Regulatory network in heat stress response in parasitoid wasp focusing on Xap5 heat stress regulator.

Insects are susceptible to elevated temperatures, resulting in impaired fertility, and shortened lifespan. This study investigated the genetic mechanisms underlying heat stress effects. We conducted RNA sequencing on Pteromalus puparum exposed to 25°C and 35°C, revealing transcriptional signatures. Weighted Gene Co-expression Network Analysis uncovered heat stress-associated modules, forming a regulatory network of 113 genes. The network is naturally divided into two subgroups, one linked to acute heat stress, including heat shock proteins (HSPs), and the other to chronic heat stress, involving lipogenesis genes. We identified an Xap5 Heat Shock Regulator (XHSR) gene as a crucial network component, validated through RNA interference and quantitative PCR assays. XHSR knockdown reduced wasps' lifespan while directly inducing HSPs and mediating lipogenesis gene induction. CRISPR/Cas9-mediated knockout of the Drosophila XHSR homolog reduced mutants' survival, highlighting its conserved role. This research sheds light on thermal tolerance mechanisms, offering potential applications in pest control amid global warming.

The larval saliva of an endoparasitic wasp, Pteromalus puparum, suppresses host immunity.

In the lengthy co-evolution between insects and their animal or plant hosts, insects have evolved a wide range of salivary strategies to help evade host defenses. Although there is a very large literature on saliva of herbivorous and hematophagous insects, little attention has been focused on the saliva of parasitoid wasps. Some parasitoid species are natural enemies that effectively regulate insect population sizes in nature that they are applied for biological control of agricultural pests. Here, we demonstrate the influence of the endoparasitoid, Pteromalus puparum, larval saliva on the cellular and humoral immunity of its host. Larval saliva increases mortality of hemocytes, and inhibits hemocyte spreading, a specific cellular immune action. We report that high saliva concentrations inhibit host cellular encapsulation of foreign invaders. The larval saliva also inhibits melanization in host hemolymph. The saliva inhibits the growth of some bacterial species, Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa in vitro. This may promote larvae fitness by protecting them from infections. Insight into such functions of parasitic wasp saliva provides a new insight into host-parasitoid relationships and possibly leads to new agricultural pest management technologies.

Cellular and humoral immune interactions between Drosophila and its parasitoids.

The immune interactions occurring between parasitoids and their host insects, especially in Drosophila-wasp models, have long been the research focus of insect immunology and parasitology. Parasitoid infestation in Drosophila is counteracted by its multiple natural immune defense systems, which include cellular and humoral immunity. Occurring in the hemocoel, cellular immune responses involve the proliferation, differentiation, migration and spreading of host hemocytes and parasitoid encapsulation by them. Contrastingly, humoral immune responses rely more heavily on melanization and on the Toll, Imd and Jak/Stat immune pathways associated with antimicrobial peptides along with stress factors. On the wasps' side, successful development is achieved by introducing various virulence factors to counteract immune responses of Drosophila. Some or all of these factors manipulate the host's immunity for successful parasitism. Here we review current knowledge of the cellular and humoral immune interactions between Drosophila and its parasitoids, focusing on the defense mechanisms used by Drosophila and the strategies evolved by parasitic wasps to outwit it.

Cell Lines for Honey Bee Virus Research.

With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of the virus load, there is an urgent need to protect honey bees (Apis mellifera) from fatal levels of virus infection and from the non-target effects of insecticides used in agricultural settings. A continuously replicating cell line derived from the honey bee would provide a valuable tool for the study of molecular mechanisms of virus-host interaction, for the screening of antiviral agents for potential use within the hive, and for the assessment of the risk of current and candidate insecticides to the honey bee. However, the establishment of a continuously replicating honey bee cell line has proved challenging. Here, we provide an overview of attempts to establish primary and continuously replicating hymenopteran cell lines, methods (including recent results) of establishing honey bee cell lines, challenges associated with the presence of latent viruses (especially Deformed wing virus) in established cell lines and methods to establish virus-free cell lines. We also describe the potential use of honey bee cell lines in conjunction with infectious clones of honey bee viruses for examination of fundamental virology.

Genes acting in longevity-related pathways in the endoparasitoid, Pteromalus puparum.

Among insects, lifespans vary over a broad range, from the short-lived mayflies to the 17-year periodical cicadas. Generally, lifespans are determined by a phase in life, the reproductive lifespan, which varies among species. Numerous pathways, such as the insulin/insulin-like growth factor signaling pathway, the target of rapamycin pathway and the mitogen-activated protein kinase/extracellular signal-regulated kinases pathways, influence aging and lifespan. Components of these pathways were identified as lifespan-related genes, including genes mediating growth, metabolism, development, resistance, and other processes. Many age-related genes have been discovered in fruit flies, honeybees, and ants among other insect species. Studies of insect aging and longevity can help understand insect biology and develop new pest management technologies. In this paper, we interrogated the new Pteromalus puparum genome, from which we predicted 133 putative lifespan-related genes based on their homology with known lifespan-related genes of Drosophila melanogaster. These genes function in five signaling pathways and three physiological processes. The conserved domain structures of these genes were predicted and their expression patterns were analyzed. Amino acid sequence alignments and domain structure analysis indicate that most components remain conserved across at least six insect orders. The data in this paper will facilitate future work on parasitoid lifespans, which may have economic value in biocontrol programs.

Identification and characterization of miRNAs in an endoparasitoid wasp, Pteromalus puparum.

MicroRNAs (miRNAs) are a form of endogenous small noncoding RNAs that regulate protein-coding gene expression at the posttranscriptional level. So far, knowledge of miRNAs in parasitoids remains rudimentary. We investigated miRNAs in Pteromalus puparum, a pupal endoparasitoid wasp with genome and transcriptome sequences completed. In this study, we constructed eight small RNA libraries from selected developmental stages and genders: male embryos, male larvae, male pupae, male adults, mixed-sex embryos, mixed-sex larvae, mixed-sex pupae, and female adults. We identified 254 mature miRNAs with 5p/3p arm features originated from 75 known and 119 novel miRNA genes in P. puparum, 88 of which reside in 26 clusters. The miRNAs in more than half of the clusters exhibit a consistent expression pattern, indicating they were co-transcribed from a long transcript. Comparing miRNA expression in the eight libraries, we found that 84 mature miRNAs were differentially expressed between embryos and larvae, 20 between larvae and pupae, and 26 between pupae and adults. We found some miRNAs were differentially expressed between sexes in embryos (10), larvae (29), pupae (8), and adults (14). Target predictions resulted in 211,571 miRNA-mRNA interactions for 254 different mature miRNAs. These miRNAs may be involved in sexual and developmental regulation of gene expression.

A digestive tract expressing α-amylase influences the adult lifespan of Pteromalus puparum revealed through RNAi and rescue analyses.

BACKGROUND: Midgut and salivary gland α-amylases are digestive enzymes required for the development of insects and have been investigated in some insect species. However, α-amylases in the endoparasitioid wasps have not been reported. Pteromalus puparum (Hymenoptera: Pteromalidae) is a dominant endoparasitioid wasp that parasitizes many butterfly species, including the Brassicaceae pest Pieris rapae (Lepidoptera: Pieridae). Here, we studied the characteristics and functions of three α-amylases in P. puparum. RESULTS: We cloned three genes encoding α-amylases in P. puparum, PpAmy1, PpAmy2 and PpAmy3. The full length of the PpAmy1 cDNA is 1872 bp, encoding 496 amino acids, the PpAmy2 cDNA is 1863 bp long, encoding 518 amino acids, and PpAmy3 cDNA consists of 1802 bp encoding 521 amino acids. PpAmys are highly similar in amino acid sequences, but they have separate tissue distributions. Phylogenetic results show that gene duplications may occur between PpAmy2 and PpAmy3. PpAmy1 and PpAmy3 are most highly expressed in the digestive tract and the venom apparatus, respectively, while PpAmy2 is broadly expressed in all tissues. We report that PpAmy1 acts in the digestive tract, where it influences lifespan as demonstrated using RNAi and α-amylase rescue analyses, and there is no significant difference in longevity when PpAmy2 and PpAmy3 are knocked down. CONCLUSION: PpAmys probably have roles in carbohydrate metabolism of P. puparum and its host/parasitoid relationships. The characterization and functional study of PpAmys lays the foundation for the protection and utilization of parasitoid resources, and the biological control of agricultural pests. © 2019 Society of Chemical Industry.

Functional Characterization of a Venom Protein Calreticulin in the Ectoparasitoid Pachycrepoideus vindemiae.

Venom proteins act in the immunological interactions between parasitoids and their host insects. The effect of venom proteins on host immunity is not fully understood in pupal parasitoids. We identified the functions of a venom protein, calreticulin (PvCRT), in the pupal ectoparasitoid Pachycrepoideus vindemiae. Here, we report that PvCRT features a signal peptide and two conserved "calreticulin" domains. Multiple sequence alignments show that PvCRT shares 83.54% amino acid identity with CRT from both Pteromalus puparum and Nasonia vitripennis, which infers a close relationship among these three species. Using qPCR analysis, we found a lower expression level of PvCRT (0.27-fold) in the venom apparatus compared to the corresponding carcass. Immunohistochemical localization revealed that PvCRT was ubiquitously expressed in venom gland. The expression of the PvCRT gene in Drosophila transgenic lines via the UAS/Gal4 binary expression system reduced the self-encapsulation phenotype of tu(1)Sz1 mutants. Additionally, studies on humoral immunity indicate that PvCRT does not affect the antimicrobial immune responses of the host. This work on an ectoparasitoid will increase our understanding of venom-mediated host-parasitoid interactions.

A Midgut Digestive Phospholipase A2 in Larval Mosquitoes, Aedes albopictus and Culex quinquefasciatus.

Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophospholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PLA2 was characterized using a microplate PLA2 assay. The enzyme showed substantial activities at 6 and 8 µg/µl of protein concentration with optimal activity at 20 and 25 µg/µl of substrate concentration in Aedes albopictus and Culex quinquefasciatus, respectively. PLA2 activity from both mosquitoes increased in a linear function up to 1 hour of the reaction time. Both enzymes were sensitive to pH and temperature. PLA2 showed higher enzyme activities in pH 8.0 and pH 9.0 from Aedes albopictus and Culex quinquefasciatus, respectively, at 40°C of incubation. The PLA2 activity decreased in the presence of 5 mM (Aedes albopictus) and 0.5 mM (Culex quinquefasciatus) site specific PLA2 inhibitor, oleyloxyethylphosphorylcholine. Based on the migration pattern of the partially purified PLA2 on SDS-PAGE, the protein mass of PLA2 is approximately 20-25 kDa for both mosquitoes. The information on PLA2 properties derived from this study may facilitate in devising mosquitoes control strategies especially in the development of inhibitors targeting the enzyme active site.

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 63,410Da 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.

Parasitization by Scleroderma guani influences expression of superoxide dismutase genes in Tenebrio molitor.

Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor. The cDNAs for ecCuZnSOD, icCuZnSOD, and MnSOD, respectively, encode 24.55, 15.81, and 23.14 kDa polypeptides, which possess structural features typical of other insect SODs. They showed 20-94% identity to other known SOD sequences from Bombyx mori, Musca domestica, Nasonia vitripennis, Pediculus humanus corporis, and Tribolium castaneum. Expression of these genes was analyzed in selected tissues and developmental stages, and following exposure to Escherichia coli and parasitization by Scleroderma guani. We recorded expression of all three SODs in cuticle, fat body, and hemocytes and in the major developmental stages. Relatively higher expressions were detected in late-instar larvae and pupae, compared to other developmental stages. Transcriptional levels were upregulated following bacterial infection. Analysis of pupae parasitized by S. guani revealed that expression of T. molitor SOD genes was significantly induced following parasitization. We infer that these genes act in immune response and in host-parasitoid interactions.

Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.

Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts.

MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori.

Insect development and metamorphosis are regulated by the coordination of ecdysone and juvenile hormones. Insect microRNAs (miRNAs) also act in insect development and metamorphosis by regulating genes in the ecdysone cascade. Although hundreds of insect miRNAs have been identified, the physiological functions of most remain poorly understood. Here, we report that a conserved insect miRNA, microRNA-281 (miR-281), regulates the ecdysone receptor (EcR), in an isoform-specific manner in the silkworm Bombyx mori. The B. mori EcR (BmEcR) gene encodes three isoforms: BmEcR-A, BmEcR-B1 and BmEcR-B2. The 3'UTR regions of A and B genes, which contain multiple potential microRNA targeting sites, are distinct. Target prediction revealed that miR-281 may specifically target the 3'UTR of BmEcR-B. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-281 suppressed transcription of BmEcR-B but not BmEcR-A. The expression of miR-281 and BmEcR-B are well coordinated in the Malpighian tubules from the fourth larval molt to pupation. In the Malpighian tubules of fifth instar larvae, BmEcR-B protein expression was down-regulated after injection of a miR-281 mimic while up-regulated after injection of a miR-281 inhibitor. miR-281 expression was suppressed by 20-hydroxyecdysone treatments but not affected by juvenile hormone treatments. Based on these findings, we propose that miR-281 participates in B. mori developmental regulation in the Malpighian tubules through suppression of BmEcR-B expression.

Genetic transformation mediated by piggyBac in the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae).

The Asian corn borer, Ostrinia furnacalis, is a serious pest of corn, sorghum, and cotton in China and other Asian countries. The present study is the first attempt to establish the transgenic line in O. furnacalis using a piggyBac transposon, which will shed light on the future genetic control of O. furnacalis. A piggyBac vector pBac[A3EGFP] was constructed to express enhanced green fluorescence protein (EGFP)under the control of Bombyx mori actin3 promoter. Transient EGFP expression was detected 48 h after preblastodermic microinjection of pBac[A3EGFP] and the excision assay showed the transgenic vector was precisely excised. In G1 animals, PCR (polymerase chain reaction)-based investigations revealed that the exogenous gene had been introduced into O. furnacalis genome and expressed at the transcriptional level. Western blot analysis showed EGFP expression at the protein level, indicating the heritability of the transgene.

Prostaglandin A2 influences gene expression in an established insect cell line (BCIRL-HzAM1) cells.

Prostaglandins (PGs) and other eicosanoids are oxygenated metabolites of arachidonic acid and two other C(20) polyunsaturated fatty acids. While most well studied in mammals, PGs exert important actions in insects and virtually all other invertebrates. We have been researching the mechanisms of PG actions in established insect cell lines and reported earlier that two PGs, PGA(1) and PGE(1), influence gene and protein expression in HzAM1 cells. Here we report on further experiments with three 2-series PGs, PGA(2), PGE(2) and PGF(2α). In separate experiments we treated cells with each of the three PGs for 12 and 24h and then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by Delta2D software showed that PGA(2) influenced expression of 60 proteins while PGE(2) and PGF(2α) treatments led to expression changes for only a few proteins. All spots representing changes in protein expression were processed for analysis by MALDI TOF/TOF mass spectrometry. Bioinformatic analysis of the resulting sequences yielded in silico identifications of all proteins. The apparent changes in some proteins were confirmed by quantitative PCR, which demonstrated that changes in protein expression were parallel to changes in mRNA expression. We assorted the proteins into functional categories, including 1/cell structure and function; 2/cell protection and immunity; 3/energetics and metabolism; 4/nucleotide processing; 5/protein action and processing and 6/signal transduction. These findings substantially extend our idea that one mechanism of PG actions in insect cells is the modulation of gene and protein expression.

Plasmatocyte-spreading peptide influences hemocyte behavior via eicosanoids.

Hemocyte-spreading behavior is required for expressing a cellular immune response, nodulation, which clears the vast majority of invading microbes from circulation. The nodulation response is completed by a layer of plasmatocytes, which spread over the nodule and initiate a malanization process leading to darkened nodules. Plasmatocyte-spreading peptide (PSP), the first reported insect cytokine, is responsible for mediating the spreading and attachment of some subclasses of plasmatocytes to nodules. Prostaglandins (PGs), one group of eicosanoids formed from arachidonic acid (AA), also mediate plasmatocyte spreading (PS), although the potential interactions between the PSP and PG signal transduction pathways have not been investigated. We tested our hypothesis that PSP acts via biosynthesis of eicosanoids, specifically PGs, in the beet armyworm, Spodoptera exigua. In this study, we report that (1) PSP and PGE(2) independently stimulated Ca(++)-dependent PS, (2) inhibitors of PG biosynthesis reversibly blocked PS, (3) dsRNA silencing the gene encoding proPSP blocked PS, which was rescued by PSP and by AA, (4) PSP-stimulated PS was reversibly impaired by inhibitors of PG biosynthesis, and (5) the inhibitor-impaired spreading was rescued by AA. Taken together, these points strongly support our model showing that PSP acts via a plasmatocyte-surface receptor, which stimulates biosynthesis of the PGs responsible for mediating plasmatocytes spreading.

Pteromalus puparum venom impairs host cellular immune responses by decreasing expression of its scavenger receptor gene.

Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Although there is a rich literature on these systems, parasitoid immune-disabling mechanisms have not been fully elucidated. Here we report on a newly discovered immune-disabling mechanism in the Pieris rapae/Pteromalus puparum host/parasitoid system. Because venom injections and parasitization suppresses host phagocytosis, we turned attention to the P. rapae scavenger receptor (Pr-SR), posing the hypothesis that P. puparum venom suppresses expression of the host Pr-SR gene. To test our hypothesis, we cloned a full-length cDNA of the Pr-SR. Multiple sequences alignment showed the deduced amino acid sequence of Pr-SR is similar to scavenger receptors of other lepidopterans. Bacterial and bead injections induced Pr-SR mRNA and protein expression, which peaked at 4h post-bead injection. Venom injection inhibited Pr-SR expression. Pr-SR was specifically expressed in granulocytes compared to plasmatocytes. We localized the Pr-SR protein in cytoplasm and cellular membrane, with no evidence of secretion into host plasma. Double-strand RNA designed to Pr-SR mRNA silenced expression of Pr-SR and significantly impaired host phagocytosis and encapsulation reactions. Venom injections similarly silenced Pr-SR expression during the first 8h post-treatment, after which the silencing effects gradually abated. We infer from these findings that one mechanism of impairing P. rapae hemocytic immune reactions is by silencing expression of Pr-SR.

Evaluation of intraneural ganglion cysts using three-dimensional fast spin echo-cube.

PURPOSE: To compare conventional two-dimensional fast spin echo (FSE) MRI sequences with a three-dimensional FSE extended echo train acquisition method, known as Cube, in the evaluation of intraneural ganglion cysts. Also, to demonstrate that Cube enables the consistent identification and thorough characterization of the cystic joint connection, and therefore improves patient care by superior preoperative planning. MATERIALS AND METHODS: Six patients with intraneural ganglia in the knee region (five involving the peroneal and one the tibial nerve) were evaluated using both conventional FSE MR sequences and the Cube sequence. Studies were interpreted by the consensus of three board certified musculoskeletal radiologists and one peripheral nerve neurosurgeon. Surgical correlation was available in five of the six cases. RESULTS: Both imaging methods demonstrated the cysts and at least part of their joint connections after variable amount of postprocessing. Cube proved superior to conventional imaging in its ability to acquire isotropic data that could easily be reconstructed in any plane and its ability to resolve fine anatomical details. CONCLUSION: Cube is a new MR pulse sequence that enables the consistent identification of the intraneural ganglion cyst joint connection. We believe that improved visualization and characterization of the entire cyst will improve patient outcomes by facilitating more accurate surgical intervention.