GENOmics of PHEnOlogy in the pine processory moth, Thaumetopoea pityocampa – GenoPheno

i- Produce new genomic resources and tools to identify polymorphic markers such as SNPs following two approches: sequencing of population transcriptomes, and high-throughput sequencing of reduced genomic librairies
ii- Genotype several natural populations using the newly developed markers
iii- Finely analyse the data to identify genomic regions under selection, using (or evenually developing) the most adapted algorithms

- Assembly and comparative analysis of population transcriptomes (454 data): done, publication in prep. We identified ca. 1450 SNP
- Illumina sequencing of transcriptomes for each developmental stage: done for 4 out of 7 expected stages, data are being assembled.
- SNP loci development through sequencing of reduced genomic libraries: choice of the RAD sequencing method, the first libraries were successfully developed. The raw data are available, and bio-informatic analyses under process.
- Thanks to a support from INRA, we have generated a first reference genome for the focus species. The pre-assembly is now available and will allow a better analysis of the data generated in the present project
- Sampling and phenotype characterization in the field obtained through pheromone trapping in Leiria and in 3 altitudinal gradients
- Rearing and characterization of F1 lines under laboratory conditions: F2 could not be obtained, and we have developed a new strategiy of phenotyping and genotyping on F1 lines. The last individuals will emerge from the rearings in summer 2013

- Bio-informatic analyses of the obtained data: assemply and annotation optimization for the reference genomic and transcriptomic resources; mapping and annotation of the developed SNPs.
- Analyses of the RAD-seq data: neutral genetic structure and potential introgression between the SP and the WP; analysis of the genetic signatures of selection along the genome.
- Test of Pool-Seq whole genome re-sequencing: developmene tof this method for the SP and WP populations, eventually extend to all populations.
- Sampling and phenotypic characterization of more populations in the field.
- Publication of the main results in peer-reviewed journals and in scientific congresses

Gautier M., Gharbi K., Cézard T., Foucaud J., Kerdelhué C., Pudlo P., Cornuet J.-M., Estoup A., 2013. The effect of RAD allele drop-out on the estimation of genetic variation within and between populations. Molecular Ecology, sous presse. doi: 10.1111/mec.12089
Gautier M., Foucaud J., Gharbi K., Cézard T., Galan M., Loiseau A., Thomson M., Pudlo P., Kerdelhué C., Estoup A., 2013. Estimation of population allele frequencies from next-generation data: pooled versus individual genotyping. Molecular Ecology. Accepté avec révisions mineures.
Gschlößl B., Vogel H., Burban C., Streiff R., Kerdelhué C., en préparation. Comparision of two phenologically divergent populations of the pine processionary moth Thaumetopoea pityocampa through de novo transcriptome sequencing. A soumettre à BMC Genomics.
Gautier M., Cornuet J.-M., Gharbi K., Estoup A., 2011. Effect on genetic variation estimates of allele drop out in RADs derived SNPs: a simulation-based survey. Plenary invited conference, 3rd RAD meeting, Edimbourgh, Scotland.
Burban C., Santos H., Landes J., Leblois R., Paiva M.-R., Branco M., Kerdelhué C., 2012. Allochronic differentiation of a pine processionary moth population in Portugal: evolutionary scenario based on neutral markers. MEDINSECT 3: Entomological research in Mediterranean forest ecosystems, Hammamet, Tunisie.

The ongoing climate change affects the distribution ranges and/or the biological cycles of many species. Phenology of development is a major life history trait, and any modification can have deep evolutionary consequences. Variations in emergence time of either adults or juveniles can i) disrupt the synchrony in biotic interactions (e.g. predator/prey, plant/pollinator, insect/host plant…); (ii) modify the assemblages of natural enemies, and therefore the selection pressure they impose; (iii) reduce gene flow between groups of individuals reproducing in different time frames, which can lead to allochronic differentiation, or even to speciation.
We propose to characterize the genetic bases of phenology of reproduction for a forest insect species in which this trait is highly variable and has a strong adaptive value. The pine processionary moth Thaumetopoea pityocampa (Lepidoptera: Notodontidae, hereafter PPM) is a major pest of Mediterranean pine forests. Its gregarious and urticating larvae further cause human and animal health concerns. Its distribution range expands northward and at higher altitudes following the increase of winter temperatures. It has an annual life cycle, and the timing of adult emergence is strongly related to local climatic conditions (reproduction takes place as early as June-July where winters are colder, whereas it takes place in September in warmer locations). In mountainous regions, phenological differences thus exist between sites of low vs. high altitudes, even when these are only a few kilometres apart. These differences are eventually maintained under laboratory conditions.
Moreover, a population with a much shifted phenology has been discovered in Portugal, in the Leiria National Park. Individuals from this "mutant" population emerge and reproduce in May, whereas individuals developing in sympatry and exhibiting a "normal" phenology reproduce in September. Consequently, larval development occurs during summer rather than winter. Genetic analyses with presumably neutral markers suggest that this atypical population has been founded locally from few individuals that underwent a major change in their life cycle.
We therefore have a remarkable biological model to identify the regions of the genome involved in phenology, both in the case of the natural continuum of phenotypic variation observed over the distribution range, and for the atypical population of Leiria. Rather than analysing a limited number of candidate genes possibly involved in the expression of phenology, we propose an ambitious project in which we will develop genome-wide scans using newly developed markers to identify signatures of adaptive selection for this life-history trait. The genome structure of the PPM has been poorly characterized so far. Yet, the development of next-generation, high-throughput genotyping and sequencing technologies enables to propose an innovative approach to meet our goals.
We thus plan: (i) to produce genomic resources to identify new polymorphic markers (mostly Single Nucleotide Polymorphisms, SNPs) using two complementary approaches, (high-throughput sequencing of transcriptomes, and of reduced genomes of individuals exhibiting contrasted phenologies); (ii) to genotype ca. 2500 of the identified SNPs in several natural populations as well as in an outbred cross to build the first genetic map; (iii) and to finely analyse data in order to identify the regions of the genome underlying adaptive selection, using, and eventually developing, the most adapted algorithms.
This project will allow to build a new team of 5 young scientists who have complementary skills. It may constitute a first step toward the construction of a wider international consortium grouping scientists interested in the ecology and evolution of the PPM.