Molecular characterization of the sans fille gene in Antheraea pernyi: A highly conserved gene during the evolution of animals

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and plays an important role in sex determination in Drosophila melanogaster. In this study, the snf gene from Antheraea pernyi (Lepidoptera: Saturniidae), an economically important insect, was isolated and characterized. The obtained 925 bp cDNA sequence contains an open reading frame of 669 bp encoding a polypeptide of 222 amino acids, showing 78% sequence identity to that from D. melanogaster. A database search revealed that SNF protein homologs are present in many animals, including invertebrates and vertebrates, with more than 70% amino acid sequence identities, suggesting that they were highly conserved during the evolution of animals. Phylogenetic analysis revealed that A. pernyi SNF was closely related to Bombyx mori SNF. Quantitative real-time PCR (qRT-PCR) analysis showed that the A. pernyi snf gene was transcribed during five larval developmental stages, and in six tested tissues (ovaries, testes, silk glands, fat body, integument, and hemolymph), with the most abundance determined in the gonads (ovaries or testes). Investigation of expression changes throughout embryonic development indicated that A. pernyi snf mRNA was expressed at a low level from days 0 to 4, and reached a maximum level at day 10, but decreased to a low level before hatching. These results suggest that the product of the snf gene may play important roles in the development of A. pernyi.     

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.     

A novel hexamerin with an unexpected contribution to the prophenoloxidase activation system of the Chinese oak silkworm,Antheraea pernyi

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein.     

Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes

Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoids mediate ROS production by activating NADPH-dependent oxidase (NOX) and tested the idea in the model insect, Spodoptera exigua. A NOX gene (we named SeNOX4) was identified and cloned, yielding a full open reading frame encoding 547 amino acid residues with a predicted molecular weight of 63,410 Da and an isoelectric point at 9.28. A transmembrane domain and a large intracellular domain containing NADPH and FAD-binding sites were predicted. Phylogenetic analysis indicated SeNOX4 clusters with other NOX4 genes. SeNOX4 was expressed in all life stages except eggs, and exclusively in hemocytes. Bacterial challenge and, separately, arachidonic acid (AA, a precursor of eicosanoid biosynthesis) injection increased its expression. The internalization step was assessed by counting hemocytes engulfing fluorescence-labeled bacteria. The phagocytic behavior was inhibited by dsRNA suppression of SeNOX4 expression and, separately by dexamethasone (DEX, a specific inhibitor of eicosanoid biosynthesis) treatments. However, injecting AA to dsSeNOX4-treated larvae did not rescue the phagocytic activity. Hemocytic ROS production increased following bacterial challenge, which was sharply reduced in dsSeNOX4-treated, and separately, in DEX-treated larvae. AA partially reversed the suppressed ROS production in dsSeNOX4-treated larvae. Treating larvae with either the ROS-suppressing dsSeNOX4 construct or DEX rendered experimental larvae unable to inhibit bacterial proliferation in their hemocoels. We infer that eicosanoids mediate ROS production during phagocytosis by inducing expression of SeNOX4.     

The influence of chronic eicosanoid biosynthesis inhibition on life history of the greater waxmoth, Galleria mellonella and its ectoparasitoid, Bracon hebetor

Eicosanoids are oxygenated metabolites of three C20 polyunsaturated fatty acids, mainly arachidonic acid (AA; 20:4n-6), but also 20:3n-6 and 20:5n-3. Aside from their importance in biomedicine, eicosanoids act in invertebrate biology. Prostaglandins (PGs) influence salt and water transport physiology in insect rectal epithelia and in Malpighian tubules. PGs also influence a few insect behaviors, including releasing oviposition behavior and behavioral fever. Eicosanoids act in ovarian development and in insect immunity. Because eicosanoids act in several areas of insect biology, we posed the hypothesis that chronic inhibition of eicosanoid biosynthesis, in the absence of microbial challenge, can influence insect life table parameters, including developmental time, survival, adult longevity and parasitoid fecundity. Here we report that inhibiting eicosanoid biosynthesis throughout the larval life exerted minor influences on some life table parameters of the greater wax moth, Galleria mellonella and its ectoparasitoid, Bracon hebetor, however, the inhibitors strongly reduced the production and hatchability of the parasitoids’ eggs. The significance of the work relates to the potentials of understanding and targeting eicosanoid systems as a platform for developing new technologies of insect pest management. As seen here, the impact of targeting eicosanoid systems is seen in crucial moments of insect life histories, such as reproduction or immune challenge rather than in overall larval development.     

Characterization of insect cytosolic juvenile hormone binding protein gene: Highly homology with vertebrate glyoxalase domain containing protein 4

Cytosolic juvenile hormone binding protein (cJHBP) is a carrier of juvenile hormone (JH) in insects, however knowledge about its evolution and expression remains extremely limited. In this study, a gene encoding for cJHBP was isolated from the Chinese oak silkmoth Antheraea pernyi. A database search showed that the homologous sequences were present in several animal species including nematodes, insects, tunicates, fish, and mammals. The A. pernyi cJHBP had 54–85% identity with its homolog from other insects, and 58–62% identity with vertebrate glyoxalase domain containing protein 4 (Glod-4). Phylogenetic analysis supported the hypothesis that insect cJHBP shares a common ancestor with vertebrate Glod-4. RT-PCR detection showed that the cJHBP gene was expressed throughout the developmental stages and in all tested tissues of A. pernyi, which agreed with the data from Bombyx mori cJHBP and Homo sapiens Glod-4. These data suggest that insect cJHBP may play a similar function as vertebrate Glod-4.     

Molecular Characterization of Two Genes Encoding Novel Ca2+-Independent Phospholipase A2s from the Silkworm,Bombyx mori

Eicosanoids are crucial downstream signals in the insect immune responses. Phospholipase A2 (PLA2) catalyzes phospholipids, the initial step in eicosanoid biosynthesis. In mammals, the biological roles of Ca²⁺-independent Phospholipase A2 (iPLA2) have been extensively studied; however, only a few studies have attempted to explore iPLA2 functions in insects. In this study, we identified two iPLA2 genes (designated as BmiPLA2A and BmiPLA2B) in the silkworm, Bombyx mori. BmiPLA2A had a 2427 base pair (bp) open reading frame (ORF) that coded for a protein with 808 amino acids. In contrast, BmiPLA2B had a 1731 bp ORF that coded for a protein with 576 amino acids. Domain analysis revealed that BmiPLA2A had six ankyrin repeat domains, but BmiPLA2B lacks these domains. BmiPLA2A and BmiPLA2B were transcribed widely in various tissues and developmental stages with different expression patterns. The administration of 20-hydroxyecdysone increased their expression levels in the epidermis and hemocytes. Furthermore, challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria induced the expression of BmiPLA2A and BmiPLA2B with variable degrees along with different time points. Our findings imply that BmiPLA2A and BmiPLA2B may have important biological roles in the development and innate immunity of B. mori.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Benzylideneacetone and other phenylethylamide bacterial metabolites induce apoptosis to kill insects

Xenorhabdus and Photorhabdus are entomopathogenic bacteria that can induce immunosuppression against target insects by suppressing eicosanoid biosynthesis, leading to fatal septicemia. These bacteria can synthesize and release secondary metabolites such as benzylideneacetone (BZA) and other phenylethylamide compounds that can inhibit phospholipase A₂ (PLA₂) and shut down eicosanoid biosynthesis. However, insecticidal activities of these bacterial metabolites remain unclear. Thus, the objective of this study was to assess cytotoxicities of BZA and seven other bacterial metabolites to insect cells. These eight bacterial metabolites exhibited significant cytotoxicities against an insect cell line Sf9 at micromolar range. Especially, BZA and cPY were highly potent at low micromolar range. When these eight bacterial metabolites were injected to hemocoels of Spodoptera exigua larvae, they significantly decreased total count of hemocytes. In Sf9 cell line and hemocytes, these bacterial metabolites induced cell membrane blebbings, apoptotic vesicles, and genomic DNA fragmentation. Terminal deoxyribonucleotidyl transferase nick end translation assay showed that these bacterial metabolites caused significant DNA breakages in cells in a dose-dependent manner. However, a pan caspase inhibitor treatment significantly rescued the cell death induced by these bacterial metabolites. Cytotoxicities of these bacterial metabolites were highly correlated with their insecticidal activities. These results indicate that the insecticidal activities of the bacterial metabolites may be induced by their apoptotic activities against hemocytes and other insect cells. Taken together, these results suggest that phenylethylamide compounds might have potential as novel insecticides.     

Five small heat shock protein genes from Chilo suppressalis: characteristics of gene, genomic organization, structural analysis, and transcription profiles

Small heat shock proteins (sHSPs) are the most diverse but also the most poorly known family of molecular chaperones, and they play essential roles in various biological processes. The striped stem borer, Chilo suppressalis (Insecta: Lepidoptera: Pyralidae), is one of the most serious pests of rice, causing extensive damage and yield loss. In this study, we isolated and characterized five members of the sHSPs family—Cshsp19.8, Cshsp21.4, Cshsp21.5, Cshsp21.7a, and Cshsp21.7b—from C. suppressalis. The cDNAs of these genes encoded proteins of 177, 187, 191, 191, and 191 amino acids with isoelectric points of 7.0, 5.6, 6.1, 6.3, and 6.3, respectively. While Cshsp19.8, Cshsp21.5, and Cshsp21.7b had no introns, Cshsp21.4 and Cshsp21.7a contained one and two introns, respectively. Structural analysis indicated that all five Cshsps possessed conserved arginine and a V/IXI/V motif, which is related to hydrophobic characteristics of sHSPs. The five heat shock proteins can be classified into two main groups: an orthologous type (Cshsp21.4 and Cshsp21.7a) and a species-specific type (Cshsp19.8, Cshsp21.5, and Cshsp21.7b). Real-time quantitative PCR analyses revealed that Cshsp19.8, Cshsp21.5, Cshsp21.7a, and Cshsp21.7b all exhibited their highest expression levels within Malpighian tubules or the hindgut, while such levels were found in the head for Cshsp21.4. The expression of Csshsps at different developmental stages revealed that the mRNA levels of Cshsp19.8, Cshsp21.4, Cshsp21.5, and Cshsp21.7b peaked in adults, whereas the highest level of Cshsp21.7a was observed in first instar larvae. Cshsp19.8 and Cshsp21.7b were both upregulated dramatically by heat and cold, and Cshsp21.5 could be induced by cold stress. Neither Cshsp21.4 nor Cshsp21.7a responded to heat or cold. These results demonstrated that different Csshsps play distinctive roles in the regulation of the physiological activities in C. suppressalis.     

The recycling of protein components of the flight-specific lipophorin in Locusta migratoria

The metabolism of locust lipophorin A⁺ during lipid delivery to the flight muscle and lipid loading at the fat body was studied in vitro. Protein C₂ was shown to be released upon hydrolysis of lipophorin A⁺-carried diacylglycerol by the flight muscle lipoprotein lipase. This in vitro released protein C₂ was shown to reassociate with lipophorin A_y upon hormone-induced lipid mobilization from fat body in vitro. These results demonstrate the reversibility of the association of protein C₂ with lipophorin A_y and support the shuttle function of the protein components of locust lipophorin A⁺ in lipid transport.     

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.     

Sequential immunosuppressive activities of bacterial secondary metabolites from the entomopahogenic bacterium Xenorhabdus nematophila

The entomopathogenic bacterium Xenorhabdus nematophila secretes at least eight bacterial metabolites that play crucial roles suppressing target insect immune responses by inhibiting eicosanoid biosynthesis. We analyzed sequential changes in bacterial metabolite production during bacterial growth and analyzed their individual immunosuppressive activities against the insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth pattern in both insect host and culture medium, and eight metabolites were secreted at different time points. At the early growth phase (6–12 h), Ac-FGV and PHPP were detected in significant amounts in the culture broth. At this early phase, both Ac-FGV (18 μg/ml) and oxindole (110 μg/ml) levels significantly inhibited phenoloxidase and phospholipase A₂ activities in S. exigua hemolymph. At the late growth phase (12–36 h), all eight metabolites were detected at significant levels (10–140 μg/ml) in the culture broth and were sufficient to induce hemocyte toxicity. These results suggest that X. nematophila sequentially produces immunosuppressive metabolites that might sequentially and cooperatively inhibit different steps of insect immune responses.     

Baculovirus p33 Binds Human p53 and Enhances p53-Mediated Apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.     

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.     

Antheraea pernyi silk fibroin maintains the immunosupressive properties of human bone marrow mesenchymal stem cells

We reported previously that regenerated Antheraea pernyi silk fibroin (A. pernyi SF) could support the attachment and growth of human bone marrow mesenchymal stem cells (hBMSCs). In this work, the immunosupressive effects of hBMSCs cultured on the A. pernyi SF films on T-cells were investigated in vitro. The production of IL-6, CD80, CD86 and HLA-DR by the hBMSCs was also observed. The study showed that hBMSCs cultured on the regenerated A. pernyi SF films still kept their immunosupression on T-cell proliferation and IL-2 secretion. Moreover, regenerated A. pernyi SF like regenerated Bombyx mori SF and collagen did not elicit T-cell proliferation but it could support the expression of IL-6 and surface antigen of hBMSCs. Regenerated A. pernyi SF can maintain the function of hBMSCs in immunomodulation and cytokines production, which has the potential utility of hBMSCs combined with A. pernyi SF in tissue replacement and repair.