EpOME mediates the immune resolution and its alkoxide analog enhances the virulence of microbial insecticides against the legume pod borer, Maruca vitrata.

Excessive and unnecessary immune responses cause serious adverse effects due to self-tissue damage and energy consumption, particularly at the late stage of infection to terminate the induced immunity. Unlike mammals, which use long-chain fatty acid oxylipins, C18 oxygenated polyunsaturated fatty acids are suggested to act as immune resolvins in insects, including two epoxyoctadecamonoenoic acids (9,10-EpOME and 12,13-EpOME). This study investigated the physiological roles of EpOMEs in immune resolution in the lepidopteran insect, Maruca vitrata. The levels of two EpOMEs in the larvae increased during the late infection stage upon immune challenge. At their peak concentrations at 96 h post-infection (pi), both EpOMEs were found at similar levels: 323.18 pg/mg body weight for 9,10-EpOME and 322.07 pg/mg body weight for 12,13-EpOME. Both EpOMEs inhibited cellular and humoral immune responses, with 12,13-EpOME being more potent than 9,10-EpOME. Genes associated with EpOME synthase and degradation, identified as Mv-CYP1 and Mv-sEH, were detected in various developmental stages and tissues of M. vitrata. RNA interference (RNAi) targeting Mv-CYP1 failed to inhibit the immune response, whereas RNAi targeting Mv-sEH enhanced the immunosuppression. In contrast to the acute (< 12 h pi) immune response involving eicosanoid biosynthesis, the expression of these two genes linked to EpOME metabolism increased significantly at the late infection stage (> 12 h pi). Several alkoxide analogs of EpOME, with the epoxide group replaced by an alkoxide group, were synthesized; one such derivative demonstrated substantially greater efficacy than the natural EpOMEs in inhibiting the immune response. Additionally, using EpOME alkoxide significantly increased the effectiveness of microbial insecticides. Moreover, exposing young larvae to sublethal doses of EpOME alkoxide or sEH inhibitor induced severe developmental delays. These results suggest a novel strategy for insect pest control using insect immune resolvin analogs.

Burkholderia gut symbiont induces insect host fecundity by modulating Kr-h1 gene expression.

Full-length cDNAs of the Broad-Complex (BR-C) from Riptortus pedestris were cloned. Moreover, Kr-h1 and BR-C expression levels in apo-symbiotic and symbiotic host insects were compared to verify whether they are modulated by Burkholderia gut symbionts. Interestingly, Kr-h1 expression level was significantly increased in symbiotic females. To determine how Kr-h1 affects fecundity in insects, the biosynthesis of two reproduction-associated proteins, hexamerin-α and vitellogenin, was investigated in R. pedestris females. Hexamerin-α and vitellogenin expression at the transcriptional and translational levels decreased in Kr-h1-suppressed symbiotic females, subsequently reduced egg production. These results suggest that Burkholderia gut symbiont modulates Kr-h1 expression to enhance ovarian development and egg production of R. pedestris by increasing the biosynthesis of the two proteins.

Large-scale lipidomic profiling identifies novel potential biomarkers for prion diseases and highlights lipid raft-related pathways.

Prion diseases are transmissible spongiform encephalopathies induced by the abnormally-folded prion protein (PrPSc), which is derived from the normal prion protein (PrPC). Previous studies have reported that lipid rafts play a pivotal role in the conversion of PrPC into PrPSc, and several therapeutic strategies targeting lipids have led to prolonged survival times in prion diseases. In addition, phosphatidylethanolamine, a glycerophospholipid member, accelerated prion disease progression. Although several studies have shown that prion diseases are significantly associated with lipids, lipidomic analyses of prion diseases have not been reported thus far. We intraperitoneally injected phosphate-buffered saline (PBS) or ME7 mouse prions into mice and sacrificed them at different time points (3 and 7 months) post-injection. To detect PrPSc in the mouse brain, we carried out western blotting analysis of the left hemisphere of the brain. To identify potential novel lipid biomarkers, we performed lipid extraction on the right hemisphere of the brain and liquid chromatography mass spectrometry (LC/MS) to analyze the lipidomic profiling between non-infected mice and prion-infected mice. Finally, we analyzed the altered lipid-related pathways by a lipid pathway enrichment analysis (LIPEA). We identified a total of 43 and 75 novel potential biomarkers at 3 and 7 months in prion-infected mice compared to non-infected mice, respectively. Among these novel potential biomarkers, approximately 75% of total lipids are glycerophospholipids. In addition, altered lipids between the non-infected and prion-infected mice were related to sphingolipid, glycerophospholipid and glycosylphosphatidylinositol (GPI)-anchor-related pathways. In the present study, we found novel potential biomarkers and therapeutic targets of prion disease. To the best of our knowledge, this study reports the first large-scale lipidomic profiling in prion diseases.

Insulin signaling mediates previtellogenic development and enhances juvenile hormone-mediated vitellogenesis in a lepidopteran insect, Maruca vitrata.

BACKGROUND: Insulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females. RESULTS: A pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis. CONCLUSIONS: IIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.

Insulin-like peptides of the legume pod borer, Maruca vitrata, and their mediation effects on hemolymph trehalose level, larval development, and adult reproduction.

Insulin-like peptides (ILPs) of insects mediate various physiological processes including hemolymph sugar level, immature growth, female reproduction, and lifespan. In target cells of ILPs, insulin/insulin-like growth factor signaling (IIS) is highly conserved in animals. IIS in the legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is known to be involved in maintaining hemolymph trehalose levels and promoting larval growth. However, ILPs in M. vitrata have not been reported yet. This study predicted two ILP genes of Mv-ILP1 and Mv-ILP2 from transcriptome of M. vitrata. Mv-ILP1 and Mv-ILP2 shared high sequence homologies and domain architecture with Drosophila ILPs. Both ILPs exhibited similar expression patterns in most developmental stages, showing high expression levels in adult stage. In the larval stage, Mv-ILP1 and Mv-IlP2 were expressed mostly in the brain and fat body. However, in the adult stage, both ILP genes were expressed more in the abdomen than those in the head containing the brain. RNA interference (RNAi) of either Mv-ILP1 or Mv-ILP2 during larval stage resulted in significant malfunctioning in regulating hemolymph trehalose titers. RNAi-treated larvae also exhibited significant retardation of larval growth. RNAi treatment in adult stage interfered with the ovarian development of females. These results suggest that Mv-ILP1 and Mv-ILP2 play crucial roles in mediating larval growth and adult reproduction.

Comparative Metabolic Profiling in Drosophila suzukii by Combined Treatment of Fumigant Phosphine and Low Temperature.

Background/Objectives: The mechanisms of action of phosphine are diverse and include neurotoxicity, metabolic inhibition, and oxidative stress; however, its efficacy at low temperatures is unclear. Methods: Comparative metabolomics is suitable for investigating the response of the spotted-wing fly Drosophila suzukii to exposure toward a combination of cold stimuli and fumigant PH3. Results: Under this combined exposure, 52 metabolites exhibiting significant differences in stress were identified and their physiological roles were analyzed in the Drosophila metabolic pathway. Most metabolites were involved in amino acids, TCA cycle, and nucleic acids. In addition, the alteration levels of cell membrane lipids, such as glycerophospholipids, sphingolipids, and glycerolipids, clearly showed changes in the combined treatment compared to PH3 and low temperatures alone. Aconitic acid, a component of the TCA cycle, was completely inhibited by the combined treatment. Conclusions: These results suggest that treatment-specific indicators could be useful biomarkers to indicate the synergistic effects of PH3 and low temperature on energy metabolism.

Metabolite changes by combined treatment, ethyl formate and low temperature, in Drosophila suzukii.

Although ethyl formate (EF) fumigant and low temperature applications are widely used for pest management, studies related to their mechanisms of action and subsequent metabolic changes in Drosophila suzukii models are still unclear. In this study, a comparative metabolome analysis was performed to investigate the major metabolites modified by EF and low temperature and how they are related to and affect insect physiology. Most of the identified metabolites function in metabolic pathways related to the biosynthesis of amino acids, nucleotides and cofactors. In addition, a combined treatment with EF and low temperature significantly altered the tricarboxylic acid cycle (TCA) and the levels of the purine and pyrimidine classes of metabolites. Interestingly, the levels of cytochrome P450 and glutathione metabolites involved in detoxification dramatically changed under stress conditions compared to those in the control group.

A combination of biochemical and proteomic analyses reveals Bx-LEC-1 as an antigenic target for the monoclonal antibody 3-2A7-2H5-D9-F10 specific to the pine wood nematode.

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Functional Analysis of Pheromone Biosynthesis Activating Neuropeptide Receptor Isoforms in Maruca vitrata.

Insect sex pheromones are volatile chemicals that induce mating behavior between conspecific individuals. In moths, sex pheromone biosynthesis is initiated when pheromone biosynthesis-activating neuropeptide (PBAN) synthesized in the suboesophageal ganglion binds to its receptor on the epithelial cell membrane of the pheromone gland. To investigate the function of PBAN receptor (PBANR), we identified two PBANR isoforms, MviPBANR-B and MviPBANR-C, in the pheromone glands of Maruca vitrata. These two genes belong to G protein-coupled receptors (GPCRs) and have differences in the C-terminus but share a 7-transmembrane region and GPCR family 1 signature. These isoforms were expressed in all developmental stages and adult tissues. MviPBANR-C had the highest expression level in pheromone glands among the examined tissues. Through in vitro heterologous expression in HeLa cell lines, only MviPBANR-C-transfected cells responded to MviPBAN (≥5 µM MviPBAN), inducing Ca2+ influx. Sex pheromone production and mating behavior were investigated using gas chromatography and a bioassay after MviPBANR-C suppression by RNA interference, which resulted in the major sex pheromone component, E10E12-16:Ald, being quantitatively reduced compared to the control, thereby decreasing the mating rate. Our findings indicate that MviPBANR-C is involved in the signal transduction of sex pheromone biosynthesis in M. vitrata and that the C-terminal tail plays an important role in its function.

A Drosophila model of GSS syndrome suggests defects in active zones are responsible for pathogenesis of GSS syndrome.

We have established a Drosophila model of Gerstmann-Sträussler-Scheinker (GSS) syndrome by expressing mouse prion protein (PrP) having leucine substitution at residue 101 (MoPrP(P101L)). Flies expressing MoPrP(P101L), but not wild-type MoPrP (MoPrP(3F4)), showed severe defects in climbing ability and early death. Expressed MoPrP(P101L) in Drosophila was differentially glycosylated, localized at the synaptic terminals and mainly present as deposits in adult brains. We found that behavioral defects and early death of MoPrP(P101L) flies were not due to Caspase 3-dependent programmed cell death signaling. In addition, we found that Type 1 glutamatergic synaptic boutons in larval neuromuscular junctions of MoPrP(P101L) flies showed significantly increased numbers of satellite synaptic boutons. Furthermore, the amount of Bruchpilot and Discs large in MoPrP(P101L) flies was significantly reduced. Brains from scrapie-infected mice showed significantly decreased ELKS, an active zone matrix marker compared with those of age-matched control mice. Thus, altered active zone structures at the molecular level may be involved in the pathogenesis of GSS syndrome in Drosophila and scrapie-infected mice.

Insecticidal Serralysin of Serratia marcescens Is Detoxified in M3 Midgut Region of Riptortus pedestris.

Riptortus pedestris insect indiscriminately acquires not only the symbiotic bacterium Burkholderia insecticola, but also entomopathogens that are abundant in the soil via feeding. However, it is unclear how the host insect survives oral infections of entomopathogens. A previous study suggested that serralysin, a potent virulence factor produced by Serratia marcescens, suppresses cellular immunity by degrading adhesion molecules, thereby contributing to bacterial pathogenesis. Here, we observed that S. marcescens orally administered to R. pedestris stably colonized the insect midgut, while not exhibiting insecticidal activity. Additionally, oral infection with S. marcescens did not affect the host growth or fitness. When co-incubated with the midgut lysates of R. pedestris, serralysin was remarkably degraded. The detoxification activity against serralysin was enhanced in the midgut extract of gut symbiont-colonizing insects. The mRNA expression levels of serralysin genes were negligible in M3-colonizing S. marcescens. M3-colonizing S. marcescens did not produce serralysin toxin. Immunoblot analyses revealed that serralysin was not detected in the M3 midgut region. The findings of our study suggest that orally infected S. marcescens lose entomopathogenicity through host-derived degrading factors and suppression of serralysin.

Suppression of pheromone biosynthesis and mating behavior by RNA interference of pheromone gland-specific fatty acyl reductase in Maruca vitrata.

In moths, various enzymes, such as fatty acid synthases, fatty acyl desaturases, and fatty acyl reductases (FARs), are involved in pheromone biosynthesis. In particular, pheromone gland-specific FAR (pgFAR) plays an important role in converting the functional group from carboxylic to alcohol during pheromone biosynthesis. A novel pgFAR of Maruca vitrata, Mvi-pgFAR, was identified through transcriptome sequencing of its pheromone gland. To investigate the involvement of Mvi-pgFAR in pheromone biosynthesis, Mvi-pgFAR was cloned from the pheromone gland and suppressed by RNA interference (RNAi). Mvi-pgFAR harbored several conserved motifs related to NAD(P)H-binding, N-glycosylation, and adenosine / guanosine triphosphate binding. Phylogenetic analysis revealed that Mvi-pgFAR with other lepidopteran pgFARs formed an independent clade. Mvi-pgFAR was specifically expressed only in the pheromone gland. Quantitative real-time polymerase chain reaction showed that the diurnal expression levels of Mvi-pgFAR in the pheromone gland were the highest at 2 h before the scotophase. After primarily confirming Mvi-pgFAR suppression by RNAi, (E,E)-10,12-hexadecadienal (E10E12-16:Ald), a major sex pheromone component, was quantified by gas chromatography. When Mvi-pgFAR was successfully suppressed, E10E12-16:Ald production was reduced by up to half of that of the control, and the mating rate was subsequently decreased. Our results demonstrate that Mvi-pgFAR downregulation can suppress mating behavior by changing the relative sex pheromone component ratio, suggesting that Mvi-pgFAR can be used as a novel control target.

Multiple Precursor Proteins of Thanatin Isoforms, an Antimicrobial Peptide Associated With the Gut Symbiont of Riptortus pedestris.

Thanatin is an antimicrobial peptide (AMP) generated by insects for defense against bacterial infections. In the present study, we performed cDNA cloning of thanatin and found the presence of multiple precursor proteins from the bean bug, Riptortus pedestris. The cDNA sequences encoded 38 precursor proteins, generating 13 thanatin isoforms. In the phylogenetic analysis, thanatin isoforms were categorized into two groups based on the presence of the membrane attack complex/perforin (MACPF) domain. In insect-bacterial symbiosis, specific substances are produced by the immune system of the host insect and are known to modulate the symbiont's population. Therefore, to determine the biological function of thanatin isoforms in symbiosis, the expression levels of three AMP genes were compared between aposymbiotic insects and symbiotic R. pedestris. The expression levels of the thanatin genes were significantly increased in the M4 crypt, a symbiotic organ, of symbiotic insects upon systemic bacterial injection. Further, synthetic thanatin isoforms exhibited antibacterial activity against gut-colonized Burkholderia symbionts rather than in vitro-cultured Burkholderia cells. Interestingly, the suppression of thanatin genes significantly increased the population of Burkholderia gut symbionts in the M4 crypt under systemic Escherichia coli K12 injection. Overgrown Burkholderia gut symbionts were observed in the hemolymph of host insects and exhibited insecticidal activity. Taken together, these results suggest that thanatin of R. pedestris is a host-derived symbiotic factor and an AMP that controls the population of gut-colonized Burkholderia symbionts.

Deletion mutant of sPLA2 using CRISPR/Cas9 exhibits immunosuppression, developmental retardation, and failure of oocyte development in legume pod borer, Maruca vitrata.

Phospholipase A2 (PLA2) catalyzes release of free fatty acids linked to phospholipids at sn-2 position. Some of these released free fatty acids are used to synthesize eicosanoids that mediate various physiological processes in insects. Although a large number of PLA2s form a superfamily consisting of at least 16 groups, few PLA2s have been identified and characterized in insects. Furthermore, physiological functions of insect PLA2s remain unclear. Clustered regularly interspaced short parlindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been a useful research tool to validate gene function. This study identified and characterized a secretory PLA2 (sPLA2) from legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), and validated its physiological functions using CRISPR/Cas9. An open reading frame of M. vitrata sPLA2 (Mv-sPLA2) encoding 192 amino acids contained signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Mv-sPLA2 was related to other Group III sPLA2s. Mv-sPLA2 was expressed in both larval and adult stages. It was inducible by immune challenge. RNA interference (RNAi) of Mv-sPLA2 significantly suppressed cellular immunity and impaired larval development. Furthermore, RNAi treatment in female adults prevented oocyte development. These physiological alterations were also observed in a mutant line of M. vitrata with Mv-sPLA2 deleted by using CRISPR/Cas9. Mv-sPLA2 was not detected in the mutant line from western blot analysis. Addition of an eicosanoid, PGE2, significantly rescued oocyte development of females of the mutant line. These results suggest that Mv-sPLA2 plays crucial role in immune, developmental, and reproductive processes of M. vitrata.

Characterization of the first insect prostaglandin (PGE2) receptor: MansePGE2R is expressed in oenocytoids and lipoteichoic acid (LTA) increases transcript expression.

In arthropods, eicosanoids derived from the oxygenated metabolism of arachidonic acid are significant in mediating immune responses. However, the lack of information about insect eicosanoid receptors is an obstacle to completely decipher immune mechanisms underlying both eicosanoid downstream signal cascades and their relationship to immune pathogen-associated molecular patterns (PAMPs). Here, we cloned and sequenced a G protein-coupled receptor (MW 46.16 kDa) from the model lepidopteran, Manduca sexta (Sphingidae). The receptor shares similarity of amino acid motifs to human prostaglandin E2 (PGE2) receptors, and phylogenetic analysis supports its classification as a prostaglandin receptor. In agreement, the recombinant receptor was activated by PGE2 resulting in intracellular cAMP increase, and therefore designated MansePGE2R. Expression of MansePGE2R in Sf9 cells in which the endogenous orthologous receptor had been silenced showed similar cAMP increase upon PGE2 challenge. Receptor transcript expression was identified in various tissues in larvae and female adults, including Malpighian tubules, fat body, gut and hemocytes, and in female ovaries. In addition to the cDNA cloned that encodes the functional receptor, an mRNA was found featuring the poly-A tail but lacking the predicted transmembrane (TM) regions 2 and 3, suggesting the possibility that internally deleted receptor proteins exist in insects. Immunocytochemistry and in situ hybridization revealed that among hemocytes, the receptor was exclusively localized in the oenocytoids. Larval immune challenges injecting bacterial components showed that lipoteichoic acid (LTA) increased MansePGE2R expression in hemocytes. In contrast, injection of LPS or peptidoglycan did not increase MansePGE2R transcript levels in hemocytes, suggesting the LTA-associated increase in receptor transcript is regulated through a distinct pathway. This study provides the first characterization of an eicosanoid receptor in insects, and paves the way for establishing the hierarchy in signaling steps required for establishing insect immune responses to infections.

DeltaFY mutation in human torsin A [corrected] induces locomotor disability and abberant synaptic structures in Drosophila.

We investigate the molecular and cellular etiologies that underlie the deletion of the six amino acid residues (DeltaF323-Y328; DeltaFY) in human torsin A (HtorA). The most common and severe mutation involved with early-onset torsion dystonia is a glutamic acid deletion (DeltaE 302/303; DeltaE) in HtorA which induces protein aggregates in neurons and cells. Even though DeltaFY HtorA forms no protein clusters, flies expressing DeltaFY HtorA in neurons or muscles manifested a similar but delayed onset of adult locomotor disability compared with flies expressing DeltaE in HtorA. In addition, flies expressing DeltaFY HtorA had fewer aberrant ultrastructures at synapses compared with flies expressing DeltaE HtorA. Taken together, the DeltaFY mutation in HtorA may be responsible for behavioral and anatomical aberrations in gDrosophila.

Octopamine and 5-hydroxytryptamine mediate hemocytic phagocytosis and nodule formation via eicosanoids in the beet armyworm, Spodoptera exigua.

Octopamine and 5-hydroxytryptamine (5-HT) have been known to mediate cellular immune responses, such as hemocytic phagocytosis and nodule formation, during bacterial invasion in some insects. In addition, eicosanoids also mediate these cellular immune reactions in various insects, resulting in clearing the bacteria circulating in the hemolymph. This study investigated a hypothesis on signal cross-talk between both types of immune mediators in the beet armyworm, Spodoptera exigua, which had been observed in the effect of eicosanoids on mediating the cellular immune responses. In response to bacterial infection, octopamine or 5-HT markedly enhanced both hemocytic phagocytosis and nodule formation in S. exigua larvae. Their specific antagonists, phentolamine (an octopamine antagonist) or ketanserin (a 5-HT antagonist) suppressed both cellular immune responses of S. exigua. These effects of biogenic monoamines on the immune mediation were expressed through eicosanoids because the inhibitory effects of both antagonists were rescued by the addition of arachidonic acid (a precursor of eicosanoid biosynthesis). Furthermore, the stimulatory effects of both monoamines on the cellular immune responses were significantly suppressed by different inhibitors acting at their specific levels of eicosanoid biosynthesis. Taken together, this study suggests that octopamine and 5-HT can mediate hemocytic phagocytosis and nodule formation through a downstream signal pathway relayed by eicosanoids in S. exigua.

Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d), a novel serogroup with a mosquitocidal activity.

The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipienspallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.

Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenée).

Acetylcholinesterase (AChE) has been known to be the target of organophosphorous and carbamate insecticides. Only a single AChE, however, existed in insects and was involved in insecticide resistance, recently another AChE is reported in mosquitoes and aphids. We have cloned cDNAs encoding two ace genes, designated as Ha-ace1 and Ha-ace2 by a combined degenerate PCR and RACE strategy from adult heads of the oriental tobacco budworm, Helicoverpa assulta. The Ha-ace1 and Ha-ace2 genes encode 664 and 647 amino acids, respectively and have conserved motifs including a catalytic triad, a choline-binding site and an acyl pocket. Both Ha-AChEs were determined to be secretory proteins based on the existence of a signal peptide. The Ha-ace1 gene, the first reported ace1 in lepidopterans, belongs to the ace1 subfamily whereas the Ha-ace2 gene showed high similarity to those in the ace2 subfamily. Phylogenetic analysis showed that the Ha-ace1 gene was completely diverged from the Ha-ace2, suggesting that the Ha-ace genes are duplicated. Quantitative real time-PCR revealed that expression level of the Ha-ace1 gene was much higher than that of the Ha-ace2 in all body parts examined. The biochemical properties of purified proteins by affinity chromatography showed substrate specificity for acetylthiocholine iodide, and inhibitor specificity for BW284C51 and eserine and their peptide sequences partially identified by a MALDI-TOF mass spectrometer demonstrated that two Ha-AChEs were expressed in vivo.

Rapid Cold-Hardening of a Subtropical Species, Maruca vitrata (Lepidoptera: Crambidae), Accompanies Hypertrehalosemia by Upregulating Trehalose-6-Phosphate Synthase.

A subtropical insect, Maruca vitrata (F.) (Lepidoptera: Crambidae), is invasive to temperate zones, in which low temperatures during winter would be a serious challenge for colonization. This study assessed cold tolerance and cold-hardening of M. vitrata to understand its overwintering mechanism. Supercooling capacity was confirmed in all developmental stages exhibiting body freezing points at lower than -10°C, in which supercooling points (SCPs) were significantly different among developmental stages, with eggs having the lowest SCP (at -22.5°C). However, all developmental stages suffered significant mortality after being exposed to low temperatures much higher than SCPs. Furthermore, nonfreezing injury increased with elapsed time at 25°C after cold shock. One of the nonfreezing symptoms was a darkening on thorax, which was explained by uncontrolled prophenoloxidase activation. Pre-exposure to 8°C for 1 h significantly increased the survival of both young and old larvae to a low-temperature treatment (-5°C for 1 h). Rapid cold-hardening (RCH) was accompanied by significant increase in hemolymph trehalose concentration. During RCH, trehalose-6-phosphate synthase was significantly upregulated in its expression level. These results suggest that M. vitrata is a freeze-susceptible species and becomes cold-hardy via hypertrehalosemia.