrobial peptide gene expression. Finally, benzylideneacetone (BZA, a specific sPLA2 inhibitor) treatment inhibited plasma sPLA2 activity of naive larvae in a dose-dependent manner. An addition of BZA significantly increased the bacterial virulence of an entomopathogen, Bacillus thuringiensis. These results suggest that Px-sPLA2 is an immune-associated factor of P. xylostella and its relatively high level of concentration in the plasma of naive larvae strongly suggests its role as a prophylactic factor in defending against pathogens at early infection stages.