Nanoparticle delivery of polynucleic acids traditionally relies on the modulation of surface interactions to achieve loading and release. This work investigates the additional role of confinement in mobility of dsRNA (84 and 282 base pair (bp) sequences of Spodoptera frugiperda) as a function of silica nanopore size (nonporous, 3.9, 8.0, and 11.3 nm). Amine-functionalized nanoporous silica microspheres (NPSMs, ∼10 μm) are used to directly visualize the loading and exchange of fluorescently labeled dsRNA. Porous particles are fully accessible to both lengths of dsRNA by passive diffusion, except for 282 bp dsRNA in 3.9 nm pores. The stiffness of dsRNA suggests that encapsulation occurs by threading into nanopores, which is inhibited when the ratio of dsRNA length to pore size is large. The mobility of dsRNA at the surface and in the core of NPSMs, as measured by fluorescence recovery after photobleaching, is similar. The mobility increases with pore size (from 0.0002 to 0.001 μm2/s for 84 bp dsRNA in 3.9-11.3 nm pores) and decreases with the length of dsRNA. However, when the dsRNA is unable to load into the pores (on nonporous particles and for 282 bp dsRNA in 3.9 nm pores), surface mobility is not detectable. The pore structure appears to serve as a "source" to provide a mobile network of dsRNA at the particle surface. The importance of mobility is demonstrated by exchange experiments, where NPSMs saturated with mobile dsRNA can exchange dsRNA with the surrounding solution, while immobile dsRNA is not exchanged. These results indicate that nanoparticle synthesis techniques that provide pores large enough to take up polynucleic acids internally (and not simply on the external surface of the particle) can be harnessed to design polynucleic acid/nanoporous silica combinations for controlled mobility as a path forward toward effective nanocarriers.

Background Polydnaviruses (PDVs) are mutualistic endogenous viruses inoculated by some lineages of parasitoid wasps into their hosts, where they facilitate successful wasp development. PDVs include the ichnoviruses and bracoviruses that originate from independent viral acquisitions in ichneumonid and braconid wasps respectively. PDV genomes are fully incorporated into the wasp genomes and consist of (1) genes involved in viral particle production, which derive from the viral ancestor and are not encapsidated, and (2) proviral segments harboring virulence genes, which are packaged into the viral particle. To help elucidating the mechanisms that have facilitated viral domestication in ichneumonid wasps, we analyzed the structure of the viral insertions by sequencing the whole genome of two ichnovirus-carrying wasp species,Hyposoter didymatorandCampoletis sonorensis. Results Assemblies with long scaffold sizes allowed us to unravel the organization of the endogenous ichnovirus and revealed considerable dispersion of the viral loci within the wasp genomes. Proviral segments contained species-specific sets of genes and occupied distinct genomic locations in the two ichneumonid wasps. In contrast, viral machinery genes were organized in clusters showing highly conserved gene content and order, with some loci located in collinear wasp genomic regions. This genomic architecture clearly differs from the organization of PDVs in braconid wasps, in which proviral segments are clustered and viral machinery elements are more dispersed. Conclusions The contrasting structures of the two types of ichnovirus genomic elements are consistent with their different functions: proviral segments are vehicles for virulence proteins expected to adapt according to different host defense systems, whereas the genes involved in virus particle production in the wasp are likely more stable and may reflect ancestral viral architecture. The distinct genomic architectures seen in ichnoviruses versus bracoviruses reveal different evolutionary trajectories that have led to virus domestication in the two wasp lineages.

Amine-functionalized mesoporous silica nanoparticles (MSNPAs) are ideal carriers for oligonucleotides for gene delivery and RNA interference. This investigation examines the thermodynamic driving force of interactions of double-stranded (ds) RNA with MSNPAs as a function of RNA length (84 and 282 base pair) and particle pore diameter (nonporous, 2.7, 4.3, and 8.1 nm) using isothermal titration calorimetry, extending knowledge of solution-based nucleic acid-polycation interactions to RNA confined in nanopores. Adsorption of RNA follows a two-step process: endothermic interactions driven by entropic contribution from counterion (and water) release and an exothermic regime dominated by short-range interactions within the pores. Evidence of hindered pore loading of the longer RNA and pore size-dependent confinement of RNA in the MSPAs is provided from the relative contributions of the endothermic and exothermic regimes. Reduction of endothermic and exothermic enthalpies in both regimes in the presence of salt for both lengths of RNA indicates the significant contribution of short-range electrostatic interactions, whereas Delta H and Delta G values are consistent with conformation changes and desolvation of nucleic acids upon binding with polycations. Knowledge of the interactions between RNA and functionalized porous nanoparticles will aid in porous nanocarrier design suitable for functional RNA delivery.

Polydnaviruses (PDVs) are mutualistic endogenous viruses associated with some lineages of parasitoid wasps that allow successful development of the wasps within their hosts. PDVs include two taxa resulting from independent virus acquisitions in braconid (bracoviruses) and ichneumonid wasps (ichnoviruses). PDV genomes are fully incorporated into the wasp genomes and comprise (1) virulence genes located on proviral segments that are packaged into the viral particle, and (2) genes involved in the production of the viral particles, which are not encapsidated. Whereas the genomic organization of bracoviruses within the wasp genome is relatively well known, the architecture of endogenous ichnoviruses remains poorly understood. We sequenced the genome of two ichnovirus-carrying wasp species, Hyposoter didymator and Campoletis sonorensis . Complete assemblies with long scaffold sizes allowed identification of the integrated ichnovirus, highlighting an extreme dispersion within the wasp genomes of the viral loci, i.e. isolated proviral segments and clusters of replication genes. Comparing the two wasp species, proviral segments harbor distinct gene content and variable genomic environment, whereas viral machinery clusters show conserved gene content and order, and can be inserted in collinear wasp genomic regions. This distinct architecture is consistent with the biological properties of the two viral elements: proviral segments producing virulence proteins allowing parasitism success are fine-tuned to the host physiology, while an ancestral viral architecture was likely maintained for the genes involved in virus particle production. Finding a distinct genomic architecture of ichnoviruses and bracoviruses highlights different evolutionary trajectories leading to virus domestication in the two wasp lineages.

Insect cell cultures are widely used in studies of insect cell physiology, toxicology, developmental biology and microbial pathology. The lethal effects of crude venom extracted from the ectoparasitic wasp Bracon hebetor were examined with three cultured insect cell lines; Spodoptera frugiperda (Sf9), Tribolium castaneum (TcA) and Aedes aegypti (Aag-2). Venom caused cells to round-up, swell and eventually die. Despite similar sensitivities and overlapping LC 50 values [(0.00125-0.00695) venom reservoir equivalents (VRE)/µl], significant differences were noted at the onset of cytotoxicity among the three insect cell lines. Cells from the Tribolium castaneum (TcA) and Aedes aegypti (Aag-2) showed little sensitivity to the venom: 0.0046 VRE were needed to induce 50% mortality in TcA [50% lethal concentration (LC 50 ) = 0.0046 VRE/µl], and 0.0069 VRE were needed to induce 50% mortality in Aag-2 [50% lethal concentration (LC 50 ) = 0.0069 VRE/µl). Over 80% of the Sf9 cells were nonviable within 1 h after the addition of an LC 99 dose of venom, whereas the other cells required a 5-10-fold longer incubation period to produce mortality above 50%.

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High FiveTM cells, and SfSWT-4 cells, which can produce glycoproteins with complex-type N-glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N-glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P-vank-1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT-4 cells were infected with a vankyrin-encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin-expressing cells were combined with a vankyrin-encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1496-1507, 2017.

Aenasius bambawalei Hayat (Encyrtidae: Hymenoptera) has been synonymized with Aenasius arizonensis (Girault) is a small, newly discovered endoparasitoid of the cotton mealybug Phenacoccuss solenopsis Tinsley (Pseudococcidae: Hemiptera), which completes its life cycle inside the body of its host and it is a potential insect control tool. Despite the acquired knowledge regarding host-parasitoid interaction, little information is available on the factors of parasitoid origin able to modulate mealybug physiology. The components of A. arizonensis venom have not been well studied but venom from other parasitoids and wasps contain biologically active proteins that have potential applications in pest management or may be of medicinal importance. To provide an insight into the transcripts expressed in the venom gland of A. arizonensis, a transcriptomic database was developed utilizing high throughput RNA sequencing approaches to analyze the genes expressed in venom glands of this endoparasitic wasp. The resulting A. arizonensis RNA sequences were assembled de-novo with contigs then blasted against the NCBI non-redundant sequence database. Contigs which matched database sequences were mostly homologous to genes from hymenopteran parasitoids such as Nasonia vitripennis, Copidosoma floridanum, Fopius arsenus and Pteromalas puparium. Further analysis of the A. arizonensis database was then performed which focused on selected genes encoding proteins potentially involved in host developmental arrest, disrupting the host immune system, host paralysis, and transcripts that support these functions. Sequenced mRNAS predicted to encode full length ORFs of Calreticulin, Serine Protease Precursor and Arginine kinase proteins were identified and the tissue specific expression of these putative venom genes was analyzed by RT-PCR. In addition, results also demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database and may provide useful information for devising control tools for insect pests and other applications.

Venom is a key-factor in the regulation of host physiology by parasitic Hymenoptera and a potentially rich source of novel bioactive substances for biotechnological applications. The limited study of venom from the ectoparasitoid Bracon hebetor, a tiny wasp that attacks larval pest insects of field and stored products and is thus a potential insect control agent, has not described the full complement and composition of these biomolecules. To have a comprehensive picture of genes expressed in the venom glands of B. hebetor, a venom gland transcriptome was assembled by using next generation sequencing technologies followed by de novo assemblies of the 10.81 M sequence reads yielded 22,425 contigs, of which 10,581 had significant BLASTx hits to know genes. The majority of hits were to Diachasma alloeum, an ectoparasitoid from same taxonomic family, as well as other wasps. Gene ontology grouped the sequences into molecular functions in which catalytic activity with 42.2% was maximum, cellular components in which cells with 33.8% and biological processes among which metabolic process with 30% had the most representatives. In this study, we highlight the most abundant sequences, and those that are likely to be functional components of the venom for parasitization. Full length ORFs of Calreticulin, Venom Acid Phosphatase Acph-1 like protein and arginine kinase proteins were isolated and their tissue specific expression was studied by RT-PCR. Our report is the first to characterize components of the B. hebetor venom glands that may be useful for developing control tools for insect pests and other applications.

Ichnoviruses (IVs), unique symbiotic viruses carried by ichneumonid campoplegine wasps, derive from integration of a paleo-ichnovirus into an ancestral wasp genome. The modern 'genome' is composed of both regions that are amplified, circularized and encapsidated into viral particles and non-encapsidated viral genomic regions involved in particle morphogenesis. Packaged genomes include multiple circular dsDNAs encoding many genes mostly organized in gene families. Virus particles are assembled in specialized ovarian cells from which they exit into the oviduct lumen; mature virions are injected during oviposition into the insect host. Expression of viral proteins in infected cells correlates with physiological alterations of the host enabling success of parasitism.

The cys‐motif gene family associated with Campoletis sonorensis ichnovirus contains 10 members, WHv1.6, WHv1.0, VHv1.1, VHv1.4, AHv1.0, A'Hv0.8, FHv1.4, LHv2.8, UHv0.8, and UHv0.8a. The results of this study indicated that, within the encapsidated virion, WHv1.6 is the most abundant cys‐motif gene, while the combined AHv genes are the least abundant. During parasitization of Heliothis virescens by Campoletis sonorenis, WHv1.6 transcripts were the mostly highly expressed, while the combined UHv genes had the lowest expression. Further proteomic analysis of WHv1.6 showed that it accumulates at high levels in parasitized plasma by 6 h, and is detectable in the haemocytes, fat body, malpighian tubules, nerve cord and epidermis by 2 days after parasitization. Localization experiments led us to conclude that WHv1.6 interacts with the cell membrane along with other organelles within a virus‐infected cell and prevents immunocytes from spreading or adhering to a foreign surface. Similarly to VHv1.4 and VHv1.1, WHv1.6 is able to inhibit the translation of haemocyte and Malpighian tubule RNAs. Our results showed that the expression of cys‐motif genes during parasitization is related to the gene copy number of each gene within the encapsidated virion and may also be dependent upon cis‐regulatory element activity in different target tissues. In addition, WHv1.6 plays a major role in inhibiting the cellular encapsulation response by H. virescens.

Nancy E. Beckage is widely recognized for her pioneering work in the field of insect host-parasitoid interactions beginning with endocrine influences of the tobacco hornworm, Manduca sexta, host and its parasitoid wasp Apanteles congregatus (now Cotesia congregata) on each other's development. Moreover, her studies show that the polydnavirus carried by the parasitoid wasp not only protects the parasitoid from the host's immune defenses, but also is responsible for some of the developmental effects of parasitism. Nancy was a highly regarded mentor of both undergraduate and graduate students and more widely of women students and colleagues in entomology. Her service both to her particular area and to entomology in general through participation on federal grant review panels and in the governance of the Entomological Society of America, organization of symposia at both national and international meetings, and editorship of several different journal issues and of several books is legendary. She has left behind a lasting legacy of increased understanding of multilevel endocrine and physiological interactions among insects and other organisms and a strong network of interacting scientists and colleagues in her area of entomology.

This datasheet on Campoletis sonorensis covers Identity, Distribution, Natural Enemies.

A large number of genes encoding structural components of polydnaviruses associated with ichneumonid wasps of the subfamily Campopleginae have recently been identified (Volkoff et al., 2010). The genes involved in the production of ichnovirus particle proteins were shown to be localized in specific regions embedded in the genome of the wasp Hyposoter didymator. These specialized regions were named 'IchnoVirus Structural Proteins Encoding Regions' (IVSPERs). Three IVSPERs, representing over 60kb in length, were identified in the H. didymator genome. Although they are not packaged in the virus particles, the IVSPERs can be considered functionally as an integral part of the viral genome and are amplified during virus replication. Their particular genomic organization (high coding sequence density, single-exon predicted genes) suggests that IVSPERs represent fingerprints of an ancestor virus that integrated its own DNA into the genome of an ancestor wasp. The IVSPER genes constitute a set of genes specific to ichnoviruses and conserved among ichnovirus-associated wasps, as shown for the nudivirus-related genes involved in bracovirus particle production. However, their lack of similarity with genes from known pathogenic viruses suggests that ichnoviruses originated from a virus family that has yet to be described. Altogether these recent findings show that ichnoviruses and bracoviruses derive from independent evolutionary events and represent an example of convergent evolution in the use of virus particles to transfer virulence genes into the host caterpillar.

The restricted host range of many baculoviruses, highly pathogenic to Lepidoptera and non-pathogenic to mammals, limits their use to single or few closely related Lepidopteran species and is an obstacle to extending their implementation for pest control. The insect immune response is a major determinant of the ability of an insect pathogen to efficiently multiply and propagate. We have developed an original model system to study the Lepidopteran antiviral immune response based on Spodoptera littoralis resistance to AcMNPV (Autographa californica multiple nucleopolyhedrovirus) infection and the fascinating immunosuppressive activity of polydnaviruses .Our aim is to elucidate the mechanisms through which the immunosuppressive insect polydnaviruses promote replication of pathogenic baculoviruses in lepidopteran hosts that are mildly or non-permissive to virus- replication. In this study we : 1- Assessed the extent to which and the mechanisms whereby the immunosuppressive Campoletis sonorensis polydnavirus (CsV) or its genes enhanced replication of a well-characterized pathogenic baculovirus AcMNPV, in polydnavirus-immunosuppressedH. zea and S. littoralis insects and S. littoralis cells, hosts that are mildly or non-permissive to AcMNPV. 2- Identified CsV genes involved in the above immunosuppression (e.g. inhibiting cellular encapsulation and disrupting humoral immunity). We showed that: 1. S. littoralis larvae mount an immune response against a baculovirus infection. 2. Immunosuppression of an insect pest improves the ability of a viral pathogen, the baculovirus AcMNPV, to infect the pest. 3. For the first time two PDV-specific genes of the vankyrin and cystein rich-motif families involved in immunosuppression of the host, namely Pvank1 and Hv1.1 respectively, enhanced the efficacy of an insect pathogen toward a semipermissive pest. 4. Pvank1 inhibits apoptosis of Spodopteran cells elucidating one functional aspect of PDVvankyrins. 5. That Pvank-1 and Hv1.1 do not show cooperative effect in S. littoralis when co-expressed during AcMNPV infection. Our results pave the way to developing novel means for pest control, including baculoviruses, that rely upon suppressing host immune systems by strategically weakening insect defenses to improve pathogen (i.e. biocontrol agent) infection and virulence. Also, we expect that the above result will help to develop systems for enhanced insect control that may ultimately help to reduce transmission of insect vectored diseases of humans, animals and plants as well as provide mechanisms for suppression of insect populations that damage crop plants by direct feeding.

A new abortigenic disease, now known as mare reproductive loss syndrome (MRLS), significantly affected the horse industry in the Ohio River Valley of the United States in late April and early May of 2001 and 2002. In 2001, approximately 25% of all pregnant mares aborted within several weeks (over 3,000 mares lost pregnancies), and abortion rates exceeded 60% on some farms. Mare reproductive loss syndrome struck hard and without warning, it was caused by something in the environment, it was not transmitted between animals, and it was not associated with any known abortigenic agent or disease. These experiments demonstrated that horses will inadvertently consume Eastern tent caterpillars (ETC) when the insects are present in the pasture or other feedstuffs, and MRLS-type abortions were induced in experimental animals (mares and pigs) by mixing ETC with the feed of the animals. Eastern tent caterpillars are hirsute (hairy) caterpillars, and the only part of the caterpillar that caused MRLS abortions was the cuticle. The experiments revealed that the setae (hairs) embed into the submucosa of the alimentary tract creating microgranulomatous lesions. It is hypothesized that the alimentary tract lesions allow bacteria from the alimentary tract of the mare, principally streptococci, actinobacilli, and to a lesser extent enterococci, to invade the circulatory system of the mare. The bacteria then establish infections in tissues where the immune surveillance of the mare is reduced, such as the fetus and placenta. Fetal and placental fluid bacterial infections lead to fetal death and abortion characteristic of MRLS. Inadvertent ingestion of ETC by pregnant mares causes MRLS. Currently the only known means to prevent MRLS is to avoid exposure of horses, particularly pregnant mares, to ETC and probably most hirsute caterpillars.

SUMMARY Parasitoid wasps produce virulence factors that bear significant resemblance to viruses and have the ability to block host defense responses. The function of these virulence factors, produced predominantly in wasp venom glands, and the ways in which they interfere with host development and physiology remain mysterious. Here, we report the discovery of a specialized system of canals in venom glands of five parasitoid wasps that differ in their infection strategies. This supracellular canal system is made up of individual secretory units, one per secretory cell. Individual units merge into the canal lumen. The membrane surface of the proximal end of each canal within the secretory cell assumes brush border morphology, lined with bundles of F-actin. Systemic administration of cytochalasin D compromises the integrity of the secretory unit. We show a dynamic and continuous association of p40, a protein of virus-like particles from a Drosophila parasitoid, L. heterotoma, with the canal and venom gland lumen. Similar structures in three Leptopilina species and Ganaspis xanthopoda,parasitoids of Drosophila spp., and Campoletis sonorenesis,a parasitoid of Heliothis virescens, suggest that this novel supracellular canal system is likely to be a common trait of parasitoid venom glands that is essential for efficient biogenesis and delivery of virulence factors.

The polydnaviruses represent an unusual example of a highly evolved symbiosis between some parasitic wasps, DNA containing particles or viruses, and lepidopteran larval hosts of the wasp. The viruses can no longer replicate independently, as genes that encode viral structural proteins are restricted to the wasp genome and are not encapsidated. Interestingly, the DNA that is encapsidated is more similar in terms of gene identity and gene density to eukaryotic genomes than viral genomes. We compare and relate this unusual example of natural genetic engineering to the well-known system of viral lysogeny. The similarities in the two systems may prove useful in understanding the replication strategy and genomic organization of polydnaviruses and provide some insight into how this unusual virus system may have evolved.

The Campoletis sonorensis ichnovirus ( Cs IV) vankyrin genes encode proteins containing truncated ankyrin repeat domains with sequence homology to the inhibitory domains of NF‐κB transcription factor inhibitors, IκBs. The Cs IV vankyrin proteins are thought to be involved in the suppression of NF‐κB activity during immune response and/or developmental events in the parasitized host. Here we report that when P‐vank‐1 was expressed stably from Sf9 cells, prolonged survival of these cells was observed after baculovirus infection, UV irradiation, and treatment with the apoptosis‐inducing chemical camptothecin compared to untransformed Sf9 cells. Furthermore, P‐vank‐1 inhibited nuclear and internucleosomal degradation and caspase activity after induction of apoptosis in Sf9 cells stably expressing P‐vank‐1. This is the first report of a polydnavirus protein with anti‐apoptotic function.