Virus-vectoring thrips regulate the excessive multiplication of tomato spotted wilt virus using their antiviral immune responses.

The tomato spotted wilt virus (TSWV) is a member of the Tospoviridae family and has an negative/ambisense single-stranded RNA genome. Frankliniella occidentalis and F. intonsa are known to be dominant pests in Capsicum annuum (hot pepper) and can cause damage to the plant either directly by feeding, or indirectly by transmitting TSWV in a persistent and propagative manner, resulting in serious economic damage. This study compared the immune responses of two different thrips species against TSWV infection by transcriptome analysis, which then allowed the assessment of antiviral responses using RNA interference (RNAi). Both adult thrips shared about 90 % of the transcripts in non-viruliferous conditions. Most signal components of the immune pathways were shared by these two thrips species, and their expression levels fluctuated differentially in response to TSWV infection at early immature stages. The functional assays using RNAi treatments indicated that the Toll and JAK/STAT pathways were associated with the antiviral responses, but the IMD pathway was not. The upregulation of dorsal switch protein one supported its physiological role in recognizing TSWV infection and triggering the eicosanoid biosynthetic pathway, which mediates melanization and apoptosis in thrips. In addition, the signal components of the RNAi pathways fluctuated highly after TSWV infection. Individual RNAi treatments specific to the antiviral signalling and response components led to significant increases in the TSWV amount in the thrips, causing virus-induced mortality. These findings suggest that immune signalling pathways leading to antiviral responses are operating in the thrips to regulate TSWV litres to prevent a fatal viral overload. This study also indicates the differential antiviral responses between the TSWV-transmitting F. occidentalis and F. intonsa.

Screening of insect immune suppressors using a recombinant phospholipase A2 of a lepidopteran insect.

Phospholipase A2 (PLA2 ) catalyzes phospholipids at the sn-2 position to release free fatty acids, including arachidonic acid (AA) or its precursor. The free AA is then oxygenated into different eicosanoids, which mediate the diverse physiological processes in insects. Any inhibition of the PLA2 catalysis would give rise to serious malfunctioning in insect growth and development. An onion moth, Acrolepiopsis sapporensis, encodes four different PLA2 genes (As-PLA2 A-As-PLA2 D), in which As-PLA2 A is dominantly expressed at all developmental stages and in different larval tissues. RNA interference of the As-PLA2 A expression significantly reduced the PLA2 activity of A. sapporensis, which suffered from immunosuppression. A recombinant As-PLA2 A protein was purified from a bacterial expression system, which exhibited a typical Michaelis-Menten kinetics and hence susceptible to a specific inhibitor to sPLA2 and dithiothreitol. A total of 19 bacterial metabolites derived from Xenorhabdus and Photorhabdus were screened against the recombinant As-PLA2 A. Five potent metabolites were highly inhibitory and followed a competitive enzyme inhibition. These five inhibitors suppressed the immune responses of A. sapporensis by inhibiting hemocyte-spreading behavior and phenoloxidase activity. However, an addition of AA could significantly rescue the immunosuppression induced by the selected inhibitors. These studies suggest that the recombinant As-PLA2 A protein can be applied for high-throughput screening of insect immunosuppressive compounds.

Enhancement of an entomopathogenic fungal virulence against the seedcorn maggot, Delia platura, by suppressing immune responses with a bacterial culture broth of Photorhabdus temperata subsp. temperata.

In Korea, there are two maggot species in the Delia genus that commonly infest the roots and stems of the Welsh onion, thus causing serious economic damage on the crop at the seedling stage. In this study, the seedcorn maggot (Delia platura) was detected in onion fields in two different localities in Korea. After overwintering, maggot infestations occurred throughout the entire growing seasons from transplantation to harvest, but their specific patterns of occurrence varied in the two localities examined. Entomopathogenic fungi induced significant virulence against the maggot larvae, in which a strain of Beauveria bassiana was effective, though it exhibited limited mortality in its insecticidal activity. To enhance this insecticidal activity, a culture broth from an entomopathogenic bacterium, Photorhabdus temperata temperata (Ptt), was added to B. bassiana treatment. The addition of Ptt broth significantly increased the insecticidal activity of B. bassiana in a dose-dependent manner. To elucidate this enhancement in insecticidal activity, the immunosuppressive activity of Ptt broth was assessed by identifying the immune responses of the seedcorn maggots. The seedcorn maggots possessed at least three different hemocytes with plasmatocytes, crystal cells, and lamellocytes. These hemocytes exhibited nodule formation in response to the fungal infection. In addition to the cellular immunity, the maggots exhibited inducible expressions of antimicrobial peptide (AMP) genes such as cecropin and defensin. The addition of Ptt broth suppressed the nodule formation and the AMP expressions in response to the fungal infection. Altogether, this study demonstrated the innate immune responses in a non-model insect, D. platura, along with the application of immunosuppression to develop a highly efficient biological control by enhancing the virulence of B. bassiana.

Enhanced baculoviral virulence by suppressing the degradation of an insect immune resolvin, epoxyoctadecamonoenoic acid, in three lepidopteran insects.

Epoxyoctadecamonoenoic acids (EpOMEs) are produced from linoleic acid by a cytochrome P450 monooxygenase (CYP) and play a crucial role in terminating excessive and unnecessary immune responses during the late infection stage in insects. This suggests that an increase in the EpOME level may enhance the virulence of insect pathogens against pests. This study tested this hypothesis using a specific inhibitor against soluble epoxide hydrolase (sEH) to degrade EpOMEs, which leads to elevated endogenous EpOME levels. A baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was used to infect three different lepidopteran insects (Spodoptera exigua, Maruca vitrata, and Plutella xylostella) by oral feeding or hemocoelic injection treatments. Within one hour, the viral infection induced the expression of three different phospholipase A2 (PLA2) genes and, after 12 h, up-regulated the expressions of CYP and sEH genes in Spodopera exigua. As expected, AcMNPV virulence was suppressed by the addition of arachidonic acid (a catalytic product of PLA2) but was enhanced by the addition of either of the EpOME regioisomers. In addition, treatment with a specific sEH inhibitor (AUDA) increased AcMNPV virulence against three different lepidopteran insects, presumably by increasing endogenous EpOME levels. This enhanced effect of EpOMEs on virulence was further supported by specific RNA interference (RNAi), in which RNAi specific to CYP expression decreased AcMNPV virulence while a specific RNAi against sEH expression significantly enhanced virulence. In response to AcMNPV infection, TUNEL assay results showed that S. exigua larvae exhibited apoptosis in the midgut, fat body, and epidermis. Inhibition of apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, significantly increased virulence. Similarly, the addition of AUDA to the viral treatment suppressed the gene expression of five inducible caspases and cytochrome C to suppress apoptosis, which led to a significant increase in the tissue viral titers. These results indicate that EpOMEs play a role in terminating excessive and unnecessary immune responses against viral infection during the late stage by down-regulating antiviral apoptosis in lepidopteran insects.

Greenhouse test of spraying dsRNA to control the western flower thrips, Frankliniella occidentalis, infesting hot peppers.

BACKGROUND: The western flower thrips Frankliniella occidentalis is an insect pest that damages various crops, including hot peppers. It is a vector of a plant pathogen, tomato spotted wilt virus. To control this pest, chemical insecticides have been used in the past, but the control efficacy is unsatisfactory owing to rapid resistance development by F. occidentalis. METHODOLOGY: This study reports a novel control technology against this insect pest using RNA interference (RNAi) of the vacuolar-type ATPase (vATPase) expression. Eight subunit genes (vATPase-A ∼ vATPase-H) of vATPase were obtained from the F. occidentalis genome and confirmed for their expressions at all developmental stages. RESULTS: Double-stranded RNAs (dsRNAs) specific to the eight subunit genes were fed to larvae and adults, which significantly suppressed the corresponding gene expressions after 24-h feeding treatment. These RNAi treatments resulted in significant mortalities, in which the dsRNA treatments at ∼2,000 ppm specific to vATPase-A or vATPase-B allowed complete control efficacy near 100% mortality in 7 days after treatment. To prevent dsRNA degradation by the digestive proteases during oral feeding, dsRNAs were formulated in a liposome and led to an enhanced mortality of the larvae and adults of F. occidentalis. The dsRNAs were then sprayed at 2,000 ppm on F. occidentalis infesting hot peppers in a greenhouse, which resulted in 53.5-55.9% control efficacy in 7 days after treatment. Even though the vATPases are conserved in different organisms, the dsRNA treatment was relatively safe for non-target insects owing to the presence of mismatch sequences compared to the dsRNA region of F. occidentalis. CONCLUSION: These results demonstrate the practical feasibility of spraying dsRNA to control F. occidentalis infesting crops.

Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs.

Xenorhabdus hominickii, an entomopathogenic bacterium, inhibits eicosanoid biosynthesis of target insects to suppress their immune responses by inhibiting phospholipase A2 (PLA2) through binding to a damage-associated molecular pattern (DAMP) molecule called dorsal switch protein 1 (DSP1) from Spodoptera exigua, a lepidopteran insect. However, the signalling pathway between DSP1 and PLA2 remains unknown. The objective of this study was to determine whether DSP1 could activate Toll immune signalling pathway to activate PLA2 activation and whether X. hominickii metabolites could inhibit DSP1 to shutdown eicosanoid biosynthesis. Toll-Spätzle (Spz) signalling pathway includes two Spz (SeSpz1 and SeSpz2) and 10 Toll receptors (SeToll1-10) in S. exigua. Loss-of-function approach using RNA interference showed that SeSpz1 and SeToll9 played crucial roles in connecting DSP1 mediation to activate PLA2. Furthermore, a deletion mutant against SeToll9 using CRISPR/Cas9 abolished DSP1 mediation and induced significant immunosuppression. Organic extracts of X. hominickii culture broth could bind to DSP1 at a low micromolar range. Subsequent sequential fractionations along with binding assays led to the identification of seven potent compounds including 3-ethoxy-4-methoxyphenol (EMP). EMP could bind to DSP1 and prevent its translocation to plasma in response to bacterial challenge and suppress the up-regulation of PLA2 activity. These results suggest that X. hominickii inhibits DSP1 and prevents its DAMP role in activating Toll immune signalling pathway including PLA2 activation, leading to significant immunosuppression of target insects.

Prostaglandin E2 mediates chorion formation of the Asian tiger mosquito, Aedes albopictus, at late oogenesis.

Chorion-i.e., the eggshell-is formed during the late stage of oogenesis by follicular epithelium in the ovary. Although the endocrine signal(s) driving choriogenesis remain unclear in mosquitoes, this process in other insects has been suspected to involve the mediation of prostaglandins (PGs). This study tested the role of PG in the choriogenesis of the Asian tiger mosquito, Aedes albopictus, and its influence on controlling the expressions of genes associated with chorion formation by a transcriptome analysis. An immunofluorescence assay showed that PGE2 is localised in follicular epithelium. With the treatment of aspirin, an inhibitor of PG biosynthesis, at mid oogenesis, the PGE2 signal disappeared in the follicular epithelium led to significantly inhibited chorion formation along with a malformed eggshell. Ovary transcriptomes were assessed by RNASeq at the mid and late ovarian developmental stages. Differentially expressed genes (DEGs) exhibiting more than twofold changes in expression levels included 297 genes at mid stage and 500 genes at late stage. These DEGs at these two developmental stages commonly included genes associated with egg and chorion proteins of Ae. albopictus. Most chorion-associated genes were clustered in the 168 Mb region on a chromosome and exhibited significantly induced expressions at both ovarian developmental stages. The inhibition of PG biosynthesis significantly suppressed the expression of the chorion-associated genes while the addition of PGE2 rescued the gene expressions and led to recovery of choriogenesis. These results suggest that PGE2 mediates the choriogenesis of Ae. albopictus.

In vivo transient expression of a viral silencing suppressor, NSs, derived from tomato spotted wilt virus decreases insect RNAi efficiencies.

Tomato spotted wilt virus is a single-stranded RNA virus and causes a serious plant disease. Its horizontal transmission depends on some thrips species including Frankliniella occidentalis. Its genome encodes a nonstructural protein, nonstructural (NSs), which acts as a silencing suppressor and plays a crucial role in the pathogenicity by defending antiviral immunity using RNA interference (RNAi) in plant hosts. However, its physiological function as a silencing suppressor was not well clarified in insect vectors. This study assessed any change of RNAi efficiencies in two other insect systems by NSs expression. To this end, the gene was cloned into a eukaryotic expression vector and transiently expressed in two different insect species via in vivo transient expression (IVTE). After feeding the recombinant construct to non-viruliferous F. occidentalis, NSs expression was observed for over 2 days in the thrips. Under this expression of NSs, thrips were rescued from a treatment of a toxic double stranded RNA specific to v-ATPase. Interestingly, the thrips treated with IVTE significantly suppressed the expression of RNAi machinery genes such as SID and Dicer-2. The recombinant vector expressing NSs was injected to a non-vector insect, Spodoptera exigua, larvae. The larvae expressing NSs by the IVTE were highly susceptible to an infection of a RNA virus called iflavirus. These suggest that NSs acts as a silencing suppressor in insects and would be used for a synergist for RNA pathogens to control insect pests.

PGE2 mediation of egg development in Western flower thrip, Frankliniella occidentalis.

Eicosanoids mediate various insect physiological processes, including reproduction. Especially, the eicosanoid prostaglandin E2  (PGE2 ) is known to mediate oocyte development in some insects. The explosive reproductive potential of the Western flower thrips, Frankliniella occidentalis, damages various agricultural crops. However, little is known about the underlying physiological processes of egg development in this pest. This study found that treatment with aspirin (ASP) (a specific cyclooxygenase (COX) inhibitor) used to inhibit PGE2 biosynthesis during ovarian development significantly suppressed the reproduction of female F. occidentalis. However, the addition of PGE2 to ASP-treated females significantly rescued the suppressed reproduction. PGE2 was detected in growing ovarian follicles in an immunofluorescence assay. The hypothetical biosynthetic machinery of PGE2 was predicted from the F. occidentalis genome and included phospholipase A2  (PLA2 ), COX-like peroxidase (POX), and PGE2 synthase (PGES). Three specific PLA2 s were highly expressed in female adults during active oogenesis. Specific POX and PGES genes also showed high expression during active oogenesis. The adverse effect of ASP treatment on oogenesis was observed in follicle formation in the germarium where the follicle numbers in an ovariole were decreased, which resulted in hypotrophied ovaries. This impairment was rescued by the addition of PGE2 . ASP treatment also significantly inhibited chorion formation and suppressed gene expression associated with choriogenesis, which included chorion protein, mucin, and yellow while it did not inhibit vitellogenin gene expression. However, the addition of PGE2 induced the expression of the target genes suppressed by ASP treatment and rescued chorion formation. These results suggest that PGE2 mediated ovarian development by affecting follicle formation and choriogenesis in F. occidentalis.

Immune responses of the Asian onion moth, Acrolepiopsis sapporensis, and their genetic factors from RNA-Seq analysis.

A nonmodel insect, Acrolepiopsis sapporensis, has been analyzed in immune responses. The total hemocytes in the fifth instar larvae were 2.33 × 106 cells/mL. These hemocytes comprised at least five different types and different relative ratios: 47% granulocytes, 26% plasmatocytes, 11% oenocytoid, 8% prohemocytes, and 5% spherulocytes. Upon bacterial challenge, some of the hemocytes exhibited typical hemocyte-spreading behaviors, such as focal adhesion, and filopodial and lamellipodial cytoplasmic extensions. The hemocyte behaviors induced cellular immune responses demonstrated by nodule formation. In addition, the plasma collected from the immune-challenged larvae exhibited humoral immune responses by bacterial growth inhibition along with enhanced phenoloxidase enzyme activity. These cellular and humoral immune responses were further analyzed by determining the immune-associated genes from a transcriptome generated by RNA-Seq. A total of about 12 Gb sequences led to about 218,116 contigs, which were predicted to encode about 46,808 genes. Comparative expression analysis showed 8392 uniquely expressed genes in the immune-challenged larvae. Differentially expressed gene (DEG) analysis among the commonly expressed genes indicated that 782 genes were upregulated and 548 genes were downregulated in the expressions after bacterial challenge. These immune-associated genes included pattern recognition receptors, immune mediation/signaling genes, and various immune effectors. Specifically, the genetic components of the Toll, IMD, and JAK/STAT immune signaling pathways were included in the DEG database. These results demonstrate the immune responses of A. sapporensis larvae and suggest the genes associated with the immune responses in this nonmodel insect.

Why can insects not biosynthesize cholesterol?

Two aspects of insect lipid biochemistry differ from the mammalian background. In one aspect, nearly a hundred years ago scientists demonstrated that the polyunsaturated fatty acid (PUFAs), linoleic acid (LA; 18:2n-6) is an essential nutrient in the diets of all mammals that have been studied in that regard. An unknown number of insect species are able to biosynthesize LA de novo. Some species take the biosynthesized LA into fatty acid elongation/desaturation pathways to produce other PUFAs, 18:3n-6, 20:3n-6 and 20:4n-6. A couple of species use the de novo produced LA to biosynthesize prostaglandins and other eicosanoids, short-lived signal moieties that mediate important physiological actions in immunity and reproduction. Insects differ from mammals, also, in their lack of genes that encode enzymes acting in biosynthesis of cholesterol. Insects require dietary cholesterol to meet their cellular, physiological, developmental, and reproductive needs. Looking at a broader view of invertebrate biochemistry, most protostomes lost all or most genes involved in cholesterol biosynthesis. The massive gene loss occurred during the Ediacaran Period, which lasted 96 million years, from the end of the Cryogenian Period (635 million years ago; MYA) to the beginning of the Cambrian Period (538.6 MYA). The key point here is that the inability to biosynthesize cholesterol is not limited to insects; it occured in most protostomes. We address the protostome need and benefits of acquiring exogenous sterols.

What is in a model?

After reading contradictory claims of model status for some insect species, we feel a brief discussion of the topic may be useful. Here, we document a few examples where clarity on model status seems to be lacking, briefly review work on widely recognized models, and offer criteria for including any given species as a model organism.

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.

Phurealipids, produced by the entomopathogenic bacteria, Photorhabdus, mimic juvenile hormone to suppress insect immunity and immature development.

Phurealipids (Photorhabdus urea lipids) are synthesized from Photorhabdus bacteria that are symbiotic to entomopathogenic nematodes. Their chemical structures are similar to that of juvenile hormone (JH) and have been suspected to mimic JH signaling in immunity and the development of insects. This study investigated the physiological roles of phurealipids with respect to their contribution to bacterial pathogenicity using four natural (HB13, HB69, HB416, and HB421) and one derivative (HB27) compound. First, phurealipids like JH suppressed insect immune responses. Overall, phurealipids showed JH like immunosuppressive behavior in a lepidopteran insect Spodoptera exigua larvae. More specifically, phurealipids significantly suppressed the hemocyte spreading behavior which is a key immune response upon immune challenge. Interestingly, the methyl urea derivatives (HB13, HB27, and HB69) were more potent than the unmethylated forms (HB416 and HB421). The inhibitory activity of phurealipids prevented the cellular immune response measured by hemocytic nodule formation in response to the bacterial challenge. Phurealipids also suppressed the expression of cecropin and gallerimycin, which are two highly inducible antimicrobial peptides, in S. exigua upon immune challenge. The immunosuppressive activity of the phurealipids significantly enhanced the bacterial pathogenicity of Bacillus thuringiensis against S. exigua. Second, phurealipids like JH prevented insect metamorphosis. Especially, the methylated urea derivatives of the phurealipids showed the JH-like function by inducing the expression of S. exigua Kr-h1, a transcriptional factor. At the pupal stage, exhibiting the lowest expression of Kr-h1, phurealipid treatments elevated the expression level of Kr-h1 and delayed the pupa-to-adult metamorphosis. These results suggest that phurealipids play crucial roles in Photorhabdus pathogenicity by suppressing host immune defenses and delaying host metamorphosis.

HMGB1-like dorsal switch protein 1 of the mealworm, Tenebrio molitor, acts as a damage-associated molecular pattern.

High-mobility group box 1 (HMGB1) is a nuclear protein highly conserved in eukaryotes and ubiquitously expressed to regulate transcription and chromatin remodeling. Dorsal switch protein 1 (DSP1) is its insect homolog. A lepidopteran DSP1 acts as a damage-associated molecular pattern (DAMP) in response to immune challenge. The objective of this study was to determine the role of DAMP in the mealworm beetle, Tenebrio molitor, a coleopteran insect. DSP1 of T. molitor (Tm-DSP1) encodes 536 amino acids and shares sequence similarities with Homo sapiens HMGB1 (56.3%) and Spodoptera exigua DSP1 (59.2%). An antisera raised against S. exigua DSP1 was cross-reactive to Tm-DSP1. Like other insect DSPs, Tm-DSP1 has a relatively long N-terminal extension in addition to two conserved HMG box domains. It was expressed in all developmental stages of T. molitor and different larval tissues. Upon immune challenge, its expression level was upregulated. Its RNA interference (RNAi) treatment resulted in a significant reduction in immune responses measured by hemocyte nodule formation against bacterial infection. In addition, the induction of some antimicrobial peptide genes to the immune challenge was suppressed by its RNAi treatment. Interestingly, phospholipase A2 associated with eicosanoid biosynthesis was significantly suppressed in its catalytic activity by the RNAi treatment specific to Tm-DSP1 expression. Without any pathogen infection, injection of a lepidopteran DSP1 induced both cellular and humoral immune responses. These results suggest that Tm-DSP1 in T. molitor can act as a DAMP molecule and mediate immune responses upon immune challenge.

An ovary-specific mucin is associated with choriogenesis mediated by prostaglandin signaling in Spodoptera exigua.

Polytrophic ovarioles of Spodoptera exigua, a lepidopteran insect, begins with the development of oocytes and differentiation of nurse cells followed by vitellogenesis and choriogenesis. Compared with previtellogenic and vitellogenic developments, choriogenesis has not been clearly understood yet in endocrine control. This study investigated the expression and function of a mucin-like structural protein of S. exigua called Se-Mucin1 in choriogenesis. It was highly expressed in ovarioles containing chorionated oocytes. The expression level of Se-Mucin1 was increased during adult stage as early as 18 h after adult emergence, reaching the maximal level at 24 h and later. Interestingly, DNA amount of Se-Mucin1 was increased by almost four folds during early adult stage while other genes (hexokinase and glyceraldehyde-3-phosphate dehydrogenase) not directly associated with chorion formation did not show genomic DNA increase, suggesting specific gene amplification of Se-Mucin1. RNA interference (RNAi) suppressed Se-Mucin1 expression by injecting 1 µg of double-strand RNA to teneral females (<5 h after emergence), which exhibited significantly impaired fecundity and egg hatching rate. Eggs laid by RNAi-treated females were malformed in eggshell structures with loss of mesh-like fibers. Treatment with aspirin, a prostaglandin (PG) biosynthesis inhibitor, suppressed the induction of Se-Mucin1 expression during early adult stage and impaired egg development. An addition of PGE2 significantly rescued such impairment in Se-Mucin1 expression and subsequent egg development. These results suggest that PGs mediate choriogenesis of S. exigua by activating the expression of chorion-associated genes including Se-Mucin1.

The first report of prostacyclin and its physiological roles in insects.

Prostaglandins (PGs) mediate physiological processes of insects as well as mammals. Prostaglandin I2 (PGI2) is a relatively well-known eicosanoid with potent hormone-like actions on various tissues of vertebrates, however, its presence and biosynthetic pathway have not been described in insects. This study demonstrated that fat bodies of the lepidopteran species, Spodoptera exigua, contained ~ 3.6 pg/g PGI2. To identify its biosynthetic pathway, a PGI2 synthase gene of S. exigua (Se-PGIS) was predicted from a transcriptome of S. exigua; 25.6% homology with human PGIS was demonstrated. Furthermore, a predicted three-dimensional structure of Se-PGIS was demonstrated to be 38.3% similar to the human PGIS ortholog, including catalytic residues. Se-PGIS was expressed in all developmental stages of S. exigua and most abundant larval and adult stages; immune challenging of larvae significantly up-regulated these expression levels. The inducible expression of Se-PGIS expression was followed by a greater than four-fold increase in the concentration of PGI2 in fat bodies 10 h after immune challenge. RNA interference (RNAi) against Se-PGIS was performed by injecting double-stranded RNA (dsRNA). Under these RNAi conditions, cellular immune responses (e.g., hemocyte-spreading behavior, nodulation, phenoloxidase activity) were not affected by bacterial challenge. The addition of PGI2 to larvae treated with an eicosanoid biosynthesis inhibitor did not rescue the immunosuppression. Interestingly, PGI2 injection significantly suppressed nodule formation in response to bacterial challenge. In addition to the negative effect of PGI2 against immunity, the Se-PGIS-RNAi treatment significantly interfered with immature development and severely impaired oocyte development in female adults; the addition of PGI2 to RNAi-treated females significantly recovered oocyte development. Se-PGIS RNAi treatment also impaired male fertility by reducing fecundity after mating with untreated females. These results suggest that PGI2 acts as a negative regulator of immune responses initiated by other factors and mediates S. exigua development and reproduction.

Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 to suppress host insect immunity.

BACKGROUND: Xenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates (species or strains) of Xenorhabdus and Photorhabdus. RESULTS: All bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities. CONCLUSION: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

Prostaglandin catabolism in Spodoptera exigua, a lepidopteran insect.

Several prostaglandins (PGs) and PG-synthesizing enzymes have been identified from insects. PGs mediate cellular and humoral immune responses. However, uncontrolled and prolonged immune responses might have adverse effects on survival. PG catabolism in insects has not been reported. Here, using a transcriptomic analysis, we predicted the presence of two PG-degrading enzymes, PG dehydrogenase (SePGDH) and PG reductase (SePGR), in Spodoptera exigua, a lepidopteran insect. SePGDH and SePGR expression levels were upregulated after immune challenge. However, their expression peaks occurred after those of PG biosynthesis genes, such as those encoding PGE2 synthase or PGD2 synthase. SePGDH and SePGR expression levels were upregulated after injection with PGE2 or PGD2 In contrast, such upregulated expression was not detected after injection with leukotriene B4, an eicosanoid inflammatory mediator. RNA interference (RNAi) using double-stranded RNAs specific to SePGDH or SePGR suppressed their expression levels. The RNAi treatment resulted in an excessive and fatal melanization of larvae even after a non-pathogenic bacterial infection. Phenoloxidase (PO) activity mediating the melanization in larval plasma was induced by bacterial challenge or PGE2 injection. Although the induced PO activity decreased after 8 h in control larvae, those treated with dsRNAs specific to PG-degrading enzyme genes kept a high PO activity for a longer period. These results suggest that SePGDH and SePGR are responsible for PG degradation at a late phase of the immune response.

Why most insects have very low proportions of C20 polyunsaturated fatty acids: The oxidative stress hypothesis.

Eicosanoids, a group of C20 oxygenated polyunsaturated fatty acids (PUFAs), mediate various physiological processes, such as immunity, reproduction, excretion, and metabolism in insects. Arachidonic acid (AA) is used for the main precursor for the production of various eicosanoids. However, most terrestrial insects possess relatively low AA levels. Insects are presumed to be evolved since the Paleozoic era, at which oxygen levels might be much higher than current conditions. Compared with other animals, they exhibit relatively high metabolic rates with the well-developed tracheal system, which directly supply enough oxygen to active tissues like flight muscles. This might allow insects to be susceptible to reactive oxygen species (ROS) generated from high oxidative catabolism. Long-chain PUFAs including AA is usually reacted with ROS and become peroxidized. Peroxidized PUFAs cause various cellular damage. Thus, we propose a hypothesis that terrestrial insects minimize AA levels to minimize oxidative stress.