Single domain von Willebrand factor type C "cytokines" and the regulation of the stress/immune response in insects.

The single domain von Willebrand factor type C (SVWC) appears in small secreted peptides that are arthropod-specific and are produced following environmental stress or pathogen exposure. Most research has focused on proteins with SVWC domain that are induced after virus infection and are hypothesized to function as "cytokines" to regulate the innate immune response. The expansion of SVWC genes in insect species indicates that many other functions remain to be discovered. Research in shrimp has elucidated the adaptability of Vago-like peptides in the innate immune response against bacteria, fungi and viruses after activation by Jak-STAT and/or Toll/Imd pathways in which they can act as pathogen-recognition receptors or cytokine-like signaling molecules. SVWC factors also appear in scorpion venoms and tick saliva, underlining their versatility to acquire new functions. This review discusses the discovery and function of SVWC peptides from insects to crustaceans and chelicerates and reveals the enormous gaps in knowledge that remain to be filled to understand this enigmatic group of secreted peptides.

Drosophila X virus-like particles as delivery carriers for improved oral insecticidal efficacy of scorpion Androctonus australis peptide against the invasive fruit fly, Drosophila suzukii.

Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects, offering a promising insecticidal component for insect pest control. However, effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph, where they can act. Here, we investigated the potential of a novel nanocarrier, Drosophila X virus-like particle (DXV-VLP), for delivering a neurotoxin from the scorpion Androctonus australis Hector (AaIT) against the invasive pest fruit fly, Drosophila suzukii. Our results show that the fusion proteins of DXV polyproteins with AaIT peptide at their C-termini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system, and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions. In addition, the AaIT peptides displayed on DXV-VLPs retained their toxicity, as demonstrated in injection bioassays that resulted in severe mortality (72%) in adults after 72 h. When fed to adults, mild mortality was observed in the group treated with DXV-AaIT (38%), while no mortality occurred in the group treated with AaIT peptide, thus indicating the significant role of DXV-VLPs in delivering AaIT peptides. Overall, this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control. Moreover, it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.

Functional characterization of putative ecdysone transporters in lepidopteran pests.

The insect steroid hormone ecdysone plays a critical role in insect development. Several recent studies have shown that ecdysone enters cells through Organic Anion Transporting Polypeptides (OATPs) in insects such as flies and mosquitoes. However, the conservation of this mechanism across other arthropods and the role of this transporter in canonical ecdysone pathways are less well studied. Herein we functionally characterized the putative ecdysone importer (EcI) from two major agricultural moth pests: Helicoverpa armigera (cotton bollworm) and Spodoptera frugiperda (fall armyworm). Phylogenetic analysis of OATP transporters across the superphylum Ecdysozoa revealed that EcI likely appeared only at the root of the arthropod lineage. Partial disruption of EcI in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential for development in vivo. Depletion and re-expression of EcI in the lepidoptera cell line RP-HzGUT-AW1(MG) demonstrated this protein's ability to control ecdysone mediated signaling in gene regulation, its role in ecdysone mediated cell death, and its sensitivity to rifampicin, a well-known organic anion transporter inhibitor. Overall, this work sheds light on ecdysone uptake mechanisms across insect species and broadens our knowledge of the physiological roles of OATPs in the transportation of endogenous substrates.

Identification of Helicoverpa armigera promoters for biotechnological applications.

Helicoverpa armigera and Helicoverpa zea are highly polyphagous major agricultural pests with a global distribution. Their control is based on insecticides, however, new, effective, and environmentally friendly control tools are required to be developed and validated. In an effort to facilitate the development of advanced biotechnological tools in these species that will take advantage of new powerful molecular biology techniques like CRISPR/Cas9, we used available transcriptomic data and literature resources, in order to identify RNA polymerase II and III promoters active in RP-HzGUT-AW1(MG), a midgut derived cell line from Helicoverpa zea. Following functional analysis in insect cell lines, four RNA polymerase II promoters from the genes HaLabial, HaTsp-2A, HaPtx-I and HaCaudal were found to exhibit high transcriptional activity in vitro. The HaTsp-2A promoter did not exhibit any activity in the non-midgut derived cell lines Sf-9 and Hi-5 despite high sequence conservation among Lepidoptera, suggesting that it may function in a gut specific manner. Furthermore, considering the utility of RNA polymerase III U6 promoters in methodologies such as RNAi and CRISPR/Cas9, we identified and evaluated four different U6 promoters of H. armigera. In vitro experiments based on luciferase and GFP reporter assays, as well as in vivo experiments targeting an essential gene of Helicoverpa, indicate that these U6 promoters are functional and can be used to experimentally silence or knockout target genes through the expression of shRNAs and sgRNAs respectively. Taking our findings together, we provide a set of promoters useful for the genetic manipulation of Helicoverpa species, that can be used in various applications in the context of agricultural biotechnology.

Chichen type III interferon produced by silkworm bioreactor induces ISG expression and restricts ALV-J infection in vitro.

Type III interferon (IFN-λ) has recently been shown to exert a significant antiviral impact against viruses in vertebrates. Avian leukosis virus subgroup J (ALV-J), which causes tumor disease and immunosuppression in infected chicken, is a retrovirus that is difficult to prevent and control because of a lack of vaccines and drugs. Here, we obtained chicken IFN-λ (chIFN-λ) using a silkworm bioreactor and demonstrated that chIFN-λ has antiviral activity against ALV-J infection of both chicken embryo fibroblast cell line (DF1) and epithelial cell line (LMH). We found that chIFN-λ triggered higher levels of particular type III interferon-stimulated genes (type III ISGs) including myxovirus resistance protein (Mx), viperin (RSAD2), and interferon-inducible transmembrane protein 3 (IFITM3) in DF1 and LMH cells. Furthermore, over-expression of Mx, viperin, and IFITM3 could inhibit ALV-J infection in DF1 and LMH cells. Therefore, these results suggested that the anti-ALV-J function of chIFN-λ was specifically implemented by induction of expression of type III ISGs. Our data identified chIFN-λ as a critical antiviral agent of ALV-J infection and provides a potentially and attractive platform for the production of commercial chIFN-λ.

An update on ecdysone signaling during insect oogenesis.

An overview is presented of the different functions of ecdysone signaling during insect oogenesis. An extensive genetic toolkit allowed analysis with unprecedented temporal and spatial detail in Drosophila where functions were revealed in stem cell proliferation and niche maintenance, germline cyst differentiation and follicle formation, integration of nutrient and lipid signaling, follicle maturation and ovulation. Besides putative autocrine/paracrine signaling, hormonal networks were identified that integrate ecdysone with other endocrine signaling pathways. In other insects, progress in oogenesis has lagged behind although recently RNAi emerged as a new tool to analyze gene function in ovaries in hemimetabolous insects and Tribolium.

How do oral insecticidal compounds cross the insect midgut epithelium?

The use of oral insecticidal molecules (small molecules, peptides, dsRNA) via spray or plant mediated applications represents an efficient way to manage damaging insect species. With the exception of Bt toxins that target the midgut epithelium itself, most of these compounds have targets that lie within the hemocoel (body) of the insect. Because of this, one of the greatest factors in determining the effectiveness of an oral insecticidal compound is its ability to traverse the gut epithelium and enter the hemolymph. However, for many types of insecticidal compounds, neither the pathway taken across the gut nor the specific genes which influence uptake are fully characterized. Here, we review how different types of insecticidal compounds enter or cross the midgut epithelium through passive (diffusion) or active (transporter based, endocytosis) routes. A deeper understanding of how insecticidal molecules cross the gut will help to best utilize current insecticides and also provide for more rational design of future ones.

Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection.

UNLABELLED: The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. IMPORTANCE: This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10.

Development of cell-based bioassay with Sf9 cells expressing TcSKR1 and TcSKR2 and differential activation by sulfated and non-sulfated SK peptides.

Insect sulfakinin receptors (SKRs) are G-protein-coupled receptors (GPCRs) that interact with sulfakinins (SKs) to modulate diverse biological processes. One of the indispensable roles of SKs is in the regulation of food intake in insects. In this project we report on the development of a cell-based receptor assay system with insect Sf9 cells, expressing TcSKR1 and TcSKR2 from the red flour beetle Tribolium castaneum, a model and important pest insect in agriculture. In this system, a stable presence of the two TcSKRs was supported by Western blotting. The expressed TcSKRs were coupled to Gαs-protein upon activation and stimulated cAMP accumulation in Sf9 cells. Exposure of the transfected cell lines to sulfated SK (sSK) activated TcSKR1 at 1 nM; the EC50 of sSK to obtain 50% of receptor activation was similar for both receptors. In contrast, µM concentrations of non-sulfated SK were necessary to activate both TcSKRs. In conclusion, this cell-based TcSKR assay system is useful to screen SK-related peptides and mimetics and to better document ligand-receptor structure-activity relationships. Given the importance of SK signaling system in insects, the present study may provide new insights on the development of new methods to control pest insects.

Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: stability and interactions with the core RNAi machinery.

While several studies have been conducted to investigate the stability of dsRNA in the extracellular medium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has been introduced into the cell. Here, we investigate the stability of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) genomic dsRNA fragments after transfection into Bombyx-derived Bm5 cells. Using RT-PCR as a detection method, we found that dsRNA could persist for long periods (up to 8 days) in the intracellular environment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence had no effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also found that transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, even when co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggesting that the neutralization by RNAi does not play a role in the establishment of an in vitro culture system. The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the establishment for an in vitro culture system for BmCPV.

Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides.

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies.

Ecdysteroid signaling in ecdysteroid-resistant cell lines from the polyphagous noctuid pest Spodoptera exigua.

Although dibenzoylhydrazine-type non-steroidal ecdysone agonists such as methoxyfenozide (RH-2485) have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, cell lines derived from the polyphagous noctuid pest Spodoptera exigua (Se4 cells) were selected for continuous growth in the presence of high concentrations of 20-hydroxyecdysone (20E) or methoxyfenozide. Here we describe an analysis of ecdysteroid receptor signaling in the ecdysteroid-resistant Se4 cell lines. In contrast to other ecdysteroid-resistant cell lines described in literature, our data support the existence of a normal functioning ecdysteroid receptor complex in the resistant Se4 cell lines: (1) using a recombinant BmNPV baculovirus as a transduction tool, activation of an ecdysone-responsive luciferase cassette was demonstrated; (2) the early gene HR3 is constitutively expressed in the resistant cell lines that are grown in the presence of 20E or methoxyfenozide. Quantitative RT-PCR experiments indicated that expression levels of SeEcR mRNA were comparable among sensitive and resistant cell lines. Sequencing of PCR fragments also revealed the presence of SeEcR mRNA with a wild-type ligand-binding domain in resistant cells. Finally, a possible role for the gene FTZ-F1, whose expression correlates with the absence of circulating ecdysteroids during insect development, in the resistance mechanism was investigated. However, it was observed that FTZ-F1, in contrast to what is observed during insect development, is constitutively expressed in Se4 cells and that its expression is not regulated by the addition of ecdysteroid. It is proposed that the resistance mechanism in Se4 cells resides at the coupling between the conserved hierarchical cascade of early and early-late gene expression and the differentiation program in the Se4 cell line. The use of insect cell lines for the investigation of resistance against dibenzoylhydrazine ecdysone agonists and their relevance for uncovering resistance mechanisms in insects during pest control programs is discussed.

Prostaglandin signaling and ovarian follicle development in the silkmoth, Bombyx mori.

Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.

Transformed lepidopteran cells expressing a protein of the silkmoth fat body display enhanced susceptibility to baculovirus infection and produce high titers of budded virus in serum-free media.

Baculovirus vectors constitute important tools for therapeutic protein production and mammalian cell transduction for gene therapy applications. A prerequisite for such applications is that the cell lines in which baculoviruses are propagated be maintained in serum-free media that are devoid of potential human pathogens. However, in serum-free media, the performance of baculovirus-based systems can be significantly reduced. In this report, we show that silkmoth-derived host cell lines for the Bombyx mori-nuclear polyhedrosis virus (BmNPV) that are transformed with the gene for the promoting protein (PP), a silkmoth-derived secreted factor containing a lipid-binding domain, display enhanced susceptibility to BmNPV infection and enhanced budded virus productivity in serum-free media. For transformed silkmoth cells maintained in serum-free media, the rate of BmNPV entry is enhanced by two orders of magnitude relative to the untransformed cells, while the rate of budded virus production is increased five-fold. The infectivity-enhancing effect can be also conferred to normal cells grown in serum-free media by addition of conditioned media from the transformed cells, which contain the secreted recombinant PP. Thus, PP substitutes for serum factors whose presence facilitates baculovirus entry into the cells. However, the effects of silkmoth-derived PP may be specific to the BmNPV-silkmoth system since little or no changes in viral infectivity are obtained by PP expression in Trichoplusia ni-derived High-Fivetrade mark cells grown in serum-free media and infected with a different baculovirus (AcNPV).