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.

Effector proteins play crucial roles in plant-parasite interactions by suppressing plant defenses and hijacking plant physiological responses to facilitate parasite invasion and propagation. Although effector proteins have been characterized in many microbial plant pathogens, their nature and role in adaptation to host plants are largely unknown in insect herbivores. Aphids rely on salivary effector proteins injected into the host plants to promote phloem sap uptake. Therefore, gaining insight into the repertoire and evolution of aphid effectors is key to unveiling the mechanisms responsible for aphid virulence and host plant specialization. With this aim in mind, we assembled catalogues of putative effectors in the legume specialist aphid, Acyrthosiphon pisum, using transcriptomics and proteomics approaches. We identified 3,603 candidate effector genes predicted to be expressed in A. pisum salivary glands (SGs), and 740 of which displayed up-regulated expression in SGs in comparison to the alimentary tract. A search for orthologs in 17 arthropod genomes revealed that SG-up-regulated effector candidates of A. pisum are enriched in aphid-specific genes and tend to evolve faster compared with the whole gene set. We also found that a large fraction of proteins detected in the A. pisum saliva belonged to three gene families, of which certain members show evidence consistent with positive selection. Overall, this comprehensive analysis suggests that the large repertoire of effector candidates in A. pisum constitutes a source of novelties promoting plant adaptation to legumes.

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.

Effector proteins play crucial roles in determining the outcome of various plant-parasite interactions. Aphids inject salivary effector proteins into plants to facilitate phloem feeding, but some proteins might trigger defense responses in certain plants. The pea aphid, Acyrthosiphon pisum, forms multiple biotypes, and each biotype is specialized to feed on a small number of closely related legume species. Interestingly, all the previously identified biotypes can feed on Vicia faba; hence, it serves as a universal host plant of A. pisum. We hypothesized that the salivary effector proteins have a key role in determining the compatibility between specific host species and A. pisum biotypes and that each biotype produces saliva containing a specific mixture of effector proteins due to differential expression of encoding genes. As the first step to address these hypotheses, we conducted two sets of RNA-seq experiments. RNA-seq analysis of dissected salivary glands (SGs) from reference alfalfa- and pea-specialized A. pisum lines revealed common and line-specific repertoires of candidate salivary effector genes. Based on the results, we created an extended catalogue of A. pisum salivary effector candidates. Next, we used aphid head samples, which contain SGs, to examine biotype-specific expression patterns of candidate salivary genes. RNA-seq analysis of head samples of alfalfa- and pea-specialized biotypes, each represented by three genetically distinct aphid lines reared on either a universal or specific host plant, showed that a majority of the candidate salivary effector genes was expressed in both biotypes at a similar level. Nonetheless, we identified small sets of genes that were differentially regulated in a biotype-specific manner. Little host plant effect (universal vs. specific) was observed on the expression of candidate salivary genes. Analysis of previously obtained genome re-sequenced data of the two biotypes revealed the copy number variations that might explain the differential expression of some candidate salivary genes. In addition, at least four candidate effector genes that were present in the alfalfa biotype but might not be encoded in the pea biotype were identified. This work sets the stage for future functional characterization of candidate genes potentially involved in the determination of plant specificity of pea aphid biotypes.

Aphids are piercing-sucking insect pests and feed on phloem sap. During feeding, aphids inject a battery of salivary proteins into host plant. Some of these proteins function like effectors of microbial pathogens and influence the outcome of plant–aphid interactions. The pea aphid (Acyrthosiphon pisum) is the model aphid and encompasses multiple biotypes each specialized to one or a few legume species, providing an opportunity to investigate the underlying mechanisms of the compatibility between plants and aphid biotypes. We aim to identify the aphid factors that determine the compatibility with host plants, hence involved in the host plant specialization process, and hypothesize that salivary proteins are one of those factors. Agrobacterium-mediated transient gene expression is a powerful tool to perform functional analyses of effector (salivary) proteins in plants. However, the tool was not established for the legume species that A. pisum feeds on. Thus, we decided to optimize the method for legume plants to facilitate the functional analyses of A. pisum salivary proteins. We screened a range of cultivars of pea (Pisum sativum) and alfalfa (Medicago sativa). None of the M. sativa cultivars was suitable for agroinfiltration under the tested conditions; however, we established a protocol for efficient transient gene expression in two cultivars of P. sativum, ZP1109 and ZP1130, using A. tumefaciens AGL-1 strain and the pEAQ-HT-DEST1 vector. We confirmed that the genes are expressed from 3 to 10 days post-infiltration and that aphid lines of the pea adapted biotype fed and reproduced on these two cultivars while lines of alfalfa and clover biotypes did not. Thus, the pea biotype recognizes these two cultivars as typical pea plants. By using a combination of ZP1109 and an A. pisum line, we defined an agroinfiltration procedure to examine the effect of in planta expression of selected salivary proteins on A. pisum fitness and demonstrated that transient expression of one candidate salivary gene increased the fecundity of the aphids. This result confirms that the agroinfiltration can be used to perform functional analyses of salivary proteins in P. sativum and consequently to study the molecular mechanisms underlying host specialization in the pea aphid complex.

17Most endogenous viruses, an important proportion of eukaryote genomes, are doomed to slowly 18 decay. Little is known, however, on how they evolve when they confer a benefit to their host. 19Bracoviruses are essential for the parasitism success of parasitoid wasps, whose genomes they 20 integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp 21 genome, for the first time at a chromosomal scale, that symbiotic bracovirus genes spread to 22 and colonized all the chromosomes. Moreover, large viral clusters are stably maintained 23 suggesting strong evolutionary constraints. Genomic comparison with another wasps revealed 24 that this organization was already established ~53 mya. Transcriptomic analyses highlight 25 temporal synchronization of viral gene expression, leading to particle production. Immune 26 genes are not induced, however, indicating the virus is not perceived as foreign by the wasp. 27This recognition suggests that no conflicts remain between symbiotic partners when benefits to 28 them converge. 29 30 31 32 33 Main 34 Cotesia wasps (Hymenoptera, Braconidae) are parasitoids of Lepidoptera. Female 35 wasps lay their eggs into caterpillars and larvae develop feeding on the host hemolymph. 36Several Cotesia species are famous for their use as biological control agents to control insect 37 pests, such as Cotesia flavipes which is massively released over several million hectares of 38 sugarcane fields in Brazil 1,2 . Parasitoid wasps evolved several strategies that increase parasitic 39 success, including a sensitive olfactory apparatus to locate their hosts 3,4 and detoxification 40 mechanisms against plant toxic compounds accumulating in their host (Fig. 1). But the most 41 original strategy is the domestication of a bracovirus (BV) shared by over 46,000 braconid wasp 42 species 5 . Bracoviruses originate from a single integration event ~103 million years ago (mya) 43 of a nudivirus in the genome of the last common ancestor of this group 6-9 . Virus domestication 44 nowadays confers a benefit to the wasps that use BVs as virulence gene delivery systems 10 . 45

Aphids secrete proteins from their stylets that evidence indicates function similar to pathogen effectors for virulence. Here, we describe two small candidate effector gene families of the pea aphid, Acyrthosiphon pisum, that share highly conserved secretory signal peptide coding regions and divergent non-secretory coding sequences derived from miniature exons. The KQY candidate effector family contains eleven members with additional isoforms, generated by alternative splicing. Pairwise comparisons indicate possible four unique KQY families based on coding regions without the secretory signal region. KQY1a, a representative of the family, is encoded by a 968 bp mRNA and a gene that spans 45.7 kbp of the genome. The locus consists of 37 exons, 33 of which are 15 bp or smaller. Additional KQY members, as well as members of the KHI family, share similar features. Differential expression analyses indicate that the genes are expressed preferentially in salivary glands. Proteomic analysis on salivary glands and saliva revealed 11 KQY members in salivary proteins, and KQY1a was detected in an artificial diet solution after aphid feeding. A single KQY locus and two KHI loci were identified in Myzus persicae, the peach aphid. Of the genes that can be anchored to chromosomes, loci are mostly scattered throughout the genome, except a two-gene region (KQY4/ KQY6). We propose that the KQY family expanded in A. pisum through combinatorial assemblies of a common secretory signal cassette and novel coding regions, followed by classical gene duplication and divergence.

Males and females share essentially the same genome but differ in their optimal values for many phenotypic traits, which can result in intra-locus conflict between the sexes. Aphids display XX/X0 sex chromosomes and combine unusual X chromosome inheritance with cyclical parthenogenesis. Theoretical and empirical works support the hypothesis that the large excess of male-biased genes observed on the aphid X chromosome compared to autosomes has evolved in response to sexual conflicts, by restricting the product of a sexually antagonistic allele to the sex it benefits. However, whether such masculinization of the X affects all tissues (as expected if it evolved in response to sexual conflicts) or reflects tissue specificities (which would contradict the sexual conflict hypothesis) remains an open question. To address it, we measured gene expression in different somatic and gonadic tissues of males, sexual females and parthenogenetic females of the pea aphid. We observed a masculinization of the X at the tissue-level, with male-biased genes being 2.5 to 3.5 more frequent on the X than expected. We also tested the hypothesis that gene duplication can facilitate the attenuation of conflicts by allowing gene copies to neo- or sub-functionalize and reach sex-specific optima. As predicted, X-linked copies of duplicated genes having their other copies on autosomes were more frequently male-biased (40.5% of the genes) than duplicated autosomal genes (6.6%) or X-linked single-copy genes (32.5%). These results highlight a peculiar pattern of expression of X-linked genes in aphids at the tissue level and provides further support for sex-biased expression as a mechanism to attenuate intra-locus sexual conflicts.