Permissiveness of lepidopteran hosts is linked to differential expression of bracovirus genes.

Polydnaviruses in the genus Bracovirus (BV) are associated with parasitoid wasps in the family Braconidae. BV-carrying wasps rely on their associated viruses to parasitize permissive hosts but also occasionally oviposit into host species that are non-permissive. Here, we studied Microplitis demolitor and M. demolitor bracovirus (MdBV) in Chrysodeixis includens, a permissive host, and Trichoplusia ni, which is usually non-permissive. M. demolitor laid eggs and injected MdBV into both hosts but almost no wasp offspring developed in T. ni. MdBV DNA similarly persisted in both host species, but deep sequencing data showed that transcript abundance for most viral genes was higher in C. includens than T. ni. Overall, our results identify lower expression of MdBV genes as an important factor in the non-permissiveness of T. ni. However, certain genes with functions in immunosuppression were sufficiently expressed to have similar effect in T. ni and C. includens.

Polydnavirus Ank proteins bind NF-κB homodimers and inhibit processing of Relish.

Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-κB (NF-κB) transcription factors, and inhibitor κB (IκB) proteins that complex with and regulate NF-κBs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs). Most PDVs encode ank genes that share features with IκBs, while our own prior studies suggested that two ank family members from Microplitis demolitor bracovirus (MdBV) (Ank-H4 and Ank-N5) behave as IκB mimics. However, the binding affinities of these viral mimics for Rel proteins relative to endogenous IκBs remained unclear. Surface plasmon resonance (SPR) and co-immunoprecipitation assays showed that the IκB Cactus from Drosophila bound Dif and Dorsal homodimers more strongly than Relish homodimers. Ank-H4 and -N5 bound Dif, Dorsal and Relish homodimers with higher affinity than the IκB domain of Relish (Rel-49), and also bound Relish homodimers more strongly than Cactus. Ank-H4 and -N5 inhibited processing of compound Relish and reduced the expression of several antimicrobial peptide genes regulated by the Imd signaling pathway in Drosophila mbn2 cells. Studies conducted in the natural host Pseudoplusia includens suggested that parasitism by M. demolitor also activates NF-κB signaling and that MdBV inhibits this response. Overall, our data provide the first quantitative measures of insect and viral IκB binding affinities, while also showing that viral mimics disable Relish processing.

The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens.

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism.

Transcriptomic profiling of Microplitis demolitor bracovirus reveals host, tissue and stage-specific patterns of activity.

The polydnaviruses (PDVs) are a family of DNA viruses that are symbiotically associated with parasitoid wasps. The transcription of particular genes or gene-family members have been reported for several PDVs, but no studies have characterized the spatio-temporal patterns of expression for the entire complement of predicted genes in the encapsidated genome of any PDV isolate. The braconid wasp Microplitis demolitor carries the PDV Microplitis demolitor bracovirus (MdBV) and parasitizes larval stage Pseudoplusia (Chrysodeixis) includens. The encapsidated genome consists of 15 genomic segments with 51 predicted ORFs encoding proteins ≥100 aa. A majority of these ORFs form four multimember gene families (ptp, ank, glc and egf) while the remaining ORFs consist of single copy (orph) genes. Here we used RT-PCR and quantitative real-time PCR methods to profile the encapsidated transcriptome of MdBV in P. includens and M. demolitor. Our results indicate that most predicted genes are expressed in P. includens. Spatial patterns of expression in P. includens differed among genes, but temporal patterns of expression were generally similar, with transcript abundance progressively declining between 24 and 120 h. A subset of ptp, ank and orph genes were also expressed in adult female but not male M. demolitor. Only one encapsidated gene (ank-H4) was expressed in all life stages of M. demolitor, albeit at much lower levels than in P. includens. However, another encapsidated gene (orph-B1) was expressed in adult M. demolitor at similar levels to those detected in P. includens.

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design.

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

The UGA-CiE1 cell line from Chrysodeixis includens exhibits characteristics of granulocytes and is permissive to infection by two viruses.

The soybean looper, Chrysodeixis (Pseudoplusia) includens (Lepidoptera: Noctuidae) is an economically important insect pest and a highly permissive host for the parasitoid Microplitis demolitor and its associated polydnavirus M. demolitor bracovirus (MdBV). Here we established a cell line from C. includens embryos designated UGA-CiE1 cells. CiE1 cells morphologically resemble granulocytes, which are a subpopulation of C. includens hemocytes. Antibody and RT-PCR analyses indicated that CiE1 cells express several molecular and functional markers that identify granulocytes. We further determined that CiE1 cells are permissive to infection by MdBV, exhibiting alterations very similar to MdBV-infected granulocytes, and Autographa californica multiple nucleopolyhedrosis virus (AcMNPV). Combined with the ability to transfect CiE1 cells with high efficiency and knock down expression of viral genes by RNA interference, we conclude this cell line has several attributes of value for studying immune interactions with polydnaviruses and potentially other pathogens.

The members of bHLH transcription factor superfamily are required for female reproduction in the red flour beetle, Tribolium castaneum.

Proteins containing the basic Helix-Loop-Helix (bHLH) domain function as transcription factors and play important roles during the development of various metazoans including insects, nematodes and vertebrates. Insect genomes contain more than 50 bHLH transcription factors, but the function of only a few of these proteins in regulation of female reproduction is known. Using RNA interference, we have tested knock-down in the expression of genes coding for bHLH transcription factors in newly emerged adult females to determine their function in regulation of female reproduction in the red flour beetle, Tribolium castaneum. Knock-down in the expression of genes coding for four bHLH transcription factors (TcSRC, TcSim1, TcAsh and TcDaughterless) caused mortality in the female beetles. In addition, knocking-down the expression of 16 bHLH genes affected oogenesis and knock-down in the expression of 13 genes affected embryogenesis. Two genes TcSide1 and TcSpineless are required for both oogenesis and embryogenesis. Thus, the data reported here showed that 31 bHLH transcription factors are required for female survival, reproduction and embryogenesis.

Functional characterization of PAS and HES family bHLH transcription factors during the metamorphosis of the red flour beetle, Tribolium castaneum.

The basic helix-loop-helix transcription factors are present in animals, plants and fungi and play important roles in the control of cellular proliferation, tissue differentiation, development and detoxification. Although insect genomes contain more than 50 helix-loop-helix transcription factors, the functions of only a few are known. RNAi has become a widely used tool to knock-down the expression to analyze the function of genes. As RNAi works well in Tribolium castaneum, we utilized this insect and RNAi to determine functions of 19 bHLH transcription factors belonging to PAS and HES families during the larval stages of the red flour beetle, T. castaneum. We searched the genome sequence of T. castaneum and identified 53 bHLH genes. Phylogenetic analyses classified these 53 genes into ten families; PAS, HES, Myc/USF, Hand, Mesp, Shout, p48, NeuroD/Neurogenin, Atonal and AS-C. In RNAi studies, knocking-down the expression of seven members of the PAS and HES families affected the growth and development of T. castaneum. An inability to grow to reach critical weight to undergo metamorphosis, failure to complete larval-pupal or pupal-adult ecdysis and abnormal wing development are among the most common phenotypes observed in RNAi insects. Among the bHLH transcription factors studied, the steroid receptor coactivator (SRC) showed the most severe phenotypes. Knock-down in the expression of the gene coding for SRC caused growth arrest by affecting the regulation of lipid metabolism. These studies demonstrate the power of RNAi for functional characterization of members of the multigene families in this model insect.

Interaction of proteins involved in ecdysone and juvenile hormone signal transduction.

Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.