Role of Genes in Regulating Host Plants Expansion in Tephritid Fruit Flies (Diptera) and Potential for RNAi-Based Control.

Host plant expansion is an important survival strategy for tephritids as they expand their range. Successful host expansion requires tephritids to adapt to the chemical and nonchemical properties of a novel host fruit, such as fruit color, phenology, and phytochemicals. These plant properties trigger a series of processes in tephritids, with each process having its own genetic basis, which means that various genes are involved in regulating host plant expansion by tephritids. This review summarizes current knowledge on the categories and roles of genes involved in host plant expansion in several important tephritid species, including genes related to chemoreception (olfactory and gustation), vision, digestion, detoxification, development, ribosomal and energy metabolism. Chemoreception- and detoxification- and digestion-related genes are stimulated by volatile chemicals and secondary chemicals of different hosts, respectively, which are involved in the regulation of nervous signal transduction that triggers behavioral, physical, and chemical responses to the novel host fruit. Vision-, nerve-, and development-related genes and metabolism-associated genes are activated in response to nonchemical stimuli from different hosts, such as color and phenology, to regulate a comprehensive adaptation of the extending host for tephritids. The chemical and nonchemical signals of hosts activate ribosomal and energy-related genes that result in the basic regulation of many processes of host expansion, including detoxification and development. These genes do not regulate novel host use individually, but multiple genes regulate multilevel adaptation to novel host fruits via multiple mechanisms. These genes may also be potential target genes for RNAi-based control of tephritid pests.

Deciphering the worldwide invasion of the Asian long-horned beetle: A recurrent invasion process from the native area together with a bridgehead effect.

Retracing introduction routes is crucial for understanding the evolutionary processes involved in an invasion, as well as for highlighting the invasion history of a species at the global scale. The Asian long-horned beetle (ALB) Anoplophora glabripennis is a xylophagous pest native to Asia and invasive in North America and Europe. It is responsible for severe losses of urban trees, in both its native and invaded ranges. Based on historical and genetic data, several hypotheses have been formulated concerning its invasion history, including the possibility of multiple introductions from the native zone and secondary dispersal within the invaded areas, but none have been formally tested. In this study, we characterized the genetic structure of ALB in both its native and invaded ranges using microsatellites. In order to test different invasion scenarios, we used an approximate Bayesian "random forest" algorithm together with traditional population genetics approaches. The strong population differentiation observed in the native area was not geographically structured, suggesting complex migration events that were probably human-mediated. Both native and invasive populations had low genetic diversity, but this characteristic did not prevent the success of the ALB invasions. Our results highlight the complexity of invasion pathways for insect pests. Specifically, our findings indicate that invasive species might be repeatedly introduced from their native range, and they emphasize the importance of multiple, human-mediated introductions in successful invasions. Finally, our results demonstrate that invasive species can spread across continents following a bridgehead path, in which an invasive population may have acted as a source for another invasion.

Dataset documenting prevalence and counts of pine processionary moth tents on local host trees in 3 regions of France with different climatic environments.

The pine processionary moth Thaumetopoea pityocampa is a defoliating lepidopter that develops during winter. The larvae are gregarious and bear urticating setae that are harmful to humans and vertebrates. They shelter in conspicuous silk tents that are easy to detect. We here present a dataset comprising tree characterization and tent counts from 3 agglomerations in France located in regions with different climatic environments. The studied trees belong to various conifer species that are potential hosts for the caterpillars. In each site, we defined clusters as one target tree and its 10-62 nearest neighbors, and surveyed each tree within the clusters by informing: tree species, coordinates, size, number of tents. We characterized a total of 3690 trees, including 2009 trees in Orléans (grouped in 68 clusters), 359 trees in La Baule (18 clusters) and 1322 trees in Montpellier (52 clusters). We provide the raw data characterizing each individual tree, graphs showing the prevalence and mean number of tents for the tree species included in the survey, and maps allowing to locate each tree. This dataset brings information about host preference of the pine processionary moth and will be useful as a baseline to study spatio-temporal variability of host-insect relationships. It can also be informative for decision-makers and managers of urban greenings to avoid trees that are likely to be heavily infested for plantation in proximity to vulnerable people.

The effect of RAD allele dropout on the estimation of genetic variation within and between populations.

Inexpensive short-read sequencing technologies applied to reduced representation genomes is revolutionizing genetic research, especially population genetics analysis, by allowing the genotyping of massive numbers of single-nucleotide polymorphisms (SNP) for large numbers of individuals and populations. Restriction site-associated DNA (RAD) sequencing is a recent technique based on the characterization of genomic regions flanking restriction sites. One of its potential drawbacks is the presence of polymorphism within the restriction site, which makes it impossible to observe the associated SNP allele (i.e. allele dropout, ADO). To investigate the effect of ADO on genetic variation estimated from RAD markers, we first mathematically derived measures of the effect of ADO on allele frequencies as a function of different parameters within a single population. We then used RAD data sets simulated using a coalescence model to investigate the magnitude of biases induced by ADO on the estimation of expected heterozygosity and F(ST) under a simple demographic model of divergence between two populations. We found that ADO tends to overestimate genetic variation both within and between populations. Assuming a mutation rate per nucleotide between 10(-9) and 10(-8), this bias remained low for most studied combinations of divergence time and effective population size, except for large effective population sizes. Averaging F(ST) values over multiple SNPs, for example, by sliding window analysis, did not correct ADO biases. We briefly discuss possible solutions to filter the most problematic cases of ADO using read coverage to detect markers with a large excess of null alleles.

Estimation of population allele frequencies from next-generation sequencing data: pool-versus individual-based genotyping.

Molecular markers produced by next-generation sequencing (NGS) technologies are revolutionizing genetic research. However, the costs of analysing large numbers of individual genomes remain prohibitive for most population genetics studies. Here, we present results based on mathematical derivations showing that, under many realistic experimental designs, NGS of DNA pools from diploid individuals allows to estimate the allele frequencies at single nucleotide polymorphisms (SNPs) with at least the same accuracy as individual-based analyses, for considerably lower library construction and sequencing efforts. These findings remain true when taking into account the possibility of substantially unequal contributions of each individual to the final pool of sequence reads. We propose the intuitive notion of effective pool size to account for unequal pooling and derive a Bayesian hierarchical model to estimate this parameter directly from the data. We provide a user-friendly application assessing the accuracy of allele frequency estimation from both pool- and individual-based NGS population data under various sampling, sequencing depth and experimental error designs. We illustrate our findings with theoretical examples and real data sets corresponding to SNP loci obtained using restriction site-associated DNA (RAD) sequencing in pool- and individual-based experiments carried out on the same population of the pine processionary moth (Thaumetopoea pityocampa). NGS of DNA pools might not be optimal for all types of studies but provides a cost-effective approach for estimating allele frequencies for very large numbers of SNPs. It thus allows comparison of genome-wide patterns of genetic variation for large numbers of individuals in multiple populations.

An extreme case of plant-insect codiversification: figs and fig-pollinating wasps.

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification. [Biogeography; coevolution; cospeciation; host switching; long-branch attraction; phylogeny.].

Sequence, assembly and count datasets of viruses associated to the pine processionary moth Thaumetopoea pityocampa (Denis & Schiffermüller) (Lepidoptera, Notodontidae) identified from transcriptomic high-throughput sequencing.

The pine processionary moth Thaumetopoea pityocampa is a Lepidopteran pest species occurring in the Western Mediterranean. It causes heavy pine defoliations and it is a public and animal health concern because of its urticating caterpillars. Very little is known about the viruses associated to this species, as only two viruses were described so far. We here present a dataset corresponding to 34 viral transcripts, among which 27 could be confidently assigned to 9 RNA and DNA viral families (Iflaviridae, Reoviridae, Partitiviridae, Permutotetraviridae, Flaviviridae, Rhabdoviridae, Parvoviridae, Baculoviridae and PolyDNAviridae). These transcripts were identified from an original transcriptome assembled for the insect host, using both blast search and phylogenetic approaches. The data were acquired from 2 populations in Portugal and 2 populations in Italy. The transcripts were de novo assembled and used to identify viral sequences by homology searches. We also provide information about the populations and life stages in which each virus was identified. The data produced will allow to enrich the virus taxonomy in Lepidopteran hosts, and to develop PCR-based diagnostic tools to screen colonies across the range and determine the distribution and prevalence of the identified viral species.

Setae from the pine processionary moth (Thaumetopoea pityocampa) contain several relevant allergens.

BACKGROUND: Pine processionary larvae produce urticating hairs (setae) that serve for protection against predators. Setae induce cutaneous reactions in animals and humans. The presence of toxic or allergic mechanisms is a matter of debate. OBJECTIVES: To detect the presence of allergens in setae and to characterize them. MATERIALS AND METHODS: Setae extracts were characterized by gel staining and immunoblot, with sera from patients with immediate reactions and positive prick test reactions, as well as a rabbit antiserum raised against setae. Setae proteins were fractionated by high-performance liquid chromatography. The most relevant allergen was analysed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), and its sequence was deduced from an expressed sequence tag bank. Results. Setae contained at least seven different allergens. The most intense detection corresponded to a protein of MW ~ 14,000 that was similar to thaumetopoein, a previously described protein with mast cell-degranulating properties. MALDI-MS-based de novo sequencing provided a partial amino acid sequence different from that of the previously described allergen Tha p 1, and it was named Tha p 2. This allergen was detected in 61% of patients, and it is therefore a new major caterpillar allergen. CONCLUSIONS: Penetration of the setae from the pine processionary caterpillar delivers their allergenic content in addition to causing mechanical or toxic injury.

Complete mitogenome data from a summer population specimen of the urticating pine defoliator Thaumetopoea pityocampa (Lepidoptera, Notodontidae, Thaumetopoeinae, Thaumetopoea).

We present a de novo mitogenome assembly obtained from specimens sampled in the so-called summer population (SP) of Thaumetopoea pityocampa (Denis and Schiffermüller, 1775) in Portugal. Contrary to the typical larval development occurring in winter in this species, the larvae of this unique population develop during summer. The sequencing data used were obtained from genomic libraries originally generated to assemble the nuclear genome of T. pityocampa[1]. We also provide a complete annotation and a phylogenetic representation which positions the Portuguese summer population of T. pityocampa and an Italian typical individual of the same species among the Notodontidae family and more distant Noctuoidea species. This data represents a valuable new resource for an expanding and urticating insect pest.

Phylogeny and evolution of life-history strategies in the Sycophaginae non-pollinating fig wasps (Hymenoptera, Chalcidoidea).

BACKGROUND: Non-pollinating Sycophaginae (Hymenoptera, Chalcidoidea) form small communities within Urostigma and Sycomorus fig trees. The species show differences in galling habits and exhibit apterous, winged or dimorphic males. The large gall inducers oviposit early in syconium development and lay few eggs; the small gall inducers lay more eggs soon after pollination; the ostiolar gall-inducers enter the syconium to oviposit and the cleptoparasites oviposit in galls induced by other fig wasps. The systematics of the group remains unclear and only one phylogeny based on limited sampling has been published to date. Here we present an expanded phylogeny for sycophagine fig wasps including about 1.5 times the number of described species. We sequenced mitochondrial and nuclear markers (4.2 kb) on 73 species and 145 individuals and conducted maximum likelihood and Bayesian phylogenetic analyses. We then used this phylogeny to reconstruct the evolution of Sycophaginae life-history strategies and test if the presence of winged males and small brood size may be correlated. RESULTS: The resulting trees are well resolved and strongly supported. With the exception of Apocrytophagus, which is paraphyletic with respect to Sycophaga, all genera are monophyletic. The Sycophaginae are divided into three clades: (i) Eukoebelea; (ii) Pseudidarnes, Anidarnes and Conidarnes and (iii) Apocryptophagus, Sycophaga and Idarnes. The ancestral states for galling habits and male morphology remain ambiguous and our reconstructions show that the two traits are evolutionary labile. CONCLUSIONS: The three main clades could be considered as tribes and we list some morphological characters that define them. The same biologies re-evolved several times independently, which make Sycophaginae an interesting model to test predictions on what factors will canalize the evolution of a particular biology. The ostiolar gall-inducers are the only monophyletic group. In 15 Myr, they evolved several morphological adaptations to enter the syconia that make them strongly divergent from their sister taxa. Sycophaginae appears to be another example where sexual selection on male mating opportunities favored winged males in species with small broods and wingless males in species with large broods. However, some species are exceptional in that they lay few eggs but exhibit apterous males, which we hypothesize could be due to other selective pressures selecting against the re-appearance of winged morphs.

Coding-Complete Genome Sequences of an Iteradensovirus and an Alphapermutotetra-Like Virus Identified from the Pine Processionary Moth (Thaumetopoea pityocampa) in Portugal.

The coding-complete genome sequences of an iteradensovirus (family Parvoviridae) and an alphapermutotetra-like virus (family Permutotetraviridae) were discovered from transcriptomic data sets obtained from Thaumetopoea pityocampa larvae collected in Portugal. Each of the coding-complete genome sequences of these viruses contains three main open reading frames (ORFs).

Coding-Complete Genome Sequence of a Partitivirus Isolated from Pine Processionary Moth Eggs.

Two coding-complete nucleotide sequences of a partitivirus (family Partitiviridae) were discovered in transcriptomic data sets obtained from eggs of the Lepidoptera Thaumetopoea pityocampa Each segment encodes a single open reading frame, and these two segments are predicted to encode an RNA-dependent RNA polymerase and a coat protein, respectively.

Monitoring of adult emergence in the pine processionary moth between 1970 and 1984 in Mont Ventoux, France.

BACKGROUND: The current climate change has marked impacts on the phenology of species, i.e. the timing of the various stages of their life cycle. Yet, to fully understand how phenological patterns can be modified according to changes in temperature regimes, it is of prime importance to rely on high quality historical data. Here, we propose a very valuable dataset including individual monitoring from pupation to adult emergence of 46 479 individuals of pine processionary moth (Thaumetopoea pityocampa) surveyed between 1970 and 1984 in southern France along an altitudinal gradient. As optional prolonged diapause occurs in this species, i.e. some individuals experience one or more years of diapause before emerging, the caterpillars sampled in any given year were monitored during up to 5 years. The goal was to give precise information about phenology in this species to further analyse its temporal patterns of variation. NEW INFORMATION: This dataset is unique by its richness and the type of data it contains. Phenology in the pine processionary moth is often monitored by the use of pheromone traps in the field, which does not provide all the necessary information, because it is then not possible to trace back the exact origin of the moth trapped, nor to characterise other steps of the life cycle. Moreover, as it corresponds to historical data dating back to the 70s and the 80s, the dataset provides a historical baseline of trends in the pre-warming period.

Population genetic structure of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) from Yunnan province (China) and nearby sites across the border.

Bactrocera dorsalis (Diptera: Tephritidae) is an important pest for many tropical and subtropical fruits. The fly is probably introduced in Yunnan, a southwestern province of China that shares borders with Vietnam, Laos and Myanmar. Depending on local environmental conditions, this species occurs either only in the most favorable seasons or year-round. To infer the genetic diversity and structure of the fly in the region, and to understand the relationships between the flies of year-round and seasonal areas, we analyzed 304 individuals from 14 populations using the mitochondrial cytochrome oxidase I gene (COI). The sampled populations were structured into four groups, probably isolated by the main natural barriers in Yunnan such as mountain ranges and rivers. Our data suggest either that B. dorsalis in Yunnan originated from multiple introductions events, even if the source populations still need to be identified; or that Yunnan is a natural origin of this species (i.e., that it is not invasive there). Finally, we found some evidences that the seasonal populations were colonized from nearby year-round populations.

From refugia to contact: Pine processionary moth hybrid zone in a complex biogeographic setting.

Contact zones occur at the crossroad between specific dispersal routes and are facilitated by biogeographic discontinuities. Here, we focused on two Lepidoptera sister species that come in contact near the Turkish Straits System (TSS). We aimed to infer their phylogeographic histories in the Eastern Mediterranean and finely analyze their co-occurrence and hybridization patterns in this biogeographic context. We used molecular mitochondrial and nuclear markers to study 224 individuals from 42 localities. We used discordances between markers and complementary assignment methods to identify and map hybrids and parental individuals. We confirmed the parapatric distribution of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) in the west and Thaumetopoea wilkinsoni in the east and identified a narrow contact zone. We identified several glacial refugia of T. wilkinsoni in southern Turkey with a strong east-west differentiation in this species. Unexpectedly, T. pityocampa crossed the TSS and occur in northern Aegean Turkey and some eastern Greek islands. We found robust evidence of introgression between the two species in a restricted zone in northwestern Turkey, but we did not identify any F1 individuals. The identified hybrid zone was mostly bimodal. The distributions and genetic patterns of the studied species were strongly influenced both by the Quaternary climatic oscillations and the complex geological history of the Aegean region. T. pityocampa and T. wilkinsoni survived the last glacial maximum in disjoint refugia and met in western Turkey at the edge of the recolonization routes. Expanding population of T. wilkinsoni constrained T. pityocampa to the western Turkish shore. Additionally, we found evidence of recurrent introgression by T. wilkinsoni males in several T. pityocampa populations. Our results suggest that some prezygotic isolation mechanisms, such as differences in timing of the adult emergences, might be a driver of the isolation between the sister species.

Quaternary history and contemporary patterns in a currently expanding species.

BACKGROUND: Quaternary climatic oscillations had dramatic effects on species evolution. In northern latitudes, populations had to survive the coldest periods in refugial areas and recurrently colonized northern regions during interglacials. Such a history usually results in a loss of genetic diversity. Populations that did not experience glaciations, in contrast, probably maintained most of their ancestral genetic diversity. These characteristics dramatically affected the present-day distribution of genetic diversity and may influence the ability of species to cope with the current global changes. We conducted a range-wide study of mitochondrial genetic diversity in the pine processionary moth (Thaumetopoea pityocampa/T. wilkinsoni complex, Notodontidae), a forest pest occurring around the Mediterranean Basin and in southern Europe. This species is responding to the current climate change by rapid natural range expansion and can also be accidentally transported by humans. Our aim was to assess if Quaternary climatic oscillations had a different effect across the species' range and to determine if genetic footprints of contemporary processes can be identified in areas of recent introduction. RESULTS: We identified three main clades that were spatially structured. In most of Europe, the genetic diversity pattern was typical for species that experienced marked glaciation cycles. Except in refugia, European populations were characterized by the occurrence of one main haplotype and by a strong reduction in genetic diversity, which is expected in regions that were rapidly re-colonized when climatic conditions improved. In contrast, all other sub-clades around the Mediterranean Basin occurred in limited parts of the range and were strongly structured in space, as is expected in regions in which the impact of glaciations was limited. In such places, genetic diversity was retained in most populations, and almost all haplotypes were endemic. This pattern was extreme on remote Mediterranean islands (Crete, Cyprus, Corsica) where highly differentiated, endemic haplotypes were found. Recent introductions were typified by the existence of closely-related haplotypes in geographically distant populations, which is difficult to detect in most of Europe because of a lack of overall genetic structure. CONCLUSION: In regions that were not prone to marked glaciations, recent moth introductions/expansions could be detected due to the existence of a strong spatial genetic structure. In contrast, in regions that experienced the most intense Quaternary climatic oscillations, the natural populations are not genetically structured, and contemporary patterns of population expansion remain undetected.

Genetic isolation through time: allochronic differentiation of a phenologically atypical population of the pine processionary moth.

Allochronic speciation refers to a mode of sympatric speciation in which the differentiation of populations is primarily due to a phenological shift without habitat or host change. However, it has been so far rarely documented. The present paper reports on a plausible case of allochronic differentiation between sympatric populations of the pine processionary moth (PPM), Thaumetopoea pityocampa. The PPM is a Mediterranean insect with winter larval development. A phenologically atypical population with early adult activity and summer larval development was detected 10 years ago in Portugal. Mitochondrial and nuclear sequences strongly suggest that the 'summer' individuals are closely related to the sympatric winter population, while microsatellite data show a reduction in allelic richness, a distortion of allelic frequencies and significant genetic differentiation. Moreover, monitoring of adult flights suggests that reproductive activity does not overlap between the summer and winter populations. We postulate that the summer population appeared after a sudden phenological shift of some individuals of the sympatric winter population, leading to a founder effect and complete reproductive isolation. Given that the individuals showing this new phenology are subject to different selection pressures, the observed allochronic differentiation may rapidly lead to deeper divergence.

Experimental evidence for heritable reproductive time in 2 allochronic populations of pine processionary moth.

Phenology allows organisms to overcome seasonally variable conditions through life-cycle adjustment. Changes in phenology can drastically modify the evolutionary trajectory of a population, while a shift in the reproductive time may cause allochronic differentiation. The hypothesis of heritable reproductive time was experimentally tested, by studying a unique population of the pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) which has a shifted phenology, and however co-occurs with the typical population following the classical life cycle. When populations of both types were reared under controlled conditions, the reproductive time was maintained asynchronous, as observed in the field. The shifted population was manipulated in the laboratory to reproduce later than usual, yet the offspring emerged in the next year at the expected dates thus "coming back" to the usual cycle. Hybrids from crosses performed between the 2 populations showed an intermediate phenology. From the emergence times of parents and offspring, a high heritability of the reproductive time (h = 0.76) was observed. The offspring obtained from each type of cross was genetically characterized using microsatellite markers. Bayesian clustering analysis confirmed that hybrids can be successfully identified and separated from the parental genetic classes by genotyping. Findings support the hypothesis that, for this particular population, incipient allochronic speciation is due to a heritable shift in the reproductive time that further causes assortative mating and might eventually cause ecological adaptation/maladaptation in response to environmental changes.

Genetic differentiation of the pine processionary moth at the southern edge of its range: contrasting patterns between mitochondrial and nuclear markers.

The pine processionary moth (Thaumetopoea pityocampa) is an important pest of coniferous forests at the southern edge of its range in Maghreb. Based on mitochondrial markers, a strong genetic differentiation was previously found in this species between western (pityocampa clade) and eastern Maghreb populations (ENA clade), with the contact zone between the clades located in Algeria. We focused on the moth range in Algeria, using both mitochondrial (a 648 bp fragment of the tRNA-cox2) and nuclear (11 microsatellite loci) markers. A further analysis using a shorter mtDNA fragment and the same microsatellite loci was carried out on a transect in the contact zone between the mitochondrial clades. Mitochondrial diversity showed a strong geographical structure and a well-defined contact zone between the two clades. In particular, in the pityocampa clade, two inner subclades were found whereas ENA did not show any further structure. Microsatellite analysis outlined a different pattern of differentiation, with two main groups not overlapping with the mitochondrial clades. The inconsistency between mitochondrial and nuclear markers is probably explained by sex-biased dispersal and recent afforestation efforts that have bridged isolated populations.

Comparative analysis of two phenologically divergent populations of the pine processionary moth (Thaumetopoea pityocampa) by de novo transcriptome sequencing.

The pine processionary moth Thaumetopoea pityocampa is a Mediterranean lepidopteran defoliator that experiences a rapid range expansion towards higher latitudes and altitudes due to the current climate warming. Its phenology - the time of sexual reproduction - is certainly a key trait for the local adaptation of the processionary moth to climatic conditions. Moreover, an exceptional case of allochronic differentiation was discovered ca. 15 years ago in this species. A population with a shifted phenology (the summer population, SP) co-exists near Leiria, Portugal, with a population following the classical cycle (the winter population, WP). The existence of this population is an outstanding opportunity to decipher the genetic bases of phenology. No genomic resources were so far available for T. pityocampa. We developed a high-throughput sequencing approach to build a first reference transcriptome, and to proceed with comparative analyses of the sympatric SP and WP. We pooled RNA extracted from whole individuals of various developmental stages, and performed a transcriptome characterisation for both populations combining Roche 454-FLX and traditional Sanger data. The obtained sequences were clustered into ca. 12,000 transcripts corresponding to 9265 unigenes. The mean transcript coverage was 21.9 reads per bp. Almost 70% of the de novo assembled transcripts displayed significant similarity to previously published proteins and around 50% of the transcripts contained a full-length coding region. Comparative analyses of the population transcriptomes allowed to investigate genes specifically expressed in one of the studied populations only, and to identify the most divergent homologous SP/WP transcripts. The most divergent pairs of transcripts did not correspond to obvious phenology-related candidate genes, and 43% could not be functionally annotated. This study provides the first comprehensive genome-wide resource for the target species T. pityocampa. Many of the assembled genes are orthologs of published Lepidoptera genes, which allows carrying out gene-specific re-sequencing. Data mining has allowed the identification of SNP loci that will be useful for population genomic approaches and genome-wide scans of population differentiation to identify signatures of selection.