Chelonus inanitus bracovirus encodes lineage-specific proteins and truncated immune IκB-like factors.

Bracoviruses and ichnoviruses are endogenous viruses of parasitic wasps that produce particles containing virulence genes expressed in host tissues and necessary for parasitism success. In the case of bracoviruses the particles are produced by conserved genes of nudiviral origin integrated permanently in the wasp genome, whereas the virulence genes can strikingly differ depending on the wasp lineage. To date most data obtained on bracoviruses concerned species from the braconid subfamily of Microgastrinae. To gain a broader view on the diversity of virulence genes we sequenced the genome packaged in the particles of Chelonus inanitus bracovirus (CiBV) produced by a wasp belonging to a different subfamily: the Cheloninae. These are egg-larval parasitoids, which means that they oviposit into the host egg and the wasp larvae then develop within the larval stages of the host. We found that most of CiBV virulence genes belong to families that are specific to Cheloninae. As other bracoviruses and ichnoviruses however, CiBV encode v-ank genes encoding truncated versions of the immune cactus/IκB factor, which suggests these proteins might play a key role in host-parasite interactions involving domesticated endogenous viruses. We found that the structures of CiBV V-ANKs are different from those previously reported. Phylogenetic analysis supports the hypothesis that they may originate from a cactus/IκB immune gene from the wasp genome acquired by the bracovirus. However, their evolutionary history is different from that shared by other V-ANKs, whose common origin probably reflects horizontal gene transfer events of virus sequences between braconid and ichneumonid wasps.

Investigating bracovirus chromosomal integration and inheritance in lepidopteran host and nontarget species.

Bracoviruses (BVs) are domesticated viruses found in braconid parasitoid wasp genomes. They are composed of domesticated genes from a nudivrius, coding viral particles in which wasp DNA circles are packaged. BVs are viewed as possible vectors of horizontal transfer of genetic material (HT) from wasp to their hosts because they are injected, together with wasp eggs, by female wasps into their host larvae, and because they undergo massive chromosomal integration in multiple host tissues. Here, we show that chromosomal integrations of the Cotesia typhae BV (CtBV) persist up to the adult stage in individuals of its natural host, Sesamia nonagrioides, that survived parasitism. However, while reproducing host adults can bear an average of nearly two CtBV integrations per haploid genome, we were unable to retrieve any of these integrations in 500 of their offspring using Illumina sequencing. This suggests either that host gametes are less targeted by CtBVs than somatic cells or that gametes bearing BV integrations are nonfunctional. We further show that CtBV can massively integrate into the chromosomes of other lepidopteran species that are not normally targeted by the wasp in the wild, including one which is divergent by at least 100 million years from the natural host. Cell entry and chromosomal integration of BVs are thus unlikely to be major factors shaping wasp host range. Together, our results shed new light on the conditions under which BV-mediated wasp-to-host HT may occur and provide information that may be helpful to evaluate the potential risks of uncontrolled HT associated with the use of parasitoid wasps as biocontrol agents.

Conserved Viral Transcription Plays a Key Role in Virus-Like Particle Production of the Parasitoid Wasp Venturia canescens.

Nudiviruses are large double-stranded DNA viruses related to baculoviruses known to be endogenized in the genomes of certain parasitic wasp species. These wasp-virus associations allow the production of viral particles or virus-like particles that ensure wasp parasitism success within lepidopteran hosts. Venturia canescens is an ichneumonid wasp belonging to the Campopleginae subfamily that has endogenized nudivirus genes belonging to the Alphanudivirus genus to produce "virus-like particles" (Venturia canescens virus-like particles [VcVLPs]), which package proteic virulence factors. The main aim of this study was to determine whether alphanudivirus gene functions have been conserved following endogenization. The expression dynamics of alphanudivirus genes was monitored by a high throughput transcriptional approach, and the functional role of lef-4 and lef-8 genes predicted to encode viral RNA polymerase components was investigated by RNA interference. As described for baculovirus infections and for endogenized nudivirus genes in braconid wasp species producing bracoviruses, a transcriptional cascade involving early and late expressed alphanudivirus genes could be observed. The expression of lef-4 and lef-8 was also shown to be required for the expression of alphanudivirus late genes allowing correct particle formation. Together with previous literature, the results show that endogenization of nudiviruses in parasitoid wasps has repeatedly led to the conservation of the viral RNA polymerase function, allowing the production of viruses or viral-like particles that differ in composition but enable wasp parasitic success. IMPORTANCE This study shows that endogenization of a nudivirus genome in a Campopleginae parasitoid wasp has led to the conservation, as for endogenized nudiviruses in braconid parasitoid wasps, of the viral RNA polymerase function, required for the transcription of genes encoding viral particles involved in wasp parasitism success. We also showed for the first time that RNA interference (RNAi) can be successfully used to downregulate gene expression in this species, a model in behavioral ecology. This opens the opportunity to investigate the function of genes involved in other traits important for parasitism success, such as reproductive strategies and host choice. Fundamental data acquired on gene function in Venturia canescens are likely to be transferable to other parasitoid wasp species used in biological control programs. This study also renders possible the investigation of other nudivirus gene functions, for which little data are available.

Transposable element repression using piRNAs, and its relevance to endogenous viral elements (EVEs) and immunity in insects.

The piRNA system controls transposable element (TE) mobility by transcriptional gene silencing and post-transcriptional gene silencing. Dispersed in insect genomes, piRNA clusters contain TE copies, from which they produce piRNAs (specific small RNAs). These piRNAs can both target the nascent transcripts produced by active TE copies and directly repress them by heterochromatinization. They can also target mature transcripts and cleave them following amplification by the so-called 'ping-pong' loop mechanism. Moreover, piRNA clusters contain endogenous viral elements (EVEs), from which they produce piRNAs. The current idea is that these piRNAs could participate in the antiviral response against exogenous viral infection. In this review, we show that among insects, to date, this antiviral response by the piRNA system appears mainly restricted to mosquitoes, but this could be due to the focus of most studies on arboviruses.

Bracoviruses recruit host integrases for their integration into caterpillar's genome.

Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we find that Cotesia vestalis bracovirus (CvBV), a polydnavirus of the parasitic wasp C. vestalis (Haliday), integrates its DNA circles into host Plutella xylostella (L.) genome by two distinct strategies, conservatively and randomly, through high-throughput sequencing analysis. We confirmed that the conservatively integrating circles contain an essential "8+5" nucleotides motif which is required for integration. Then we find CvBV circles are integrated into the caterpillar's genome in three temporal patterns, the early, mid and late stage-integration. We further identify that three CvBV-encoded integrases are responsible for some, but not all of the virus circle integrations, indeed they mainly participate in the processes of early stage-integration. Strikingly, we find two P. xylostella retroviral integrases (PxIN1 and PxIN2) are highly induced upon wasp parasitism, and PxIN1 is crucial for integration of some other early-integrated CvBV circles, such as CvBV_04, CvBV_12 and CvBV_24, while PxIN2 is important for integration of a late-integrated CvBV circle, CvBV_21. Our data uncover a novel mechanism in which CvBV integrates into the infected host genome, not only by utilizing its own integrases, but also by recruiting host enzymes. These findings will strongly deepen our understanding of how bracoviruses regulate and integrate into their hosts.

Genome-Wide Patterns of Bracovirus Chromosomal Integration into Multiple Host Tissues during Parasitism.

Bracoviruses are domesticated viruses found in parasitic wasp genomes. They are composed of genes of nudiviral origin that are involved in particle production and proviral segments containing virulence genes that are necessary for parasitism success. During particle production, proviral segments are amplified and individually packaged as DNA circles in nucleocapsids. These particles are injected by parasitic wasps into host larvae together with their eggs. Bracovirus circles of two wasp species were reported to undergo chromosomal integration in parasitized host hemocytes, through a conserved sequence named the host integration motif (HIM). Here, we used bulk Illumina sequencing to survey integrations of Cotesia typhae bracovirus circles in the DNA of its host, the maize corn borer (Sesamia nonagrioides), 7 days after parasitism. First, assembly and annotation of a high-quality genome for C. typhae enabled us to characterize 27 proviral segments clustered in proviral loci. Using these data, we characterized large numbers of chromosomal integrations (from 12 to 85 events per host haploid genome) for all 16 bracovirus circles containing a HIM. Integrations were found in four S. nonagrioides tissues and in the body of a caterpillar in which parasitism had failed. The 12 remaining circles do not integrate but are maintained at high levels in host tissues. Surprisingly, we found that HIM-mediated chromosomal integration in the wasp germ line has occurred accidentally at least six times during evolution. Overall, our study furthers our understanding of wasp-host genome interactions and supports HIM-mediated chromosomal integration as a possible mechanism of horizontal transfer from wasps to their hosts. IMPORTANCE Bracoviruses are endogenous domesticated viruses of parasitoid wasps that are injected together with wasp eggs into wasp host larvae during parasitism. Several studies have shown that some DNA circles packaged into bracovirus particles become integrated into host somatic genomes during parasitism, but the phenomenon has never been studied using nontargeted approaches. Here, we use bulk Illumina sequencing to systematically characterize and quantify bracovirus circle integrations that occur in four tissues of the Mediterranean corn borer (Sesamia nonagrioides) during parasitism by the Cotesia typhae wasp. Our analysis reveals that all circles containing a HIM integrate at substantial levels (from 12 to 85 integrations per host cell, in total) in all tissues, while other circles do not integrate. In addition to shedding new light on wasp-bracovirus-host interactions, our study supports HIM-mediated chromosomal integration of bracovirus as a possible source of wasp-to-host horizontal transfer, with long-term evolutionary consequences.

Chromosomal scale assembly of parasitic wasp genome reveals symbiotic virus colonization.

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

Salivary proteins of Phloeomyzus passerinii, a plant-manipulating aphid, and their impact on early gene responses of susceptible and resistant poplar genotypes.

Successful plant colonization by parasites requires the circumvention of host defenses, and sometimes a reprogramming of host metabolism, mediated by effector molecules delivered into the host. Using transcriptomic and enzymatic approaches, we characterized salivary glands and saliva of Phloeomyzus passerinii, an aphid exhibiting an atypical feeding strategy. Plant responses to salivary extracts of P. passerinii and Myzus persicae were assessed with poplar protoplasts of a susceptible and a resistant genotype, and in a heterologous Arabidopsis system. We predict that P. passerinii secretes a highly peculiar saliva containing effectors potentially interfering with host defenses, biotic stress signaling and plant metabolism, notably phosphatidylinositol phosphate kinases which seemed specific to P. passerinii. Gene expression profiles indicated that salivary extracts of M. persicae markedly affected host defenses and biotic stress signaling, while salivary extracts of P. passerinii induced only weak responses. The effector-triggered susceptibility was characterized by downregulations of genes involved in cytokinin signaling and auxin homeostasis. This suggests that P. passerinii induces an intracellular accumulation of auxin in susceptible host genotypes, which is supported by histochemical assays in Arabidopsis. This might in turn affect biotic stress signaling and contribute to host tissue manipulation by the aphid.

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Gall Wasp Transcriptomes Unravel Potential Effectors Involved in Molecular Dialogues With Oak and Rose.

To gain insight into wasp factors that might be involved in the initial induction of galls on woody plants, we performed high throughput (454) transcriptome analysis of ovaries and venom glands of two cynipid gall wasps, Biorhiza pallida and Diplolepis rosae, inducing galls on oak and rose, respectively. De novo assembled and annotated contigs were compared to sequences from phylogenetically related parasitoid wasps. The relative expression levels of contigs were estimated to identify the most expressed gene sequences in each tissue. We identify for the first time a set of maternally expressed gall wasp proteins potentially involved in the interaction with the plant. Some genes highly expressed in venom glands and ovaries may act to suppress early plant defense signaling. We also identify gall wasp cellulases that could be involved in observed local lysis of plant tissue following oviposition, and which may have been acquired from bacteria by horizontal gene transfer. We find no evidence of virus-related gene expression, in contrast to many non-cynipid parasitoid wasps. By exploring gall wasp effectors, this study is a first step toward understanding the molecular mechanisms underlying cynipid gall induction in woody plants, and the recent sequencing of oak and rose genomes will enable study of plant responses to these factors.

Cotesia congregata Bracovirus Circles Encoding PTP and Ankyrin Genes Integrate into the DNA of Parasitized Manduca sexta Hemocytes.

Polydnaviruses (PDVs) are essential for the parasitism success of tens of thousands of species of parasitoid wasps. PDVs are present in wasp genomes as proviruses, which serve as the template for the production of double-stranded circular viral DNA carrying virulence genes that are injected into lepidopteran hosts. PDV circles do not contain genes coding for particle production, thereby impeding viral replication in caterpillar hosts during parasitism. Here, we investigated the fate of PDV circles of Cotesia congregata bracovirus during parasitism of the tobacco hornworm, Manduca sexta, by the wasp Cotesia congregata Sequences sharing similarities with host integration motifs (HIMs) of Microplitis demolitor bracovirus (MdBV) circles involved in integration into DNA could be identified in 12 CcBV circles, which encode PTP and VANK gene families involved in host immune disruption. A PCR approach performed on a subset of these circles indicated that they persisted in parasitized M. sexta hemocytes as linear forms, possibly integrated in host DNA. Furthermore, by using a primer extension capture method based on these HIMs and high-throughput sequencing, we could show that 8 out of 9 circles tested were integrated in M. sexta hemocyte genomic DNA and that integration had occurred specifically using the HIM, indicating that an HIM-mediated specific mechanism was involved in their integration. Investigation of BV circle insertion sites at the genome scale revealed that certain genomic regions appeared to be enriched in BV insertions, but no specific M. sexta target site could be identified.IMPORTANCE The identification of a specific and efficient integration mechanism shared by several bracovirus species opens the question of its role in braconid parasitoid wasp parasitism success. Indeed, results obtained here show massive integration of bracovirus DNA in somatic immune cells at each parasitism event of a caterpillar host. Given that bracoviruses do not replicate in infected cells, integration of viral sequences in host DNA might allow the production of PTP and VANK virulence proteins within newly dividing cells of caterpillar hosts that continue to develop during parasitism. Furthermore, this integration process could serve as a basis to understand how PDVs mediate the recently identified gene flux between parasitoid wasps and Lepidoptera and the frequency of these horizontal transfer events in nature.

Impact of Lateral Transfers on the Genomes of Lepidoptera.

Transfer of DNA sequences between species regardless of their evolutionary distance is very common in bacteria, but evidence that horizontal gene transfer (HGT) also occurs in multicellular organisms has been accumulating in the past few years. The actual extent of this phenomenon is underestimated due to frequent sequence filtering of "alien" DNA before genome assembly. However, recent studies based on genome sequencing have revealed, and experimentally verified, the presence of foreign DNA sequences in the genetic material of several species of Lepidoptera. Large DNA viruses, such as baculoviruses and the symbiotic viruses of parasitic wasps (bracoviruses), have the potential to mediate these transfers in Lepidoptera. In particular, using ultra-deep sequencing, newly integrated transposons have been identified within baculovirus genomes. Bacterial genes have also been acquired by genomes of Lepidoptera, as in other insects and nematodes. In addition, insertions of bracovirus sequences were present in the genomes of certain moth and butterfly lineages, that were likely corresponding to rearrangements of ancient integrations. The viral genes present in these sequences, sometimes of hymenopteran origin, have been co-opted by lepidopteran species to confer some protection against pathogens.

Endogenous viruses of parasitic wasps: variations on a common theme.

In a remarkable example of convergent evolution, several parasitic wasp lineages have independently captured and maintained complex DNA virus machineries, used to transfer virulence factors. Parasitic wasps, which develop inside the larvae of their insect hosts, may inject Polydnaviruses (PDVs) or Virus-Like particles (VLPs), derived from the recurrent endogenization of several large DNA viruses. PDVs evolved from the domestication in braconid and ichneumonid wasps of viruses from different families and function as gene transfer agents. In contrast, the independent domestication of nudiviruses led to the evolution of both PDV and VLP strategies. In Venturia canescens, the endogenous nudivirus has lost the ability to encapsidate DNA, instead VLPs cargo virulence molecules of wasp origin to the parasitized host.

Dynamics and origin of cytokinins involved in plant manipulation by a leaf-mining insect.

Several herbivorous insects and plant-associated microorganisms control the phytohormonal balance, thus enabling them to successfully exploit the plant by inhibiting plant defenses and withdrawing plant resources for their own benefit. The leaf-mining moth Phyllonorycter blancardella modifies the cytokinin (CK) profile of mined leaf-tissues, and the insect symbiotic bacteria Wolbachia is involved in the plant manipulation to the benefit of the insect host. To gain a deeper understanding into the possible origin and dynamics of CKs, we conducted an extensive characterization of CKs in larvae and in infected apple leaves. Our results show the enhanced CK levels in mines, both on green and yellow leaves, allowing insects to control their nutritional supply under fluctuating environmental conditions. The spatial distribution of CKs within the mined leaves shows that hormone manipulation is strictly limited to the mine suggesting the absence of CK translocation from distant leaf areas toward the insect feeding site. Mass spectrometry analyses reveal that major CK types accumulating in mines and larvae are similar to what is observed for most gall-inducers, suggesting that strategies underlying the plant manipulation may be shared between herbivorous insects with distinct life histories. Results further show that CKs are detected in the highest levels in larvae, reinforcing our hypothesis that CKs accumulating in the mines originate from the insect itself. Presence of bacteria-specific methylthio-CKs is consistent with previous results suggesting that insect bacterial symbionts contribute to the observed phenotype. Our study provides key findings toward the understanding of molecular mechanisms underlying this intricate plant-insect-microbe interaction.

Could Preoperative Neck Anatomy Influence Follow-up of EVAR?

BACKGROUND: The aim of the study was to assess the clinical utility of strict CT scan surveillance after endovascular abdominal aneurysm repair (EVAR) and evaluate whether the anatomy of abdominal aortic aneurysm (AAA) neck (favorable/hostile) influences regular imaging control. METHODS: A retrospective study of AAA patients who underwent EVAR with aortobi-iliac endoprostheses during 2006-2013 was conducted. Exclusion criteria included other types of devices. Variables analyzed were technical and clinical success, morbimortality, complications (such as endoleaks, sac enlargement), reinterventions, reintervention-free survival, and survival rate. Preoperative CT scans were performed and repeated at 1, 6 (in selective cases), 12, and 24 months postoperatively. Patients were divided into two groups according to preoperative anatomic characteristics: group I (favorable neck) and group II (hostile neck: angle > 60°, length < 15 mm, diameter > 28 mm, and calcification or circumference thrombus ≥50%). RESULTS: A total of 127 patients with AAA (96.8% male) were included in the study. The mean age of the patients was 75.9 years (range: 51-90 years). The mean AAA diameter was 62.1 mm. Hostile neck was found in 52 patients (40.9%). The technical and clinical success rate was 100% and 30-day mortality was 0.8%. The reintervention-free survival rate was 97.6%, 96.1%, and 93.7% and the survival rate was 97.6%, 96.9%, and 91.3%, during follow-up at 6, 12, and 24 months, respectively. Accumulated complications in proximal sealing occurred in 0%, 0%, and 1.6% in group I and 1.9%, 6.1%, and 7.7% in group II at 1, 12, and 24 months, respectively. Type II endoleaks occurred in 24.3%, 14.3%, and 11.4% in group I and 9.8%, 6.3%, and 6.8% in group II at 1, 12, and 24 months, respectively. No increased diameter was detected at 6 and 12 months. No differences were observed in reinterventions and mortality rate depending on anatomy. CONCLUSIONS: CT scans performed at 6 and 12 months postoperatively did not detect complications or need for reintervention in patients with favorable necks, even in the presence of endoleaks type II, and could therefore be omitted. Hostile necks may compromise proximal sealing and require regular imaging follow-ups.

Folivory elicits a strong defense reaction in Catharanthus roseus: metabolomic and transcriptomic analyses reveal distinct local and systemic responses.

Plants deploy distinct secondary metabolisms to cope with environment pressure and to face bio-aggressors notably through the production of biologically active alkaloids. This metabolism-type is particularly elaborated in Catharanthus roseus that synthesizes more than a hundred different monoterpene indole alkaloids (MIAs). While the characterization of their biosynthetic pathway now reaches completion, still little is known about the role of MIAs during biotic attacks. As a consequence, we developed a new plant/herbivore interaction system by challenging C. roseus leaves with Manduca sexta larvae. Transcriptomic and metabolic analyses demonstrated that C. roseus respond to folivory by both local and systemic processes relying on the activation of specific gene sets and biosynthesis of distinct MIAs following jasmonate production. While a huge local accumulation of strictosidine was monitored in attacked leaves that could repel caterpillars through its protein reticulation properties, newly developed leaves displayed an increased biosynthesis of the toxic strictosidine-derived MIAs, vindoline and catharanthine, produced by up-regulation of MIA biosynthetic genes. In this context, leaf consumption resulted in a rapid death of caterpillars that could be linked to the MIA dimerization observed in intestinal tracts. Furthermore, this study also highlights the overall transcriptomic control of the plant defense processes occurring during herbivory.

Insect-induced effects on plants and possible effectors used by galling and leaf-mining insects to manipulate their host-plant.

Gall-inducing insects are iconic examples in the manipulation and reprogramming of plant development, inducing spectacular morphological and physiological changes of host-plant tissues within which the insect feeds and grows. Despite decades of research, effectors involved in gall induction and basic mechanisms of gall formation remain unknown. Recent research suggests that some aspects of the plant manipulation shown by gall-inducers may be shared with other insect herbivorous life histories. Here, we illustrate similarities and contrasts by reviewing current knowledge of metabolic and morphological effects induced on plants by gall-inducing and leaf-mining insects, and ask whether leaf-miners can also be considered to be plant reprogrammers. We review key plant functions targeted by various plant reprogrammers, including plant-manipulating insects and nematodes, and functionally characterize insect herbivore-derived effectors to provide a broader understanding of possible mechanisms used in host-plant manipulation. Consequences of plant reprogramming in terms of ecology, coevolution and diversification of plant-manipulating insects are also discussed.

Leaf-mining by Phyllonorycter blancardella reprograms the host-leaf transcriptome to modulate phytohormones associated with nutrient mobilization and plant defense.

Phytohormones have long been hypothesized to play a key role in the interactions between plant-manipulating organisms and their host-plants such as insect-plant interactions that lead to gall or 'green-islands' induction. However, mechanistic understanding of how phytohormones operate in these plant reconfigurations is lacking due to limited information on the molecular and biochemical phytohormonal modulation following attack by plant-manipulating insects. In an attempt to fill this gap, the present study provides an extensive characterization of how the leaf-miner Phyllonorycter blancardella modulates the major phytohormones and the transcriptional activity of plant cells in leaves of Malus domestica. We show here, that cytokinins strongly accumulate in mined tissues despite a weak expression of plant cytokinin-related genes. Leaf-mining is also associated with enhanced biosynthesis of jasmonic acid precursors but not the active form, a weak alteration of the salicylic acid pathway and a clear inhibition of the abscisic acid pathway. Our study consolidates previous results suggesting that insects may produce and deliver cytokinins to the plant as a strategy to manipulate the physiology of the leaf to create a favorable nutritional environment. We also demonstrate that leaf-mining by P. blancardella leads to a strong reprogramming of the plant phytohormonal balance associated with increased nutrient mobilization, inhibition of leaf senescence and mitigation of plant direct and indirect defense.

Recurrent Domestication by Lepidoptera of Genes from Their Parasites Mediated by Bracoviruses.

Bracoviruses are symbiotic viruses associated with tens of thousands of species of parasitic wasps that develop within the body of lepidopteran hosts and that collectively parasitize caterpillars of virtually every lepidopteran species. Viral particles are produced in the wasp ovaries and injected into host larvae with the wasp eggs. Once in the host body, the viral DNA circles enclosed in the particles integrate into lepidopteran host cell DNA. Here we show that bracovirus DNA sequences have been inserted repeatedly into lepidopteran genomes, indicating this viral DNA can also enter germline cells. The original mode of Horizontal Gene Transfer (HGT) unveiled here is based on the integrative properties of an endogenous virus that has evolved as a gene transfer agent within parasitic wasp genomes for ≈100 million years. Among the bracovirus genes thus transferred, a phylogenetic analysis indicated that those encoding C-type-lectins most likely originated from the wasp gene set, showing that a bracovirus-mediated gene flux exists between the 2 insect orders Hymenoptera and Lepidoptera. Furthermore, the acquisition of bracovirus sequences that can be expressed by Lepidoptera has resulted in the domestication of several genes that could result in adaptive advantages for the host. Indeed, functional analyses suggest that two of the acquired genes could have a protective role against a common pathogen in the field, baculovirus. From these results, we hypothesize that bracovirus-mediated HGT has played an important role in the evolutionary arms race between Lepidoptera and their pathogens.