7th International Workshop on the Molecular Biology and Genetics of the Lepidoptera: August 20–26, 2006 Orthodox Academy of Crete, Kolympari, Crete, Greece

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Improvement of the assembly of heterozygous genomes of non-model organisms, a case study of the genomes of two Spodoptera frugiperda host strains

International audienceThe extraction of biological information from the draft genomes of non-model organisms may result in unattainable, incomplete, or even wrong conclusions. In particular, the combination of a high level of heterozygosity and short reads sequencing may have major impact in the annotation of genes [1,2]. This wrong gene content assessment is usually the consequence of the high fragmentation of the genome sequence but it may also come from an overestimation of the genome size. The latter because the assembly of an heterozygous region for which there is a significant divergence between the two haplotypes leads sometimes to the construction of two different contigs, instead of one consensus sequence. To date, new assemblers such as Platanus [3], have been developed in regard to heterozygous data. But, the complete re-assembly of a genome leading to new automatic and manual annotations process is very cost-effective, and may still produce erroneous scaffolds and annotations. Thus, we set up a « soft » method to detect and correct false duplications due to heterozygosity in draft assemblies. In addition, to the identification and removal of the allelic regions (i.e. unmerged haplotypes), our protocol is able to relocate and merge supernumerary gene annotations.We applied this method as a pre-requisite for the comparison of the genomes of 2 Spodoptera frugiperda (Lepidoptera: Noctuidae) strains, in the frame of the WGS project supported by the Fall armyworm International Public Consortium (FAW-IPC). This moth is a well-known pest of crops throughout the Western hemisphere. This species consists of two strains adapted to different larval host-plants: the first feeds preferentially on corn, cotton and sorghum whereas the second is more associated with rice and several pasture grasses. While, the paired-end reads of the rice-variant have been directly assembled using Platanus [3], we cleaned up and corrected the first release of the corn-variant, leading to a drastic reduction of the genome assembly, with the removal of 88Mbp (17%) and the increase of the N50 from 39,593 to 52,781bp. The suppressed fragments included 3,746 gene predictions; about 80% of them have been either relocated or merged with their complementary allele. Subsequently, in order to identify new candidate genes or genomic regions involved in the host-plant adaptation, we compared the genomes and proteomes of the 2 different strains in order to identify orthologous genes, collinear regions and genome rearrangements, taking into consideration the inflated occurrence of splitted genes due to the high fragmentation of the genome

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

International audienceThe 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

SPODOBASE : an EST database for the lepidopteran crop pest Spodoptera

BACKGROUND: The 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. DESCRIPTION: The 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. CONCLUSION: The 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

Establishment and analysis of a reference transcriptome for Spodoptera frugiperda

International audienceBackground Spodoptera 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

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

Genetic structure and insecticide resistance characteristics of fall armyworm populations invading China

The rapid wide‐scale spread of fall armyworm (Spodoptera frugiperda ) has caused serious crop losses globally. However, differences in the genetic background of subpopulations and the mechanisms of rapid adaptation behind the invasion are still not well understood. Here we report the assembly of a 390.38Mb chromosome‐level genome of fall armyworm derived from south‐central Africa using Pacific Bioscience (PacBio) and Hi‐C sequencing technologies, with scaffold N50 of 12.9 Mb and containing 22260 annotated protein‐coding genes. Genome‐wide resequencing of 103 samples and strain identification were conducted to reveal the genetic background of fall armyworm populations in China. Analysis of genes related to pesticide‐ and Bt‐resistance showed that the risk of fall armyworm developing resistance to conventional pesticides is very high. Laboratory bioassay results showed that insects invading China carry resistance to organophosphate and pyrethroid pesticides, but are sensitive to genetically modified maize expressing the Bacillus thuringiensis (Bt) toxin Cry1Ab in field experiments. Additionally, two mitochondrial fragments were found to be inserted into the nuclear genome, with the insertion event occurring after the differentiation of the two strains. This study represents a valuable advance toward improving management strategies for fall armyworm

An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research

<p>Abstract</p> <p>Background</p> <p>Nocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, <it>Spodoptera littoralis</it>, and to identify candidate genes involved in odour/pheromone detection.</p> <p>Results</p> <p>By targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the <it>Bombyx mori </it>proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.</p> <p>Conclusions</p> <p>Our project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in <it>S. littoralis</it>, and for ultimately identifying original targets to fight against moth herbivorous pests.</p

Molecular determinism of ecological adaptation and speciation in two strains of the Lepidoptera Spodoptera frugiperda

Molecular determinism of ecological adaptation and speciation in two strains of the Lepidoptera [i]Spodoptera frugiperda[/i]. Congrès de génomique environnemental