Multigenic Families in Ichnovirus: A Tissue and Host Specificity Study through Expression Analysis of Vankyrins from Hyposoter didymator Ichnovirus

The viral ankyrin (vankyrin) gene family is represented in all polydnavirus (PDVs) genomes and encodes proteins homologous to I-kappaBs, inhibitors of NF-kappaB transcription factors. The structural similarities led to the hypothesis that vankyrins mimic eukaryotic factors to subvert important physiological pathways in the infected host. Here, we identified nine vankyrin genes in the genome of the Hyposoter didymator Ichnovirus (HdIV). Time-course gene expression experiments indicate that all members are expressed throughout parasitism of Spodoptera frugiperda, as assessed using RNA extracted from whole larvae. To study tissue and/or species specificity transcriptions, the expression of HdIV vankyrin genes were compared between HdIV-injected larvae of S. frugiperda and S. littoralis. The transcriptional profiles were similar in the two species, including the largely predominant expression of Hd27-vank1 in all tissues examined. However, in various insect cell lines, the expression patterns of HdIV vankyrins differed according to species. No clear relationship between vankyrin expression patterns and abundance of vankyrin-bearing genomic segments were found in the lepidopteran cell lines. Moreover, in these cells, the amount of vankyrin-bearing genomic segments differed substantially between cytosol and nuclei of infected cells, implying the existence of an unexpected step regulating the copy number of HdIV segments in cell nuclei. Our in vitro results reveal a host-specific transcriptional profile of vankyrins that may be related to the success of parasitism in different hosts. In Spodoptera hosts, the predominant expression of Hd27-vank1 suggests that this protein might have pleiotropic functions during parasitism of these insect species

Characterization and complementarity of the virulence factors in the ichneumonid wasp Hyposoter didymator

Les Hyménoptères parasitoïdes ont un développement larvaire s'effectuant au détriment d'un organisme hôte. Pour exploiter au mieux la ressource que représente un hôte arthropode dont la biologie peut présenter certains obstacles tels que la mobilité et le système immunitaire, les parasitoïdes ont développé une diversité modes de vie et de stratégies de virulence. Ce manuscrit replace les parasitoïdes dans leur contexte évolutif afin de mieux comprendre la diversité surprenante de leurs modes de vie. Ces modes de vie conditionnent la nature des interactions dans les systèmes hôte/parasitoïde. Nous verrons comment, par l'utilisation de nombreux facteurs de virulence tel que le venin, les polydnavirus et bien d'autres encore, les parasitoïdes manipulent la physiologie de leur hôte afin de le rendre adéquat à leur propre développement. Ce travail s'est intéressé au modèle endoparasitoïde Hyposoter didymator (Hym., Ichneumonidae). Nous avons ainsi caractérisé les protéines produites dans la glande à venin des femelles et identifié l'ensemble des gènes du polydnavirus associé (HdIV; H. didymator Ichnovirus), grâce à des techniques de protéomique, génomique et transcriptomique. Nous avons également suivi et quantifié les altérations de la physiologie de l'hôte Spodoptera frugiperda au cours du parasitisme et évalué le rôle relatif de différents facteurs dans ces perturbations et dans la réussite parasitaire. Nos résultats ont permis de montrer que seul le fluide du calice contenant HdIV est nécessaire au développement du parasitoïde. En parallèle, nous avons mis à jour une propriété immuno-évasive des œufs d'H. didymator liée à des protéines associées à l'exochorion. L'ensemble de ce travail a permis de dessiner un élégant schéma expliquant la complémentarité spatio-temporelle des facteurs de virulence durant le parasitisme. Finalement, nous avons cherché à mieux comprendre le déterminisme du spectre d'hôte d'H. didymator, ce qui nous a conduit à montrer que les deux stratégies de contournement de la réponse immunitaire (immuno-évasion et infection virale) se révèlent inefficaces chez les hôtes non-permissifs.Parasitic wasps must deal with physiological features of their host such as mobility, an efficient immune system and a variable metabolism. To ensure successful parasitism in a large range of arthropod hosts, parasitoids display a huge diversity of lifestyle and rely in a variety of virulence factors. In this document, we introduce parasitoid lifestyle in an evolutionary context in order to better understand the parasitoid complexity. As the parasitoid lifestyle drives the host/parasitoid interaction outcome, we discuss for all how the virulence factors such as venom, polydnaviruses and many others are used to ensure successful development of the parasitoid. In this study, we focused on the endoparasitoid Hyposoter didymator (Hym., Ichneumonidae) virulence factors. We thus identified venom proteins and the genes from the associated polydnavirus, HdIV using proteomics, genomics and transcriptomics approaches. Studies on the effect of the venom and the calyx fluid containing the polydnavirus HdIV, reveal that only the calyx fluid is necessary for Spodoptera frugiperda host physiological alteration and parasitism success. Futhermore, this work presents the discovery of a local immune-evasive property of the H. didymator egg exochorion. All these data permitted us to design an effective spatio-temporal model of the virulence factor complementarity used by H. didymator during the parasitism time course. Finally, studies on the H. didymator host range reveals the inefficiency of the different virulence factors in non-permissive hosts, opening insights on the host permissiveness molecular mechanisms

Extensive transcription analysis of the Hyposoter didymator Ichnovirus genome in permissive and non-permissive lepidopteran host species.

Ichnoviruses are large dsDNA viruses that belong to the Polydnaviridae family. They are specifically associated with endoparasitic wasps of the family Ichneumonidae and essential for host parasitization by these wasps. We sequenced the Hyposoter didymator Ichnovirus (HdIV) encapsidated genome for further analysis of the transcription pattern of the entire set of HdIV genes following the parasitization of four different lepidopteran host species. The HdIV genome was found to consist of at least 50 circular dsDNA molecules, carrying 135 genes, 98 of which formed 18 gene families. The HdIV genome had general features typical of Ichnovirus (IV) genomes and closely resembled that of the IV carried by Hyposoter fugitivus. Subsequent transcriptomic analysis with Illumina technology during the course of Spodoptera frugiperda parasitization led to the identification of a small subset of less than 30 genes with high RPKM values in permissive hosts, consisting with these genes encoding crucial virulence proteins. Comparisons of HdIV expression profiles between host species revealed differences in transcript levels for given HdIV genes between two permissive hosts, S. frugiperda and Pseudoplusia includens. However, we found no evident intrafamily gene-specific transcription pattern consistent with the presence of multigenic families within IV genomes reflecting an ability of the wasps concerned to exploit different host species. Interestingly, in two non-permissive hosts, Mamestra brassiccae and Anticarsia gemmatalis (most of the parasitoid eggs were eliminated by the host cellular immune response), HdIV genes were generally less strongly transcribed than in permissive hosts. This suggests that successful parasitism is dependent on the expression of given HdIV genes exceeding a particular threshold value. These results raise questions about the mecanisms involved in regulating IV gene expression according to the nature of the lepidopteran host species encountered

The HdIV transcriptome in different lepidopteran host species (6 h p.p.).

<p>RPKM levels are indicated for genes belonging to the <i>Vank</i> family (<b>A</b>), the <i>Cys-motif</i> family (<b>B</b>), the <i>Vinx</i> family (<b>C</b>), the <i>PRRP</i> family (<b>D</b>), the <i>Rep</i> family (<b>E</b>), the <i>N-gene</i> family (<b>F</b>), the <i>GlyPro</i> family (<b>G)</b>, the known single-copy gene (<b>H</b>), the F1 to F11 family (<b>I</b>) and the newly characterized single-copy gene (<b>J</b>). For each HdIV gene, different lowercase letters indicate a significant difference (<i>p</i><0.01).</p

Graphic representation of the HdIV genome and transcribed regions.

<p>The 50 HdIV segments are shown in linear form, from the smallest (Hd45, 2548 bp) to the largest (Hd1, 36006 bp). Pairs of HdIV segments with similar sequences were designated, the two segments of each pair being named a and b. Colored boxes show the open reading frames (ORFs). Refer to the legend for color correspondences. Introns within ORFs are indicated by a line between two colored boxes (corresponding to exons). A “transcript coverage curve” is shown above each HdIV segment (i.e. the number of Illumina reads mapping to the segment sequence; for the sake of simplicity, only data from one of the 3 “72 h p.p.” replicates were used to draw the curve). Provisional GenBank accession numbers: Hd45:KJ586284; Hd44:KJ586285; Hd43:KJ586286; Hd42:KJ586287; Hd41:KJ586288; Hd40:KJ586289; Hd39: KJ586290; Hd38:KJ586291; Hd37:KJ586292; Hd36:KJ586293; Hd35:KJ586294; Hd34:KJ586295; Hd33: KJ586296; Hd32:KJ586298, Hd31:KJ586299; Hd30:KJ586300; Hd29:KJ586303; Hd28:KJ586304; Hd27:KJ586305; Hd26b:KJ586306; Hd26a:KJ586301; Hd25:KJ586307; Hd24:KJ586308; Hd23:KJ586309; Hd22:KJ586310; Hd21:KJ586311; Hd20b:KJ586297; Hd20a:KJ586312; Hd19:KJ586313; Hd18:KJ586315; Hd17b:KJ586316; Hd17a:KJ586314; Hd16:KJ586317; Hd15:KJ586318; Hd14:KJ586319; Hd13:KJ586320; Hd12:KJ586321; Hd11b:KJ586302; Hd11a:KJ586322; Hd10:KJ586323; Hd9:KJ586324; Hd8:KJ586325; Hd7:KJ586326; Hd6:KJ586328; Hd5:KJ586329; Hd4:KJ586330; Hd3:KJ586331; Hd2b:KJ586327; Hd2a:KJ586332; Hd1:KJ586333.</p

Profile of HdIV transcript levels during the time-course of <i>S. frugiperda</i> parasitization.

<p>Each row represents the mean RPKM value for each of the 135 HdIV genes analyzed in <i>S. frugiperda</i> at 6 h, 24 h and 72 h p.p. The color scale (black to yellow) indicates RPKM level. The RPKM values were used to cluster genes into classes (A, B and C) with the AutoClass algorithm available from the <a href="mailto:AutoClass@IJM" target="_blank">AutoClass@IJM</a> website. Asterisks indicate significant (<i>p</i>>0.05) RPKM fold-changes with respect to the arbitrary chosen reference (<i>Sf</i> 6 h p.p. sample). White asterisks indicate a significant decrease and black asterisks indicate a significant increase in transcript levels (fold-changes ranging from 2 to 67).</p

Encapsulation response to parasitization by <i>H. didymator</i> in four lepidopteran host species.

<p><b>A</b>. Percentage of larvae from which <i>H. didymator</i> eggs were recovered following host dissection 12–36 h p.p. <b>B. and C</b>. Examples of <i>H. didymator</i> eggs recovered between 12–36 h p.p from a permissive host (<i>S. frugiperda</i> or <i>P. includens</i>) and a non-permissive host (<i>M. brassicae</i> or <i>A. gemmatalis</i>), respectively. <b>D</b>. Percentage of <i>H. didymator</i> larvae recovered following host dissection 72 h p.p. <b>E. and F</b>. Example of an <i>H. didymator</i> larva recovered at 72 h p.p. from a permissive host and a non-permissive host, respectively. Note that, in <b>C</b>. and <b>F</b>., a layer of host immune cells surrounds the parasitoid; this is known as encapsulation. In <b>A</b>. and <b>D</b>., different lowercase letters indicate significantly different (<i>p</i><0.05) results. <b>G</b>. Weight of lepidopteran larvae at 3 times p.p. depending on the species. C: control host larvae, P: host larvae parasitized by <i>H. didymator</i>. Asterisks indicate that, for one species and one time, body weight differed significantly between control and parasitized hosts (<i>p</i><0.05). Sf: <i>S. frugiperda</i>, Pi: <i>P. includens</i>, Mb: M. <i>brassicae</i> and Ag: <i>A. gemmatalis</i>.</p