Sylvie Gimenez

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world’s worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains (“C” and “R”) that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high rates of local genome rearrangements

The recent assembly of the silkworm Bombyx mori genome with 432 Mb on 28 holocentric chromosomes has become a reference in the genomic analysis of the very diverse Order of Lepidoptera. We sequenced BACs from two major pests, the noctuid moths Helicoverpa armigera and Spodoptera frugiperda, corresponding to 15 regions distributed on 11 B. mori chromosomes, each BAC/region being anchored by known orthologous gene(s) to analyze syntenic relationships and genome rearrangements among the three species. Nearly 300 genes and numerous transposable elements were identified, with long interspersed nuclear elements and terminal inverted repeats the most abundant transposable element classes. There was a high degree of synteny conservation between B. mori and the two noctuid species. Conserved syntenic blocks of identified genes were very small, however, approximately 1.3 genes per block between B. mori and the two noctuid species and 2.0 genes per block between S. frugiperda and H. armigera. This corresponds to approximately two chromosome breaks per Mb DNA per My. This is a much higher evolution rate than among species of the Drosophila genus and may be related to the holocentric nature of the lepidopteran genomes. We report a large cluster of eight members of the aminopeptidase N gene family that we estimate to have been present since the Jurassic. In contrast, several clusters of cytochrome P450 genes showed multiple lineage-specific duplication events, in particular in the lepidopteran CYP9A subfamily. Our study highlights the value of the silkworm genome as a reference in lepidopteran comparative genomics.

SPODOBASE : an EST database for the lepidopteran crop pest Spodoptera

BackgroundThe Lepidoptera Spodoptera frugiperda is a pest which causes widespread economic damage on a variety of crop plants. It is also well known through its famous Sf9 cell line which is used for numerous heterologous protein productions. Species of the Spodoptera genus are used as model for pesticide resistance and to study virus host interactions. A genomic approach is now a critical step for further new developments in biology and pathology of these insects, and the results of ESTs sequencing efforts need to be structured into databases providing an integrated set of tools and informations.DescriptionThe ESTs from five independent cDNA libraries, prepared from three different S. frugiperda tissues (hemocytes, midgut and fat body) and from the Sf9 cell line, are deposited in the database. These tissues were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction. So far, the SPODOBASE contains 29,325 ESTs, which are cleaned and clustered into non-redundant sets (2294 clusters and 6103 singletons). The SPODOBASE is constructed in such a way that other ESTs from S. frugiperda or other species may be added. User can retrieve information using text searches, pre-formatted queries, query assistant or blast searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary.ConclusionThe SPODOBASE database provides integrated access to expressed sequence tags (EST) from the lepidopteran insect Spodoptera frugiperda. It is a publicly available structured database with insect pest sequences which will allow identification of a number of genes and comprehensive cloning of gene families of interest for scientific community. SPODOBASE is available from URL: http://bioweb.ensam.inra.fr/spodobase

Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).

Junonia coenia Densovirus-Based Vectors for Stable Transgene Expression in Sf9 Cells: Influence of the Densovirus Sequences on Genomic Integration

ABSTRACT The invertebrate parvovirus Junonia coenia densovirus (JcDNV) shares similarities with terminal hairpins and nonstructural (NS) protein activities of adeno-associated virus (AAV) despite their evolutionary divergence (B. Dumas, M. Jourdan, A. M. Pascaud, and M. Bergoin, Virology, 191:202-222, 1992, and C. Ding, M. Urabe, M. Bergoin, and R. M. Kotin, J. Virol. 76:338-345, 2002). We demonstrate here that persistent transgene expression in insect cells results from stable integration of transfected JcDNV-derived vectors into the host genome. To assess the integrative properties of JcDNV vectors, the green fluorescent protein (GFP) gfp marker gene was fused in frame into the major open reading frame (ORF1) of the viral sequence under the control of the P9 capsid protein promoter. In addition, the influence of the nonstructural proteins on the posttransfection maintenance of the vectors was examined by interruption of one or all three NS ORFs. Following transfection of Sf9 cells with each of the JcDNV constructs, clones showing persistent GFP expression were isolated. Structural analyses revealed that the majority of the JcDNV plasmid sequence was integrated into the genome of the fluorescent clones. Integration was observed whether or not NS proteins were expressed. However, the presence of NS genes in the constructs greatly influenced the number of integrated copies and their distribution in the host genome. Disruption of NS genes expression resulted in integration of head-to-tail concatemers at multiple sites within the genome. Further analyses demonstrated that the cis JcDNV 5′ inverted terminal repeat region was the primary site of recombination. Sequence analyses of integration junctions showed rearrangements of both flanking and internal sequences for most integrations. These findings demonstrate that JcDNV vectors integrate into insect cells in a manner similar to AAV plasmids in mammalian cells.

Spodoptera frugiperda (Lepidoptera: Noctuidae) host-plant variants: two host strains or two distinct species?

Abstract The moth Spodoptera frugiperda is a well-known pest of crops throughout the Americas, which consists of two strains adapted to different host-plants: the first feeds preferentially on corn, cotton and sorghum whereas the second is more associated with rice and several pasture grasses. Though morphologically indistinguishable, they exhibit differences in their mating behavior, pheromone compositions, and show development variability according to the host-plant. Though the latter suggest that both strains are different species, this issue is still highly controversial because hybrids naturally occur in the wild, not to mention the discrepancies among published results concerning mating success between the two strains. In order to clarify the status of the two host-plant strains of S. frugiperda, we analyze features that possibly reflect the level of post-zygotic isolation: (1) first generation (F1) hybrid lethality and sterility; (2) patterns of meiotic segregation of hybrids in reciprocal second generation (F2), as compared to the meiosis of the two parental strains. We found a significant reduction of mating success in F1 in one direction of the cross and a high level of microsatellite markers showing transmission ratio distortion in the F2 progeny. Our results support the existence of post-zygotic reproductive isolation between the two laboratory strains and are in accordance with the marked level of genetic differentiation that was recovered between individuals of the two strains collected from the field. Altogether these results provide additional evidence in favor of a sibling species status for the two strains.

Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda

Repeat-associated small interfering RNAs (rasiRNAs) are derived from various genomic repetitive elements and ensure genomic stability by silencing endogenous transposable elements. Here we describe a novel subset of 46 rasiRNAs named LNCR rasiRNAs due to their homology with one long non-coding RNA (LNCR) of Spodoptera frugiperda. LNCR operates as the intermediate of an unclassified transposable element (TE-LNCR). TE-LNCR is a very invasive transposable element, present in high copy numbers in the S. frugiperda genome. LNCR rasiRNAs are single-stranded RNAs without a prominent nucleotide motif, which are organized in two distinct, strand-specific clusters. The expression of LNCR and LNCR rasiRNAs is developmentally regulated. Formation of heterochromatin in the genomic region where three copies of the TE-LNCR are embedded was followed by chromatin immunoprecipitation (ChIP) and we observed this chromatin undergo dynamic changes during development. In summary, increased LNCR expression in certain developmental stages is followed by the appearance of a variety of LNCR rasiRNAs which appears to correlate with subsequent accumulation of a heterochromatic histone mark and silencing of the genomic region with TE-LNCR. These results support the notion that a repeat-associated small interfering RNA pathway is linked to heterochromatin formation and/or maintenance during development to establish repression of the TE-LNCR transposable element. This study provides insights into the rasiRNA silencing pathway and its role in the formation of fluctuating heterochromatin during the development of one holocentric organism.

Evolutionary history of x-tox genes in three lepidopteran species: origin, evolution of primary and secondary structure and alternative splicing, generating a repertoire of immune-related proteins.

The proteins of the X-tox family have imperfectly conserved tandem repeats of several defensin-like motifs known as cysteine-stabilized αβ (CS-αβ) motifs. These immune-related proteins are inducible and expressed principally in hemocytes, but they have lost the antimicrobial properties of the ancestral defensins from which they evolved. We compared x-tox gene structure and expression in three lepidopteran species (Spodoptera frugiperda, Helicoverpa armigera and Bombyx mori). Synteny and phylogenetic analyses showed that the x-tox exons encoding CS-αβ motifs were phylogenetically closely related to defensin genes mapping to chromosomal positions close to the x-tox genes. We were able to define two groups of paralogous x-tox exons (three in Noctuids) that each followed the expected species tree. These results suggest that the ancestor of the three species already possessed an x-tox gene with at least two proto-domains, and an additional duplication/fusion should have occurred in the ancestor of the two noctuid species. An expansion of the number of exons subsequently occurred in each lineage. Alternatively, the proto x-tox gene possessed more copy and each group of x-tox domains might undergo concerted evolution through gene conversion. Accelerated protein evolution was detected in x-tox domains when compared to related defensins, concomitantly to multiplication of exons and/or the possible activation of concerted evolution. The x-tox genes of the three species have similar structural organizations, with repeat motifs composed of CS-αβ-encoding exons flanked by introns in phase 1. Diverse mechanisms underlie this organization: (i) the acquisition of new repeat motifs, (ii) the duplication of preexisting repeat motifs and (iii) the duplication of modules. A comparison of gDNA and cDNA structures showed that alternative splicing results in the production of multiple X-tox protein isoforms from the x-tox genes. Differences in the number and sequence of CS-αβ motifs in these isoforms were found between species, but also between individuals of the same species. Thus, our analysis of the genetic organization and expression of x-tox genes in three lepidopteran species suggests a rapid evolution of the organization of these genes.

Establishment and analysis of a reference transcriptome for Spodoptera frugiperda

BackgroundSpodoptera frugiperda (Noctuidae) is a major agricultural pest throughout the American continent. The highly polyphagous larvae are frequently devastating crops of importance such as corn, sorghum, cotton and grass. In addition, the Sf9 cell line, widely used in biochemistry for in vitro protein production, is derived from S. frugiperda tissues. Many research groups are using S. frugiperda as a model organism to investigate questions such as plant adaptation, pest behavior or resistance to pesticides.ResultsIn this study, we constructed a reference transcriptome assembly (Sf_TR2012b) of RNA sequences obtained from more than 35 S. frugiperda developmental time-points and tissue samples. We assessed the quality of this reference transcriptome by annotating a ubiquitous gene family - ribosomal proteins - as well as gene families that have a more constrained spatio-temporal expression and are involved in development, immunity and olfaction. We also provide a time-course of expression that we used to characterize the transcriptional regulation of the gene families studied.ConclusionWe conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome. While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts. That allowed us to explore the spatial and temporal expression of genes and to observe that some olfactory receptors are expressed in antennae and palps but also in other non related tissues such as fat bodies. Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae.

Transcriptional plasticity evolution in two strains of Spodoptera frugiperda (Lepidoptera: Noctuidae) feeding on alternative host-plants

Spodoptera frugiperda, the fall armyworm (FAW), is an important agricultural pest in the Americas and an emerging pest in sub-Saharan Africa, India, East-Asia and Australia, causing damage to major crops such as corn, sorghum and soybean. While FAW larvae are considered polyphagous, differences in diet preference have been described between two genetic variants: the corn strain (sf-C) and the rice strain (sf-R). These two strains are sometimes considered as distinct species, raising the hypothesis that ost plant specialization might have driven their divergence. To test this hypothesis, we irst performed controlled reciprocal transplant (RT) experiments to address the impact of plant diet on several traits linked to the fitness of the sf-C and sf-R strains. The phenotypical data suggest that sf-C is specialized to corn. We then used RNA-Se to identify constitutive transcriptional differences between strains, regardless of diet, in laboratory as well as in natural populations. We found that variations in mitochon rial transcription levels are among the most substantial and consistent differences between the two strains. Since mitochondrial genotypes also vary between the strains, we believe the mitochondria may have a significant role in driving strain divergence.Spodoptera frugiperda, the fall armyworm (FAW), is an important agricultural pest in the Americas and an emerging pest in sub-Saharan Africa, causing damage to major crops such as corn, sorghum and soybean. While FAW larvae are considered polyphagous, differences in diet preference have been described between two genetic variants: the Corn strain (sf-C) and the Rice strain (sf-R). These two strains are sometimes considered as distinct species, raising the hypothesis that host plant specialization might have driven their divergence. Ecological speciation takes place when the selection of divergent traits leads to the reproductive isolation of two populations. Under this hypothesis, we expect that the transcriptional response to the host plants should affect differently the fitness of the two FAW strains. We also expect that these genes should also be linked to a reproductive isolation mechanism between the strains. In this study, we performed controlled reciprocal transplant (RT) experiments to address the impact of plant diet on several traits linked to the fitness of the sf-C and sf-R strains. The phenotypical data suggest that sf-C is specialized to corn. We then used RNA-Seq to analyze the gene expression of FAW larvae from RT experiments. We show that each strain has a different response to the same plant diets. However, we also found constitutive transcriptional differences between strains in laboratory and in natural populations. In particular, we show that mitochondrial transcription is the main difference between strains. A difference in mitochondrial function may be the basis for a shift in host plant and could be involved in hybrid incompatibility, raising the hypothesis that mitochondrial genome is the main target of selection between the two strains.