Epigenetic regulation mecanisms in holocentric pest crop Spdoptera frugiperda, Lepidoptera, Noctuidae

Chez les eucaryotes, l’ADN est empaqueté dans des complexes protéiques d’histones nommés nucléosomes qui assurent sa conformation. Cet arrangement est hétérogène à travers le génome et peut être dynamiquement modifié. La régulation de l’architecture chromatinienne joue un rôle essentiel dans la stabilité des génomes ainsi que la dynamique transcriptionnelle. Certaines régions qualifiées d’ ‘’heterochromatine constitutive’’ sont toutefois connues pour être maintenues à l’état condensé. Régionalisées aux extrémités et centres des chromosomes, l’hétérochromatine constitutive participe des fonctions télomériques et centromériques.Spodoptera frugiperda (S.fru, Lépidoptère, Noctuelle) est un ravageur de culture endémique du continent américain, récemment invasif dans le continent africain. Comme tous les Lépidoptères, S.fru est une espèce holocentrique dont le centromère est réparti le long des chromosomes et non restreint en un point unique. Cette disposition interroge sur l’établissement, la distribution ainsi que la fonction conservée de l’HC puisque cette dernière est principalement décrite pour être majoritairement localisée dans de larges régions péricentriques. Comprendre l’architecture chromatinienne chez S.fru peut avoir un intérêt en lutte biologique mais également permettre d’approfondir les connaissances en épigénétique chez un organisme non-modèle.Dans le cadre de la thèse, nous nous sommes demandés si la diméthylation de la lysine 9 de l’histone 3 (H3K9me2), marqueur de l’hétérochromatine constitutive, possédait un rôle conservé chez S.fru. Pour ce faire, nous avons comparé des données de ChIP-seq d’H3K9me2 sur cellules et larves entières après avoir annoté les gènes et l’ensemble des éléments répétés du génome, susceptibles d’être enrichis par cette marque. Parallèlement, des échantillons d’ARN-seq ont été étudiés afin de questionner le statut répressif de l’hétérochromatine constitutive. Nos résultats suggèrent un invariable maintien d’H3K9me2 dans les régions (sub)télomériques transcriptionnellement inactives ainsi qu’une forte association aux locus répétés d'ADN ribosomal (rDNA). Ces séquences ne constituent toutefois qu’une minorité des régions enrichies, le reste étant retrouvé dans des séquences répétées ainsi que dans le corps des gènes, indifféremment de leur état transcriptionnel. La persistante association d’H3K9me2 aux télomères et rDNA présagerait d’un maintien de la marque à proximité des centromères dont nous proposons un modèle d’établissement.La disposition de l’hétérochromatine constitutive questionne celle des régions euchromatiniennes, pauvres en nucléosomes, transcriptionnellement active et dynamiquement modifiées au cours du développement, du cycle cellulaire et des conditions environnementales. Afin de tester l’antagonisme de ces conformations, nous avons respectivement étudié la répartition des zones ouvertes et fermées du génome de la larve au stade L4 par approches de FAIRE-seq et de MAINE-seq. Ces structures ont été décrites dans la littérature pour être enrichies par de spécifiques modifications d’histones. Ainsi nous avons mis au point le protocole de native ChIP-seq d’H3K4me3 (marque active) et H3K9me2, H3K9me3, H3K27me3 (marques répressives). L’analyse en cours de l’ensemble de ces données de séquençages permettra d’avoir une vue intégrée de l’architecture chromatinienne au stade ravageur.In eukaryotes, DNA is arranged in histones proteins complexes called nucleosomes that shape its conformation. This arrangement is heterogeneous across genomes and can be dynamically modified. Regulation of chromatin architecture plays an essential role in genome stability and transcription dynamics. Some regions named ‘’constitutive heterochromatin’’ are nonetheless known to remain highly condensed, regardless of conditions. Regionalized at extremities and chromosomes centers, constitutive heterochromatin contributes to telomeric and centromeric functions.Spodoptera frugiperda (S.fru, Lepidoptera, Noctuidae) is major crop pest in the Americas that recently invaded Africa. Like all Lepidopteran, S.fru is holocentric which means that its centromere is spread along chromosome and not restricted to a uniq point. This disposition question about establishment, distribution but also conserved function of constitutive heterochromatin since its usually and mainly localized in large pericentric regions.Deciphering chromatinian architecture in S.fru can be of interest in biological control but also allow to deepen epigenetic knowledge in a non-model organism.During my phD, we questionned the role of histone 3 lysine 9 demethylated (H3K9me2) in S.fru, a histone modification known in other yet described organisms to be a constitutive constitutive heterochromatinian hallmark.We compared H3K9me2 ChIP-seq data on cells and larvae after overall genomic functional annotation, potentially enriched for this mark. In parallel, RNA-seq samples were analyzed to question the putative repressive status of constitutive heterochromatin.Our results suggest an invariant retention of H3K9me2 in (sub)telomeric regions transcriptionally inactive but also a strong association of this mark in repeated ribosomal DNA locus (rDNA).These sequences constitutes nonetheless a minority of enriched regions since most of them regionalize in repeated sequences like transposons and tandem array but also gene bodies, independently of their transcriptional states.Persistent H3K9me2 association to telomeres and rDNA could predict of the conserved expression of this mark near centromeres. Based on literature and bioinformatics analysis, we proposed a model for S.fru holocentromeres.Constitutive heterochromatin questions euchromatin arrangement, described to be nucleosome poor, transcriptionally active and dynamically modified across development, cell cycle and environmental conditions. In order to test these structural antagonisms, we respectively studied open and closed genome conformations by FAIRE-seq and MAINE in larvae. These structures are reported to be associated to specific histones marks. We developed a native ChIP-seq protocol on H3K4me3 (active mark) and H3K9me2, H3K9me3, H3K27me3 (repressives marks). Overall analysis of these NGS data would help to picture an integrative view of chromatin architecture during larval pest stage

Positive selection alone is sufficient for whole genome differentiation at the early stage of speciation process in the fall armyworm

International audienceBackground: The process of speciation involves differentiation of whole genome sequences between a pair of diverging taxa. In the absence of a geographic barrier and in the presence of gene flow, genomic differentiation may occur when the homogenizing effect of recombination is overcome across the whole genome. The fall armyworm is observed as two sympatric strains with different host-plant preferences across the entire habitat. These two strains exhibit a very low level of genetic differentiation across the whole genome, suggesting that genomic differentiation occurred at an early stage of speciation. In this study, we aim at identifying critical evolutionary forces responsible for genomic differentiation in the fall armyworm. Results: These two strains exhibit a low level of genomic differentiation (F ST = 0.0174), while 99.2% of 200 kb windows have genetically differentiated sequences (F ST > 0). We found that the combined effect of mild positive selection and genetic linkage to selectively targeted loci are responsible for the genomic differentiation. However, a single event of very strong positive selection appears not to be responsible for genomic differentiation. The contribution of chromosomal inversions or tight genetic linkage among positively selected loci causing reproductive barriers is not supported by our data. Phylogenetic analysis shows that the genomic differentiation occurred by sub-setting of genetic variants in one strain from the other. Conclusions: From these results, we concluded that genomic differentiation may occur at the early stage of a speciation process in the fall armyworm and that mild positive selection targeting many loci alone is sufficient evolutionary force for generating the pattern of genomic differentiation. This genomic differentiation may provide a condition for accelerated genomic differentiation by synergistic effects among linkage disequilibrium generated by following events of positive selection. Our study highlights genomic differentiation as a key evolutionary factor connecting positive selection to divergent selection

Macrophage plasticity is Rac signalling and MMP9 dependant

In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilisation haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation

Macrophage morphological plasticity and migration is Rac signalling and MMP9 dependant

In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilise haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation

Characterization and expression profiling of microRNAs in response to plant feeding in two host-plant strains of the lepidopteran pest Spodoptera frugiperda

International audienceBackground - A change in the environment may impair development or survival of living organisms leading them to adapt to the change. The resulting adaptation trait may reverse, or become fixed in the population leading to evolution of species. Deciphering the molecular basis of adaptive traits can thus give evolutionary clues. In phytophagous insects, a change in host-plant range can lead to emergence of new species. Among them, Spodoptera frugiperda is a major agricultural lepidopteran pest consisting of two host-plant strains having diverged 3 MA, based on mitochondrial markers. In this paper, we address the role of microRNAs, important gene expression regulators, in response to host-plant change and in adaptive evolution. Results - Using small RNA sequencing, we characterized miRNA repertoires of the corn (C) and rice (R) strains of S. frugiperda, expressed during larval development on two different host-plants, corn and rice, in the frame of reciprocal transplant experiments. We provide evidence for 76 and 68 known miRNAs in C and R strains and 139 and 171 novel miRNAs. Based on read counts analysis, 34 of the microRNAs were differentially expressed in the C strain larvae fed on rice as compared to the C strain larvae fed on corn. Twenty one were differentially expressed on rice compared to corn in R strain. Nine were differentially expressed in the R strain compared to C strain when reared on corn. A similar ratio of microRNAs was differentially expressed between strains on rice. We could validate experimentally by QPCR, variation in expression of the most differentially expressed candidates. We used bioinformatics methods to determine the target mRNAs of known microRNAs. Comparison with the mRNA expression profile during similar reciprocal transplant experiment revealed potential mRNA targets of these host-plant regulated miRNAs. Conclusions - In the current study, we performed the first systematic analysis of miRNAs in Lepidopteran pests feeding on host-plants. We identified a set of the differentially expressed miRNAs that respond to the plant diet, or differ constitutively between the two host plant strains. Among the latter, the ones that are also deregulated in response to host-plant are molecular candidates underlying a complex adaptive trait

Transcriptional differences between the two host strains of Spodoptera frugiperda (Lepidoptera: Noctuidae)

International audienceSpodoptera 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 host plant specialization might have driven their divergence. To test this hypothesis, we first 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 mitochondrial 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

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, 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