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Narrow-spectrum insecticides are currently used to control populations of spruce budworm, Choristoneura fumiferana Clemens (Lepidoptera: Tortricidae), in eastern Canada. However, these could have nontarget impacts on other caterpillars – some of which may serve as alternative or alternate hosts to key parasitoids – that are also susceptible to control tactics. This study was conducted to determine how the insecticides, Bacillus thuringiensis variety kurstaki (Btk) and tebufenozide, used to control spruce budworm populations, impact caterpillar communities and associated parasitism rates. Post-treatment field sampling of caterpillars was conducted in 2018 and 2019 in New Brunswick, Canada, at sites treated with either Btk or tebufenozide and at control sites. Caterpillar species richness and abundance, community structure, and parasitism rates were assessed using molecular analyses for 659 collected caterpillars. We found that insecticide applications had no significant impact on abundance, species richness, or parasitism rate relative to the measurements made in the control sites. Nonetheless, a significantly higher caterpillar abundance and lower parasitism rate occurred in Btk-treated sites than in tebufenozide-treated sites. Overall, however, Btk and tebufenozide treatments did not negatively affect the non-budworm caterpillar community under the present conditions of low caterpillar densities, suggesting that parasitoids have alternative and alternate hosts after treatments that target the spruce budworm.

The Siberian silk moth, Dendrolimus sibiricus Tschetverikov, is a very serious pest of conifers in Russia and is an emerging threat in North America where an accidental introduction could have devastating impacts on native forest resources. Other Dendrolimus Germar species and related Eurasian lasiocampids in the genus Malacosoma (Hubner) could also present a risk to North America’s forests. Foreign vessels entering Canadian and U.S. ports are regularly inspected for Lymantria dispar (Linnaeus) and for the presence of other potentially invasive insects, including suspicious lasiocampid eggs. However, eggs are difficult to identify based on morphological features alone. Here, we report on the development of two TaqMan (Roche Molecular Systems, Inc., Rotkreuz, Switzerland) assays designed to assist regulatory agencies in their identification of these insects. Developed using the barcode region of the cytochrome c oxidase I (COI) gene and run in triplex format, the first assay can detect Dendrolimus and Malacosoma DNA, and can distinguish North American from Eurasian Malacosoma species. The second assay is based on markers identified within the internal transcribed spacer 2 (ITS2) region and was designed to specifically identify D. sibiricus, while discriminating closely related Dendrolimus taxa. In addition to providing direct species identification in the context of its use in North America, the D. sibiricus assay should prove useful for monitoring the spread of this pest in Eurasia, where its range overlaps with those of the morphologically identical D. superans (Butler) and similar D. pini (Linnaeus). The assays described here can be performed either in the lab on a benchtop instrument, or on-site using a portable machine.

BACKGROUND In eastern Canada, surveys of overwintering 2nd instar spruce budworm (Choristoneura fumiferana) larvae ('L2s') are carried out each fall to guide insecticide application decisions in the following spring. These surveys involve the collection of fir and spruce branches in selected stands, followed by the mechanical/chemical removal of larvae. The latter then are counted manually on filter papers, using a stereomicroscope. Considering the significant effort and difficulties which this manual counting entails, we developed a quantitative (q)PCR-based 'molecular counting' approach designed to make this step less tedious. RESULTS Using the C. fumiferana mitochondrial cytochrome c oxidase 1 (COI) gene as a target for qPCR DNA quantification, we show that the amount of DNA in a larval extract is strongly correlated with the number of larvae used to generate that extract, and that molecular estimates of L2 counts are comparable to those generated using the manual approach. In addition, we used the same DNA extracts to monitor the microsporidian pathogen Nosema fumiferanae, and the hymenopteran parasitoids Glypta fumiferanae and Apanteles fumiferanae in overwintering L2s employing a subset of a TaqMan assay developed by Nisole et al. (2020) for the identification of budworm natural enemies. We show that the proportion of individuals affected by each natural enemy in samples containing a known number of larvae can be estimated from presence/absence data through the binomial probability distribution. CONCLUSION The present proof-of-principle study shows that a molecular approach for counting L2s and assessing their natural enemy load is clearly possible and is expected to generate reliable results.

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce “antifreeze proteins” (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below −30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here, we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a “zonadhesin-like” protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the “zonadhesin-like” protein as precursor of tortricid AFPs.

Global ecosystem functions, services, and commodities are increasingly threatened by biological invasions. As a result, there is an urgent need to manage invasive species through global collaborative research. We propose an ‘applied empirical framework’ (AEF) to aggressively confront the current global biological invasion crisis. The AEF builds on existing models for invasion science that advocate 1) standardized research designs to reveal key aspects of biological invasion, and 2) collaborative research to facilitate the sharing of resources and information. The AEF further emphasizes the need for 3) the production of research ‘tools’ (e.g., data, methodologies, technical instruments) designed for direct uptake by agencies that manage biological invasion, and 4) a taxonomically targeted approach in which task forces conduct rapid, in-depth research on top-priority invasive species across their entire geographic range. We review collaborative science and the distinctive roles played by different collaborator types. We then provide an example of the AEF in action through the BioSAFE initiative (Biosurveillance of Alien Forest Enemies), a highly collaborative project aimed at developing genomic research tools to facilitate biosurveillance and intervention for forest invasive species. We illustrate the BioSAFE approach through our research on two polyphagous insect species: the wood-borer Anoplophora glabripennis, Motschusky (Coleoptera: Cerambycidae; Asian longhorned beetle) and the defoliator Lymantria dispar, Linnaeus spp. (Lepidoptera: Lymantriidae; gypsy moth). These examples illustrate how the AEF can focus and accelerate our response to the global biological invasion crisis by applying the resource capabilities of collaborative research groups to generate management tools for top-priority invasive species.

The spruce budworm, Choristoneura fumiferana, is presumed to be panmictic across vast regions of North America. We examined the extent of panmixia by genotyping 3,650 single nucleotide polymorphism (SNP) loci in 1975 individuals from 128 collections across the continent. We found three spatially structured subpopulations: Western (Alaska, Yukon), Central (southeastern Yukon to the Manitoba–Ontario border), and Eastern (Manitoba–Ontario border to the Atlantic). Additionally, the most diagnostic genetic differentiation between the Central and Eastern subpopulations was chromosomally restricted to a single block of SNPs that may constitute an island of differentiation within the species. Geographic differentiation in the spruce budworm parallels that of its principal larval host, white spruce (Picea glauca), providing evidence that spruce budworm and spruce trees survived in the Beringian refugium through the Last Glacial Maximum and that at least two isolated spruce budworm populations diverged with spruce/fir south of the ice sheets. Gene flow in the spruce budworm may also be affected by mountains in western North America, habitat isolation in West Virginia, regional adaptations, factors related to dispersal, and proximity of other species in the spruce budworm species complex. The central and eastern geographic regions contain individuals that assign to Eastern and Central subpopulations, respectively, indicating that these barriers are not complete. Our discovery of previously undetected geographic and genomic structure in the spruce budworm suggests that further population modelling of this ecologically important insect should consider regional differentiation, potentially co‐adapted blocks of genes, and gene flow between subpopulations.

The elm zigzag sawfly, Aproceros leucopoda Takeuchi (Hymenoptera: Argidae), was reported for the first time in North America during the summer of 2020. Characteristic zigzag defoliation was reported in the province of Québec, Canada, on the community science website, iNaturalist. Field trips conducted to the site resulted in the collection of live specimens (a few larvae and a cocoon from which an adult emerged) and onsite observation of diagnostic defoliation and empty cocoons, confirming the presence of this exotic species in Canada. Subsequent inspection of elm trees by naturalists and scientists in the south of the province led to the conclusion that the species is more widely distributed than first expected and that the invasion is not localised to a small area. Preliminary genetic data pointed to a possible European origin of the Canadian population, but conclusive assignment to source will require examination of more specimens and the collection of reference sequences from different European and Asian populations. This is a good example of the importance of community science in the detection of new invasive species.

For regulatory purposes, the name ‘Asian gypsy moth’ refers to a group of closely related Asian Lymantria species and subspecies whose female moths display flight capability, a trait believed to confer enhanced invasiveness relative to the European gypsy moth, Lymantria dispar dispar, whose females are flightless. Lymantria albescens and Lymantria postalba are Asian gypsy moths occurring in the southern Ryukyu Islands and in the northern Ryukyu and adjacent Kyushu and Shikoku Islands of Japan, respectively. Although once considered subspecies of L. dispar, their status as distinct species, relative to the latter, is now well established. While postalba was subsequently considered a subspecies of L. albescens, largely on the basis of differences in forewing ground colour in males, both taxa were later given distinct species status by Pogue & Schaefer (2007) following their revision of the genus Lymantria. Here, we re‐examined the validity of this revised status through the sequencing of a large portion of the mitochondrial genome (c. 60%) and multiple nuclear marker genes [elongation factor 1‐alpha (Ef‐1α), wingless (Wgl), internal transcribed spacer 2 (ITS‐2), ribosomal protein S5 (RpS5)] in representative specimens of both taxa and other Lymantria species, including L. monacha, L. xylina, L. mathura and members of the L. dispar + L. umbrosa clade. A comparison of the number of substitutions in these genomic regions among the taxa we considered showed lower or equivalent variation between L. albescens and L. postalba compared with subspecies of L. dispar, for mitochondrial and nuclear sequences, respectively. This finding was reflected in the maximum likelihood trees generated independently for mitochondrial and nuclear data, where L. albescens and L. postalba formed, in both analyses, a short‐branch sister clade basal to the L. dispar + L. umbrosa clade. We further sequenced three markers [cytochrome c oxydase 1 (COI), EF‐1α, Wgl] in multiple L. albescens–L. postalba specimens collected along a south‐to‐north transect across the Ryukyu Arc and observed no clear distinction among the sampled specimens as a function of taxonomic designation. We conclude that L. albescens and L. postalba form a single species, with postalba representing a darker‐winged morph along an apparent south‐to‐north wing colour cline. Accordingly, L. postalba is relegated to synonymy under L. albescens (syn.n.).

A total of 385 Euphaedra eberti Aurivillius, 1898, adults collected between 2012 and 2018 in the vicinity of Bangui, Central African Republic, were examined for intraspecific morphological variability, genetic diversity and genitalia structure. The species shows significant wing pattern variability. Two main morphotypes were identified in the set: the nominate form eberti, and the one comprising specimens with a red patch, form rubromaculata. However, both forms had similar genitalic structures and shared some specific wing marks, in addition to displaying the same COI (i.e., barcode region of the mitochondrial cytochrome c oxidase subunit I gene) haplotype, strongly suggesting that the two morphologically distinct forms belong to the same species, E. eberti. The causes of this variability remain unclear.

The Asian gypsy moth (AGM) is considered a very serious invasive threat in North America. For this reason, it is subjected to a bio-surveillance program that includes an extensive network of pheromone traps. For regulatory purposes, the term “AGM” designates a group of Asian Lymantria species and subspecies, comprising two L. dispar subspecies (L. d. asiatica and L. d. japonica), and three closely related species (L. umbrosa, L. albescens and L. postalba). These moths are attracted to the same pheromone as the European gypsy moth (EGM), L. dispar dispar, which is already established in North America and typically makes up the bulk of moths caught in gypsy moth pheromone traps. These different Lymantria taxa are difficult to distinguish from one another using morphological characters alone. Here, we designed a TaqMan triplex assay capable of detecting AGM in bulk pheromone trap samples. The assay targets SNPs found in three different mitochondrial genes. Using a DNA dilution series, we show that the assay can detect AGM taxa at AGM:EGM dilution ratios ≥ 1:1000. The assay was validated using batch DNA extractions of moth legs tested at a 1:100 AGM:EGM leg ratio, a proportion that is around the operational limit for a single pheromone trap. The assay provided correct identification for all AGM taxa tested. An experiment examining the integrity of DNA extracted from gypsy moths left in pheromone traps under field conditions for up to 4 months indicated that DNA quality remains sufficient, during that period, for the present assay to remain accurate.

The European gypsy moth, Lymantria dispar dispar (LDD), is an invasive insect and a threat to urban trees, forests and forest-related industries in North America. For use as a comparator with a previously published genome based on the LD652 pupal ovary-derived cell line, as well as whole-insect genome sequences obtained from the Asian gypsy moth subspecies L. dispar asiatica and L. dispar japonica, the whole-insect LDD genome was sequenced, assembled and annotated. The resulting assembly was 998 Mb in size, with a contig N50 of 662 Kb and a GC content of 38.8%. Long interspersed nuclear elements constitute 25.4% of the whole-insect genome, and a total of 11,901 genes predicted by automated gene finding encoded proteins exhibiting homology with reference sequences in the NCBI NR and/or UniProtKB databases at the most stringent similarity cutoff level (i.e., the gold tier). These results will be especially useful in developing a better understanding of the biology and population genetics of L. dispar and the genetic features underlying Lepidoptera in general.

Reducing the use of broad-spectrum insecticides is one of the many challenges currently faced by insect pest management practitioners. For this reason, efforts are being made to develop environmentally benign pest-control products through bio-rational approaches that aim at disrupting physiological processes unique to specific groups of pests. Perturbation of hormonal regulation of insect development and reproduction is one such strategy. It has long been hypothesized that some enzymes in the juvenile hormone biosynthetic pathway of moths, butterflies and caterpillars (order Lepidoptera) display unique structural features that could be targeted for the development of Lepidoptera-specific insecticides, a promising avenue given the numerous agricultural and forest pests belonging to this order. Farnesyl diphosphate synthase, FPPS, is one such enzyme, with recent work suggesting that it has structural characteristics that may enable its selective inhibition. This review synthesizes current knowledge on FPPS and summarizes recent advances in its use as a target for insecticide development.

International audience

Annual monitoring of mortality agents in the course of a spruce budworm (Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)) population cycle is essential to understanding the factors governing the rise and collapse of outbreaks. To date, assessments of causes of budworm mortality have relied on laboratory rearing of field-collected larvae, followed by visual identification of emerging parasitoids and/or microscopic analysis of pathogens in larval carcasses. Although this approach has provided vital information on the abundance and identity of mortality agents, the procedure is labor-intensive and has limits in terms of accuracy. To overcome these shortcomings, we developed a molecular identification tool that makes use of real-time quantitative PCR (qPCR) and TaqMan&reg, technologies. The tool relies on taxon-specific molecular variants (single nucleotide polymorphism [SNP] markers) found in mitochondrial (COI) and nuclear (28S rDNA) genes, for parasitoids, and in the nuclear SSU rDNA gene for microsporidian pathogens, these are then used as molecular signatures targeted by qPCR primers and TaqMan probes. Thus, the design of several sets of primers and probes deployed in multiplex format enables the identification of natural enemies via a molecular sorting process, bypassing barcode sequencing. Crude budworm DNA extracts are processed through a first module that detects dipteran and hymenopteran parasitoids, and microsporidian infections. Positive samples are then processed for species determination using three additional modules, enabling the identification of 20 common natural enemies of the spruce budworm. The tool has been fully validated using DNA samples from all comprised taxa, and both its sensitivity and accuracy compared favorably with the rearing-based method in an analysis of field-collected budworms. Using this tool, sample processing can be completed within two days, does not require larval rearing, provides accurate species identification, and can be conducted by technical staff without extensive molecular biology or insect taxonomy training.

Global trade and climate change are responsible for a surge in foreign invasive species and emerging pests and pathogens across the world. Early detection and surveillance activities are essential to monitor the environment and prevent or mitigate future ecosystem impacts. Molecular diagnostics by DNA testing has become an integral part of this process. However, for environmental applications, there is a need for cost-effective and efficient point-of-use DNA testing to obtain accurate results from remote sites in real-time. This requires the development of simple and fast sample processing and DNA extraction, room-temperature stable reagents and a portable instrument. We developed a point-of-use real-time Polymerase Chain Reaction system using a crude buffer-based DNA extraction protocol and lyophilized, pre-made, reactions for on-site applications. We demonstrate the use of this approach with pathogens and pests covering a broad spectrum of known undesirable forest enemies: the fungi Sphaerulina musiva, Cronartium ribicola and Cronartium comandrae, the oomycete Phytophthora ramorum and the insect Lymantria dispar. We obtained positive DNA identification from a variety of different tissues, including infected leaves, pathogen spores, or insect legs and antenna. The assays were accurate and yielded no false positive nor negative. The shelf-life of the lyophilized reactions was confirmed after one year at room temperature. Finally, successful tests conducted with portable thermocyclers and disposable instruments demonstrate the suitability of the method, named in Situ Processing and Efficient Environmental Detection (iSPEED), for field testing. This kit fits in a backpack and can be carried to remote locations for accurate and rapid detection of pests and pathogens.

Background Transposable elements (TEs) are common and often present with high copy numbers in cellular genomes. Unlike in cellular organisms, TEs were previously thought to be either rare or absent in viruses. Almost all reported TEs display only one or two copies per viral genome. In addition, the discovery of pandoraviruses with genomes up to 2.5-Mb emphasizes the need for biologists to rethink the fundamental nature of the relationship between viruses and cellular life. Results Herein, we performed the first comprehensive analysis of miniature inverted-repeat transposable elements (MITEs) in the 5170 viral genomes for which sequences are currently available. Four hundred and fifty one copies of ten miniature inverted-repeat transposable elements (MITEs) were found and each MITE had reached relatively large copy numbers (some up to 90) in viruses. Eight MITEs belonging to two DNA superfamilies (hobo/Activator/Tam3 and Chapaev–Mirage–CACTA) were for the first time identified in viruses, further expanding the organismal range of these two superfamilies. TEs may play important roles in shaping the evolution of pandoravirus genomes, which were here found to be very rich in MITEs. We also show that putative autonomous partners of seven MITEs are present in the genomes of viral hosts, suggesting that viruses may borrow the transpositional machinery of their cellular hosts’ autonomous elements to spread MITEs and colonize their own genomes. The presence of seven similar MITEs in viral hosts, suggesting horizontal transfers (HTs) as the major mechanism for MITEs propagation. Conclusions Our discovery highlights that TEs contribute to shape genome evolution of pandoraviruses. We concluded that as for cellular organisms, TEs are part of the pandoraviruses’ diverse mobilome. Electronic supplementary material The online version of this article (10.1186/s13100-018-0125-4) contains supplementary material, which is available to authorized users.

Genome structure characterization can contribute to a better understanding of processes such as adaptation, speciation, and karyotype evolution, and can provide useful information for refining genome assemblies. We studied the genome of an important North American boreal forest pest, the spruce budworm, Choristoneura fumiferana, through a combination of molecular cytogenetic analyses and construction of a high-density linkage map based on single nucleotide polymorphism (SNP) markers obtained through a genotyping-by-sequencing (GBS) approach. Cytogenetic analyses using fluorescence in situ hybridization methods confirmed the haploid chromosome number of n = 30 in both sexes of C. fumiferana and showed, for the first time, that this species has a WZ/ZZ sex chromosome system. Synteny analysis based on a comparison of the Bombyx mori genome and the C. fumiferana linkage map revealed the presence of a neo-Z chromosome in the latter species, as previously reported for other tortricid moths. In this neo-Z chromosome, we detected an ABC transporter C2 (ABCC2) gene that has been associated with insecticide resistance. Sex-linkage of the ABCC2 gene provides a genomic context favorable to selection and rapid spread of resistance against Bacillus thuringiensis serotype kurstaki (Btk), the main insecticide used in Canada to control spruce budworm populations. Ultimately, the linkage map we developed, which comprises 3586 SNP markers distributed over 30 linkage groups for a total length of 1720.41 cM, will be a valuable tool for refining our draft assembly of the spruce budworm genome.

Forest invasive alien species are a major threat to ecosystem stability and can have enormous economic and social impacts. For this reason, preventing the introduction of Asian gypsy moths (AGM; Lymantria dispar asiatica and L. d. japonica) into North America has been identified as a top priority by North American authorities. The AGM is an important defoliator of a wide variety of hardwood and coniferous trees, displaying a much broader host range and an enhanced dispersal ability relative to the already established European gypsy moth (L. d. dispar). Although molecular assays have been developed to help distinguish gypsy moth subspecies, these tools are not adequate for tracing the geographic origins of AGM samples intercepted on foreign vessels. Yet, this type of information would be very useful in characterizing introduction pathways and would help North American regulatory authorities in preventing introductions. The present proof-of- concept study assessed the potential of single nucleotide polymorphism (SNP) markers, obtained through genotyping by sequencing (GBS), to identify the geographic origins of gypsy moth samples. The approach was applied to eight laboratory-reared gypsy moth populations, whose original stocks came from locations distributed over the entire range of L. dispar, comprising representatives of the three recognized subspecies. The various analyses we performed showed strong differentiation among populations (FST ≥ 0.237), enabling clear distinction of subspecies and geographic variants, while revealing introgression near the geographic boundaries between subspecies. This strong population structure resulted in 100% assignment success of moths to their original population when 2,327 SNPs were used. Although the SNP panels we developed are not immediately applicable to contemporary, natural populations because of distorted allele frequencies in the laboratory-reared populations we used, our results attest to the potential of genomewide SNP markers as a tool to identify the geographic origins of intercepted gypsy moth samples.

The gypsy moth, Lymantria dispar L., is one of the most destructive forest pests in the world. While the subspecies established in North America is the European gypsy moth (L. dispar dispar), whose females are flightless, the two Asian subspecies, L. dispar asiatica and L. dispar japonica, have flight-capable females, enhancing their invasiveness and warranting precautionary measures to prevent their permanent establishment in North America. Various molecular tools have been developed to help distinguish European from Asian subspecies, several of which are based on the mitochondrial barcode region. In an effort to identify additional informative markers, we undertook the sequencing and analysis of the mitogenomes of 10 geographic variants of L. dispar, including two or more variants of each subspecies, plus the closely related L. umbrosa as outgroup. Several regions of the gypsy moth mitogenomes displayed nucleotide substitutions with potential usefulness for the identification of subspecies and/or geographic origins. Interestingly, the mitogenome of one geographic variant displayed significant divergence relative to the remaining variants, raising questions about its taxonomic status. Phylogenetic analyses placed this population from northern Iran as basal to the L. dispar clades. The present findings will help improve diagnostic tests aimed at limiting risks of AGM invasions.

The increase in global trade is responsible for a surge in foreign invasive species introductions across the world. Early detection and surveillance activities are essential to prevent future invasions. Molecular diagnostics by DNA testing has become an integral part of this process. However, for environmental applications, there is a need for cost-effective and efficient point-of-use DNA testing that would allow for the collection of results in real-time away from laboratory facilities. To achieve this requires the development of simple and fast sample processing and DNA extraction, room-temperature stable reagents and a portable instrument. We conducted a series of tests using a crude buffer-based DNA extraction protocol and lyophilized, pre-made, reactions to address the first two requirements. We chose to demonstrate the use of this approach with organisms that cover a broad spectrum of known undesirable insects and pathogens: the ascomycete Sphaerulina musiva, the oomycete Phytophthora ramorum, the basidiomycetes Cronartium ribicola and Cronartium comandrae and the insect Lymantria dispar. Tests performed from either infected leaf material or spores (pathogens), or legs and antenna (insects). We were able to obtain positive amplification for the targeted species in all the samples tested. The shelf-life of the lyophilized reactions was assessed, confirming the stability of over a year at room temperature. Finally, successful tests conducted with portable thermocyclers and disposable plastics, demonstrating the suitability of the method, named in Situ Processing and Efficient Environment Detection (iSPEED), for field testing. This kit is ideally adapted to field testing as it fits in a backpack and can be carried to remote locations.

The spruce budworm, Choristoneura fumiferana, is presumed to be panmictic across vast regions of North America. We examined the extent of panmixia by genotyping 3,650 single nucleotide polymorphism (SNP) loci in 1975 individuals from 128 collections across the continent. We found three spatially structured subpopulations: Western (Alaska, Yukon), Central (southeastern Yukon to the Manitoba–Ontario border), and Eastern (Manitoba–Ontario border to the Atlantic). Additionally, the most diagnostic genetic differentiation between the Central and Eastern subpopulations was chromosomally restricted to a single block of SNPs that may constitute an island of differentiation within the species. Geographic differentiation in the spruce budworm parallels that of its principal larval host, white spruce (Picea glauca), providing evidence that spruce budworm and spruce trees survived in the Beringian refugium through the Last Glacial Maximum and that at least two isolated spruce budworm populations diverged with spruce/fir south of the ice sheets. Gene flow in the spruce budworm may also be affected by mountains in western North America, habitat isolation in West Virginia, regional adaptations, factors related to dispersal, and proximity of other species in the spruce budworm species complex. The central and eastern geographic regions contain individuals that assign to Eastern and Central subpopulations, respectively, indicating that these barriers are not complete. Our discovery of previously undetected geographic and genomic structure in the spruce budworm suggests that further population modelling of this ecologically important insect should consider regional differentiation, potentially co‐adapted blocks of genes, and gene flow between subpopulations., We examined the population genomic structure of the spruce budworm, Choristoneura fumiferana, and found three spatially structured subpopulations: Western (Alaska, Yukon), Central (southeastern Yukon to the Manitoba–Ontario border) and Eastern (Manitoba–Ontario border to the Atlantic). Geographic differentiation in the spruce budworm parallels that of its principal larval host, white spruce (Picea glauca), providing evidence that spruce budworm and spruce trees survived in the Beringian refugium through the Last Glacial Maximum and that at least two isolated spruce budworm populations diverged with spruce/fir south of the ice sheets. Gene flow in the spruce budworm may also be affected by mountains in western North America, habitat isolation in West Virginia, regional adaptations, factors related to dispersal, and proximity of other species in the spruce budworm complex.

Cecropins form a family of amphipathic α-helical cationic peptides with broad-spectrum antibacterial properties and potent anticancer activity. The emergence of bacteria and cancer cells showing resistance to cationic antimicrobial peptides (CAMPs) has fostered a search for new, more selective and more effective alternatives to CAMPs. With this goal in mind, we looked for cecropin homologs in the genome and transcriptome of the spruce budworm, Choristoneura fumiferana. Not only did we find paralogs of the conventional cationic cecropins (Cfcec+), our screening also led to the identification of previously uncharacterized anionic cecropins (Cfcec−), featuring a poly-L-aspartic acid C-terminus. Comparative peptide analysis indicated that the C-terminal helix of Cfcec− is amphipathic, unlike that of Cfcec+, which is hydrophobic. Interestingly, molecular dynamics simulations pointed to the lower conformational flexibility of Cfcec− peptides, relative to that of Cfcec+. Phylogenetic analysis suggests that the evolution of distinct Cfcec+ and Cfcec− peptides may have resulted from an ancient duplication event within the Lepidoptera. Our analyses also indicated that Cfcec− shares characteristics with entericidins, which are involved in bacterial programmed cell death, lunasin, a peptide of plant origins with antimitotic effects, and APC15, a subunit of the anaphase-promoting complex. Finally, we found that both anionic and cationic cecropins contain a BH3-like motif (G-[KQR]-[HKQNR]-[IV]-[KQR]) that could interact with Bcl-2, a protein involved in apoptosis; this observation is congruent with previous reports indicating that cecropins induce apoptosis. Altogether, our observations suggest that cecropins may provide templates for the development of new anticancer drugs.Graphical abstractHighlightsGenes encoding novel anionic cecropins (Cfcec−), featuring a C-terminal poly-L-aspartic acid, were found in the genome of the spruce budworm, Choristoneura fumiferana.Divergence between Cfcec+ and Cfcec− could be the result of an ancient duplication event within the Lepidoptera.There is an apparent relationship between motifs observed in cecropin peptides and apoptosis.Anionic cecropins from the spruce budworm display characteristics suggesting they could have anticancer activity

Farnesyl diphosphate synthase (FPPS) is an enzyme from the class of short chain ( E)-prenyltransferases that catalyzes the condensation of two molecules of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) to generate the C15 product FPP. In insects, FPPS plays a key role in the biosynthesis of the morphogenetic and gonadotropic “juvenile hormone” (JH). Lepidopteran genomes encode two very distinct FPPS paralogs , one of which (“type-II”) is expressed almost exclusively in the JH-producing glands, the corpora allata . This paralog has been hypothesized to display structural features that enable the binding of the bulkier precursors required for the biosynthesis of lepidopteran ethyl-branched JHs. Here, we report on the first crystal structures of an insect FPPS solved to date. Apo, ligand-bound, and inhibitor-bound structures of type-II FPPS (FPPS2) from the spruce budworm , Choristoneura fumiferana (Order: Lepidoptera), were obtained. Comparison of apo and inhibitor-bound enzymes revealed differences in both inhibitor binding and structural plasticity of CfFPPS2 compared to other FPPSs. Our data showed that IPP is not essential to the closure of the C-terminal tail . Ortho-substituted pyridinium bisphosphonates, previously shown to inhibit CfFPPS2, bound to the allylic site, as predicted; however, their alkyl groups were oriented towards the homoallylic binding site, with the bulkier propyl-substituted inhibitor penetrating deeply into the IPP binding pocket. The current study sheds light on the structural basis of substrate specificity of type-II FPPS of the spruce budworm. Through a comparison with other inhibitor-bound FPPSs, we propose several approaches to improve inhibitor selectivity and potency.

International audience; Mitogenomes are useful markers for phylogenetic studies across a range of taxonomic levels. Here, we focus on mitogenome variation across the tortricid moth genus Choristoneura and particularly the spruce budworm (Choristoneura fumiferana) species complex, a notorious pest group of North American conifer forests. Phylogenetic relationships of Tortricidae, representing two subfamilies, four tribes and nine genera, were analyzed using 21 mitogenomes. These included six newly-sequenced mitogenomes for species in the spruce budworm complex plus three additional Choristoneura species and 12 previously published mitogenomes from other tortricids and one from the Cossidae. We evaluated the phylogenetic informativeness of the mitogenomes and reconstructed a time-calibrated tree with fossil and secondary calibrations. We found that tortricid mitogenomes had conserved protein and ribosomal regions, and analysis of all protein-coding plus ribosomal genes together provided an efficient marker at any taxonomic rank. The time-calibrated phylogeny showed evolutionary convergence of conifer feeding within Choristoneura, with two independent lineages, the Nearctic spruce budworm complex and the Palearctics pecies Choristoneura murinana, both shifting onto conifers about 11 million years ago from angiosperms. These two host-plant shifts both occurred after the formation of boreal forest in the late Miocene. Haplotype diversification within the spruce budworm complex occurred in the last 4 million years, and is probably linked to the initial cooling cycles of the Northern Hemisphere in the Pliocene.

Polydnaviruses (PDVs) are symbiotic viruses carried by endoparasitic wasps and transmitted to caterpillar hosts during parasitization. Although they share several features, including a segmented dsDNA genome, a unique life cycle where replication is restricted to the wasp host, and immunodepressive/developmental effects on the caterpillar host, PDVs carried by ichneumonid and braconid wasps (referred to as ichnoviruses and bracoviruses, respectively) have different evolutionary origins. In addition, ichnoviruses (IVs) form two distinct lineages, with viral entities found in wasps belonging to the subfamilies Campopleginae and Banchinae displaying strikingly different virion morphologies and genomic features. However, the current description for banchine IVs is based on the characterization of a single species, namely that of the Glypta fumiferanae IV (GfIV). Here we provide an ultrastructural and genomic analysis of a second banchine IV isolated from the wasp Apophua simplicipes, and we show that this virus shares many features with GfIV, including a multi-nucleocapsid virion, an aggregate genome size of ~300 kb, genome segments

Tranosema rostrale ichnovirus (TrIV) is a polydnavirus (PDV) transmitted by the endoparasitic wasp T. rostrale to its host Choristoneura fumiferana during oviposition. PDV genes are expressed in infected caterpillars, causing physiological disturbances that promote the survival of the developing endoparasite. The previously sequenced genome of TrIV contains ~86 genes organized in multigene families and distributed on multiple segments of circular dsDNA. Among these, the ‘T. rostrale virus’ (TrV) family comprises seven genes that are absent in other PDV genomes examined to date and whose function(s) remain(s) unknown. Here, we initiated a functional analysis of the TrV family using qPCR, transfection and RNAi approaches. TrV family genes were weakly expressed in wasp ovaries, but some displayed high transcript abundance in parasitized caterpillars. Whilst TrV1 was the most highly transcribed TrV gene in infected caterpillars, transcript levels for TrV5 and TrV6 were nearly undetectable, indicating that they may be pseudogenes. Temporal and tissue-specific patterns of transcript abundance were similar for all expressed TrV family genes, indicative of an apparent lack of difference in function or tissue specificity. Infection of Cf-203 and Sf-21 insect cells with TrIV led to a dose-dependent inhibition of cell proliferation with no sign of apoptosis. Whilst similar inhibition was observed following transfection of cells with a cloned genome segment carrying the TrV1 gene, RNA interference targeting TrV1 largely restored cell growth in TrIV-infected cells, indicating that TrV1 expression was responsible for the observed inhibition. We suggest that TrV genes may contribute to host developmental disruption by interfering with host-cell proliferation during parasitism.

Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species (L. umbrosa, L. albescens, L. postalba), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous Lymantria species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three L. dispar subspecies (including the European gypsy moth, L. dispar dispar), the three other Lymantria species comprising the AGM complex, plus five additional Lymantria species that pose a threat to forests in North America. The suite of assays is built as a "molecular key" (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5' region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3' region, as well as on the presence of an indel in the "FS1" nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into L. dispar dispar. These assays have the advantage of providing rapid and accurate identification of ten Lymantria species and subspecies considered potential FIAS.

The complete mitogenome of the Emerald Ash Borer (EAB, Agrilus planipennis) was obtained by gleaning mitochondrial sequences from whole-genome Illumina sequencing data. The circular genome has 15,942 base pairs and contains 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and an A–T-rich region. All PCGs begin with ATN codons. The nucleotide composition is highly asymmetric (31.65% A, 40.25% T, 17.39% G, 10.71% C), with an overall A–T content of 71.9%. Phylogenetic analysis based on insect mitogenomes indicated that EAB is closely related to other Buprestoidea species, clustering most closely with Chrysochroa fulgidissima.

Isoprenoids form an extensive group of natural products involved in a number of important biological processes. Their biosynthesis proceeds through sequential 1'-4 condensations of isopentenyl diphosphate (C5) with an allylic acceptor, the first of which is dimethylallyl diphosphate (C5). The reactions leading to the production of geranyl diphosphate (C10), farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20), which are the precursors of mono-, sesqui- and diterpenes, respectively, are catalyzed by a group of highly conserved enzymes known as short-chain isoprenyl diphosphate synthases, or prenyltransferases. In recent years, the sequences of many new prenyltransferases have become available, including those of several plant and animal geranyl diphosphate synthases, revealing novel mechanisms of product chain-length selectivity and an intricate evolutionary path from a putative common ancestor. Finally, there is considerable interest in designing inhibitors specific to short-chain prenyltransferases, for the purpose of developing new drugs or pesticides that target the isoprenoid biosynthetic pathway.

The endoparasitic wasp Tranosema rostrale transmits an ichnovirus to its lepidopteran host, Choristoneura fumiferana, during parasitization. As shown for other ichnoviruses, the segmented dsDNA genome of the T. rostrale ichnovirus (TrIV) features several multi-gene families, including the repeat element (rep) family, whose products display no known similarity to non-ichnovirus proteins, except for a homologue encoded by the genome of the Helicoverpa armigera granulovirus; their functions remain unknown. This study applied linear regression of efficiency analysis to real-time PCR quantification of transcript abundance for all 17 TrIV rep open reading frames (ORFs) in parasitized and virus-injected C. fumiferana larvae, as well as in T. rostrale ovaries and head–thorax complexes. Although transcripts were detected for most rep ORFs in infected caterpillars, two of them clearly outnumbered the others in whole larvae, with a tendency for levels to drop over time after infection. The genome segments bearing the three most highly expressed rep genes in parasitized caterpillars were present in higher proportions than other rep-bearing genome segments in TrIV DNA, suggesting a possible role for gene dosage in the regulation of transcription level. TrIV rep genes also showed important differences in the relative abundance of their transcripts in specific tissues (cuticular epithelium, the fat body, haemocytes and the midgut), implying tissue-specific roles for individual members of this gene family. Significantly, no rep transcripts were detected in T. rostrale head–thorax complexes, whereas some were abundant in ovaries. There, the transcription pattern was completely different from that observed in infected caterpillars, suggesting that some rep genes have wasp-specific functions.

This overview examines recent progress in the application of molecular tools to the study of insect biology and the development of pest management strategies. The sequencing and annotation of insect genomes, coupled with analyses using comparative genomics, are providing new insights into the molecular underpinnings of insect-specific processes and shedding light on their evolutionary relationships. Researchers investigate the functions of insect genes using indirect approaches such as expression profiling, and direct methods such as insertional mutagenesis and RNA interference. Biotechnological applications to pest management include the development of resistant crops and trees that express insect-specific toxins, the design of microbial agents with enhanced insecticidal potency, and the engineering of insects that can transfer lethal genes to natural populations following their mass release in the field. Comparative genomics analyses also make it possible to identify insect-specific genes that ...