Differential gene expression profiling of one- and two-dimensional apogamous gametophytes of the fern Dryopteris affinis ssp. affinis.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.

Consistent Reanalysis of Genome-wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets.

Genomic imprinting leads to different expression levels of maternally and paternally derived alleles. Over the last years, major progress has been made in identifying novel imprinted candidate genes in plants, owing to affordable next-generation sequencing technologies. However, reports on sequencing the transcriptome of hybrid F1 seed tissues strongly disagree about how many and which genes are imprinted. This raises questions about the relative impact of biological, environmental, technical, and analytic differences or biases. Here, we adopt a statistical approach, frequently used in RNA-seq data analysis, which properly models count overdispersion and considers replicate information of reciprocal crosses. We show that our statistical pipeline outperforms other methods in identifying imprinted genes in simulated and real data. Accordingly, reanalysis of genome-wide imprinting studies in Arabidopsis and maize shows that, at least for Arabidopsis, an increased agreement across datasets could be observed. For maize, however, consistent reanalysis did not yield a larger overlap between the datasets. This suggests that the discrepancy across publications might be partially due to different analysis pipelines but that technical, biological, and environmental factors underlie much of the discrepancy between datasets. Finally, we show that the set of genes that can be characterized regarding allelic bias by all studies with minimal confidence is small (~8,000/27,416 genes for Arabidopsis and ~12,000/39,469 for maize). In conclusion, we propose to use biologically replicated reciprocal crosses, high sequence coverage, and a generalized linear model approach to identify differentially expressed alleles in developing seeds.

STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets.

Proteins and their functional interactions form the backbone of the cellular machinery. Their connectivity network needs to be considered for the full understanding of biological phenomena, but the available information on protein-protein associations is incomplete and exhibits varying levels of annotation granularity and reliability. The STRING database aims to collect, score and integrate all publicly available sources of protein-protein interaction information, and to complement these with computational predictions. Its goal is to achieve a comprehensive and objective global network, including direct (physical) as well as indirect (functional) interactions. The latest version of STRING (11.0) more than doubles the number of organisms it covers, to 5090. The most important new feature is an option to upload entire, genome-wide datasets as input, allowing users to visualize subsets as interaction networks and to perform gene-set enrichment analysis on the entire input. For the enrichment analysis, STRING implements well-known classification systems such as Gene Ontology and KEGG, but also offers additional, new classification systems based on high-throughput text-mining as well as on a hierarchical clustering of the association network itself. The STRING resource is available online at https://string-db.org/.

Probing the evolutionary robustness of two repurposed drugs targeting iron uptake in Pseudomonas aeruginosa.

LAY SUMMARY: We probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen Pseudomonas aeruginosa. Using an experimental evolution approach in human serum, we showed that antivirulence treatments are not evolutionarily robust per se, but vary in their propensity to select for resistance. BACKGROUND AND OBJECTIVES: Treatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that antivirulence treatments disarm rather than kill pathogens, is controversial. Here, we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa. METHODOLOGY: We subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance. RESULTS: We found that mutants resistant against antivirulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp, a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance was presumably hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike. CONCLUSIONS AND IMPLICATIONS: Our work highlights that antivirulence treatments are not evolutionarily robust per se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

Environmental determinants of pyoverdine production, exploitation and competition in natural Pseudomonas communities.

Many bacteria rely on the secretion of siderophores to scavenge iron from the environment. Laboratory studies revealed that abiotic and biotic factors together determine how much siderophores bacteria make, and whether siderophores can be exploited by non-producing cheaters or be deployed by producers to inhibit competitors. Here, we explore whether these insights apply to natural communities, by comparing the production of the siderophore pyoverdine among 930 Pseudomonas strains from 48 soil and pond communities. We found that pH, iron content, carbon concentration and community diversity determine pyoverdine production levels, and the extent to which strains are either stimulated or inhibited by heterologous (non-self) pyoverdines. While pyoverdine non-producers occurred in both habitats, their prevalence was higher in soils. Environmental and genetic analyses suggest that non-producers can evolve as cheaters, exploiting heterologous pyoverdine, but also due to pyoverdine disuse in environments with increased iron availability. Overall, we found that environmental factors explained between-strain variation in pyoverdine production much better in soils than in ponds, presumably because high strain mixing in ponds impedes local adaption. Our study sheds light on the complexity of natural bacterial communities, and provides first insights into the multivariate nature of siderophore-based iron acquisition and competition among environmental pseudomonads.

Siderophore cheating and cheating resistance shape competition for iron in soil and freshwater Pseudomonas communities.

All social organisms experience dilemmas between cooperators performing group-beneficial actions and cheats selfishly exploiting these actions. Although bacteria have become model organisms to study social dilemmas in laboratory systems, we know little about their relevance in natural communities. Here, we show that social interactions mediated by a single shareable compound necessary for growth (the iron-scavenging pyoverdine) have important consequences for competitive dynamics in soil and pond communities of Pseudomonas bacteria. We find that pyoverdine non- and low-producers co-occur in many natural communities. While non-producers have genes coding for multiple pyoverdine receptors and are able to exploit compatible heterologous pyoverdines from other community members, producers differ in the pyoverdine types they secrete, offering protection against exploitation from non-producers with incompatible receptors. Our findings indicate that there is both selection for cheating and cheating resistance, which could drive antagonistic co-evolution and diversification in natural bacterial communities.Lab strains of Pseudomonas are model systems for the evolution of cooperation over public goods (iron-scavenging siderophores). Here, Butaite et al. add ecological and evolutionary insight into this system by showing that cheating and resistance to cheating both shape competition for iron in natural Pseudomonas communities.

The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible.

A system-wide understanding of cellular function requires knowledge of all functional interactions between the expressed proteins. The STRING database aims to collect and integrate this information, by consolidating known and predicted protein-protein association data for a large number of organisms. The associations in STRING include direct (physical) interactions, as well as indirect (functional) interactions, as long as both are specific and biologically meaningful. Apart from collecting and reassessing available experimental data on protein-protein interactions, and importing known pathways and protein complexes from curated databases, interaction predictions are derived from the following sources: (i) systematic co-expression analysis, (ii) detection of shared selective signals across genomes, (iii) automated text-mining of the scientific literature and (iv) computational transfer of interaction knowledge between organisms based on gene orthology. In the latest version 10.5 of STRING, the biggest changes are concerned with data dissemination: the web frontend has been completely redesigned to reduce dependency on outdated browser technologies, and the database can now also be queried from inside the popular Cytoscape software framework. Further improvements include automated background analysis of user inputs for functional enrichments, and streamlined download options. The STRING resource is available online, at http://string-db.org/.

Hybridization of powdery mildew strains gives rise to pathogens on novel agricultural crop species.

Throughout the history of agriculture, many new crop species (polyploids or artificial hybrids) have been introduced to diversify products or to increase yield. However, little is known about how these new crops influence the evolution of new pathogens and diseases. Triticale is an artificial hybrid of wheat and rye, and it was resistant to the fungal pathogen powdery mildew (Blumeria graminis) until 2001 (refs. 1,2,3). We sequenced and compared the genomes of 46 powdery mildew isolates covering several formae speciales. We found that B. graminis f. sp. triticale, which grows on triticale and wheat, is a hybrid between wheat powdery mildew (B. graminis f. sp. tritici) and mildew specialized on rye (B. graminis f. sp. secalis). Our data show that the hybrid of the two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts. We conclude that hybridization between mildews specialized on different species is a mechanism of adaptation to new crops introduced by agriculture.

STRING v10: protein-protein interaction networks, integrated over the tree of life.

The many functional partnerships and interactions that occur between proteins are at the core of cellular processing and their systematic characterization helps to provide context in molecular systems biology. However, known and predicted interactions are scattered over multiple resources, and the available data exhibit notable differences in terms of quality and completeness. The STRING database (http://string-db.org) aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The new version 10.0 of STRING covers more than 2000 organisms, which has necessitated novel, scalable algorithms for transferring interaction information between organisms. For this purpose, we have introduced hierarchical and self-consistent orthology annotations for all interacting proteins, grouping the proteins into families at various levels of phylogenetic resolution. Further improvements in version 10.0 include a completely redesigned prediction pipeline for inferring protein-protein associations from co-expression data, an API interface for the R computing environment and improved statistical analysis for enrichment tests in user-provided networks.

The FgfrL1 receptor is required for development of slow muscle fibers.

FgfrL1, which interacts with Fgf ligands and heparin, is a member of the fibroblast growth factor receptor (Fgfr) family. FgfrL1-deficient mice show two significant alterations when compared to wildtype mice: They die at birth due to a malformed diaphragm and they lack metanephric kidneys. Utilizing gene arrays, qPCR and in situ hybridization we show here that the diaphragm of FgfrL1 knockout animals lacks any slow muscle fibers at E18.5 as indicated by the absence of slow fiber markers Myh7, Myl2 and Myl3. Similar lesions are also found in other skeletal muscles that contain a high proportion of slow fibers at birth, such as the extraocular muscles. In contrast to the slow fibers, fast fibers do not appear to be affected as shown by expression of fast fiber markers Myh3, Myh8, Myl1 and MylPF. At early developmental stages (E10.5, E15.5), FgfrL1-deficient animals express slow fiber genes at normal levels. The loss of slow fibers cannot be attributed to the lack of kidneys, since Wnt4 knockout mice, which also lack metanephric kidneys, show normal expression of Myh7, Myl2 and Myl3. Thus, FgfrL1 is specifically required for embryonic development of slow muscle fibers.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression.

BACKGROUND: Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS: In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION: Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling.

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.

Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review.

As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

A teratocarcinoma-like human embryonic stem cell (hESC) line and four hESC lines reveal potentially oncogenic genomic changes.

The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.

Functional characterization of transcription factor motifs using cross-species comparison across large evolutionary distances.

We address the problem of finding statistically significant associations between cis-regulatory motifs and functional gene sets, in order to understand the biological roles of transcription factors. We develop a computational framework for this task, whose features include a new statistical score for motif scanning, the use of different scores for predicting targets of different motifs, and new ways to deal with redundancies among significant motif-function associations. This framework is applied to the recently sequenced genome of the jewel wasp, Nasonia vitripennis, making use of the existing knowledge of motifs and gene annotations in another insect genome, that of the fruitfly. The framework uses cross-species comparison to improve the specificity of its predictions, and does so without relying upon non-coding sequence alignment. It is therefore well suited for comparative genomics across large evolutionary divergences, where existing alignment-based methods are not applicable. We also apply the framework to find motifs associated with socially regulated gene sets in the honeybee, Apis mellifera, using comparisons with Nasonia, a solitary species, to identify honeybee-specific associations.

Sociality is linked to rates of protein evolution in a highly social insect.

Eusocial insects exhibit unparalleled levels of cooperation and dominate terrestrial ecosystems. The success of eusocial insects stems from the presence of specialized castes that undertake distinct tasks. We investigated whether the evolutionary transition to societies with discrete castes was associated with changes in protein evolution. We predicted that proteins with caste-biased gene expression would evolve rapidly due to reduced antagonistic pleiotropy. We found that queen-biased proteins of the honeybee Apis mellifera did indeed evolve rapidly, as predicted. However, worker-biased proteins exhibited slower evolutionary rates than queen-biased or nonbiased proteins. We suggest that distinct selective pressures operating on caste-biased genes, rather than a general reduction in pleiotropy, explain the observed differences in evolutionary rates. Our study highlights, for the first time, the interaction between highly social behavior and dynamics of protein evolution.

Expression profiles of urbilaterian genes uniquely shared between honey bee and vertebrates.

BACKGROUND: Large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of Bilateria (Urbilateria) is lost in each animal lineage. This event could be one of the underlying mechanisms involved in generating metazoan diversity. However, the present functions of these ancient genes have not been addressed extensively. To understand the functions and evolutionary mechanisms of such ancient Urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes (honey bee-vertebrate specific, HVS genes) as a model. RESULTS: We identified 30 honey bee and 55 mouse HVS genes. Many HVS genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee HVS genes was found to be brain enriched, and 24% of mouse HVS genes were highly expressed in either or both the brain and testis. Moreover, a minimum of 38% of mouse HVS genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. Furthermore, gene ontology (GO) analysis of HVS genes predicted that 35% of genes are associated with DNA transcription and RNA processing. CONCLUSION: These results suggest that HVS genes include genes that are biased towards expression in the brain and gonads. They also demonstrate that at least some of Urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes.

Quantification of ortholog losses in insects and vertebrates.

BACKGROUND: The increasing number of sequenced insect and vertebrate genomes of variable divergence enables refined comparative analyses to quantify the major modes of animal genome evolution and allows tracing of gene genealogy (orthology) and pinpointing of gene extinctions (losses), which can reveal lineage-specific traits. RESULTS: To consistently quantify losses of orthologous groups of genes, we compared the gene repertoires of five vertebrates and five insects, including honeybee and Tribolium beetle, that represent insect orders outside the previously sequenced Diptera. We found hundreds of lost Urbilateria genes in each of the lineages and assessed their phylogenetic origin. The rate of losses correlates well with the species' rates of molecular evolution and radiation times, without distinction between insects and vertebrates, indicating their stochastic nature. Remarkably, this extends to the universal single-copy orthologs, losses of dozens of which have been tolerated in each species. Nevertheless, the propensity for loss differs substantially among genes, where roughly 20% of the orthologs have an 8-fold higher chance of becoming extinct. Extrapolation of our data also suggests that the Urbilateria genome contained more than 7,000 genes. CONCLUSION: Our results indicate that the seemingly higher number of observed gene losses in insects can be explained by their two- to three-fold higher evolutionary rate. Despite the profound effect of many losses on cellular machinery, overall, they seem to be guided by neutral evolution.

Cloning, characterization and analysis by RNA interference of various genes of the Chelonus inanitus polydnavirus.

Successful parasitism of some endoparasitic wasps depends on an obligately symbiotic association with polydnaviruses. These unique viruses have a segmented genome consisting of circles of double-stranded (ds) DNA and do not replicate in the parasitized host. They are produced in the wasp's ovary and injected into the host along with the egg. Chelonus inanitus is an egg-larval parasitoid; its polydnavirus (CiV) has been shown to protect the parasitoid larva from the host's immune system and to induce developmental arrest in the prepupal stage. The genome of CiV consists of at least 10-12 segments and five have been sequenced up to now. Here, the complete (CiV12g2) or partial (CiV12g1, CiV16.8g1) cloning of three new CiV genes is reported. All three occur only on one viral segment and have no similarity to other known polydnavirus genes, with the exception of a high similarity of CiV12g1 to CiV14g1 and CiV12g2 to CiV14g2. Furthermore, the first attempt of in vivo application of RNA interference to study the function of polydnavirus genes is shown. Injection of dsRNA of two late- and one early- and late-expressed CiV genes into CiV/venom-containing host eggs partially rescued last-instar larvae from developmental arrest. Injection of the same dsRNAs into parasitized eggs partially reduced parasitoid survival, mainly by preventing the successful emergence of the parasitoid from the host. These viral genes thus seem to be involved in inducing developmental arrest and in keeping the cuticle soft, which appears to be necessary for parasitoid emergence and host feeding.

Stage-dependent expression of Chelonus inanitus polydnavirus genes in the host and the parasitoid.

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid of Spodoptera littoralis (Noctuidae). Along with the egg it also injects polydnaviruses (CiV) and venom, which are prerequisites for successful parasitoid development. CiV protects the parasitoid from encapsulation by the host's immune system and induces a developmental arrest in the prepupal stage. The polydnavirus genome consists of several double-stranded circular DNA segments. Proviral DNA is integrated in the wasp's genome and virus replication is restricted to the wasp's ovary. Here, the analysis of eight CiV genes located on five different segments revealed four patterns of expression in the course of parasitization: early, late, persistent but variable, and early and late. The comparison between parasitized and CiV/venom only containing hosts indicated that the presence of the parasitoid larva modulates transcript levels. Haemocytes, fat body and nervous tissue contained viral transcripts, values being highest in haemocytes. Small amounts of CiV transcripts were also observed in parasitoid larvae and pupae, suggesting transcription from the proviral integrated form of viral DNA. This is the first comparative analysis of the expression patterns of several viral genes in both parasitized and CiV/venom only containing hosts over the entire period of parasitization, and it reveals intricate interactions between the parasitoid, the polydnavirus and the host.