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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.

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.

Periodic and spatially synchronous outbreaks of insect pests have dramatic consequences for boreal and sub-boreal forests. Within these multitrophic systems, parasitoids can be stabilizing agents by dispersing toward patches containing higher host density (the so-called birdfeeder effect). However, we know little about the dispersal abilities of parasitoids in continuous forested landscapes, limiting our understanding of the spatiotemporal dynamics of host-parasitoid systems, and constraining our ability to predict forest resilience in the context of global changes. In this study, we investigate the spatial genetic structure and spatial variation in genetic diversity of two important species of spruce budworm larval parasitoids during outbreaks: Apanteles fumiferanae Viereck (Braconidae) and Glypta fumiferanae (Viereck) (Ichneumonidae). Using parasitoids sampled in 2014 from 26 and 29 locations across a study area of 350,000 km2 , we identified 1,012 and 992 neutral SNP loci for A. fumiferanae (N = 279 individuals) and G. fumiferanae (N = 382), respectively. Using DAPC, PCA, AMOVA, and IBD analyses, we found evidence for panmixia and high genetic connectivity for both species, matching the previously described genetic structure of the spruce budworm within the same context, suggesting similar effective dispersal during outbreaks and high parasitoid population densities between outbreaks. We also found a significant negative relationship between genetic diversity and latitude for A. fumiferanae but not for G. fumiferanae, suggesting that northern range limits may vary by species within the spruce budworm parasitoid community. These spatial dynamics should be considered when predicting future insect outbreak severities in boreal landscapes.

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.).

Spatial synchrony is a common characteristic of spatio‐temporal population dynamics across many taxa. While it is known that both dispersal and spatially autocorrelated environmental variation (i.e., the Moran effect) can synchronize populations, the relative contributions of each, and how they interact, are generally unknown. Distinguishing these mechanisms and their effects on synchrony can help us to better understand spatial population dynamics, design conservation and management strategies, and predict climate change impacts. Population genetic data can be used to tease apart these two processes as the spatio‐temporal genetic patterns they create are expected to be different. A challenge, however, is that genetic data are often collected at a single point in time, which may introduce context‐specific bias. Spatio‐temporal sampling strategies can be used to reduce bias and to improve our characterization of the drivers of spatial synchrony. Using spatio‐temporal analyses of genotypic data, our objective was to identify the relative support for these two mechanisms to the spatial synchrony in population dynamics of the irruptive forest insect pest, the spruce budworm (Choristoneura fumiferana), in Quebec (Canada). AMOVA, cluster analysis, isolation by distance, and sPCA were used to characterize spatio‐temporal genomic variation using 1,370 SBW larvae sampled over four years (2012–2015) and genotyped at 3,562 SNP loci. We found evidence of overall weak spatial genetic structure that decreased from 2012 to 2015 and a genetic diversity homogenization among the sites. We also found genetic evidence of a long‐distance dispersal event over >140 km. These results indicate that dispersal is the key mechanism involved in driving population synchrony of the outbreak. Early intervention management strategies that aim to control source populations have the potential to be effective through limiting dispersal. However, the timing of such interventions relative to outbreak progression is likely to influence their probability of success.

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.

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. 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. We also estimated the antibacterial activity of Cfcec-2 and a ∆Cfce-2 peptide as AMPs by testing directly their ability in inhibiting bacterial growth in a disk diffusion assay and their potential for development of novel therapeutics.

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.

Encapsulation and melanisation are innate immune reactions of insects against foreign intruders such as parasitoids. In an earlier study, we observed that immature life stages of the endoparasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae) parasitizing Choristoneura fumiferana (Lepidoptera: Tortricidae) larvae experienced higher mortality due to encapsulation and melanisation when reared at high (30 °C) than at lower (10 °C, 20 °C) temperatures. Downregulation of T. rostrale polydnavirus genes in parasitized hosts and upregulation of two genes involved in the spruce budworm's melanisation process were identified as likely contributors to parasitoid mortality at high temperature. However, levels of transcripts of genes involved in the spruce budworm's cellular encapsulation process were not measured inasmuch as candidate genes, in the spruce budworm, had not yet been identified. In addition, our assessment of temperature-dependent encapsulation and melanisation of foreign objects in spruce budworm larvae was only partial. To fill these knowledge gaps, we injected Sephadex™ beads into unparasitized spruce budworm larvae and assessed their encapsulation/melanisation after the insects had been held at three different temperatures (10, 20, and 30 °C), and we identified spruce budworm genes putatively involved in the encapsulation process and quantified their transcripts at the same three temperatures, using a qPCR approach. As expected, both encapsulation and melanisation of Sephadex™ beads increased as a function of temperature. At the molecular level, three of the five genes examined (Integrin β1, Hopscotch, Stat92E) clearly displayed temperature-dependent upregulation. The results of this study further support the hypothesis that a temperature-dependent increase in the encapsulation response of C. fumiferana against T. rostrale is due to the combined effects of reduced expression of polydnavirus genes and enhanced expression of host immune genes.

Populations are often exposed to multiple sources of gene flow, but accounts are lacking of the population genetic dynamics that result from these interactions or their effects on local evolution. Using a genomic clines framework applied to 1195 SNPs, we documented genome-wide, locus-specific patterns of introgression between Choristoneura occidentalis biennis spruce budworms and two ecologically divergent relatives, C. o. occidentalis and C. fumiferana, that it interacts with at alternate boundaries of its range. We observe contrasting hybrid indexes between these hybrid zones, no overlap in ‘gene flow outliers’ (clines showing relatively extreme extents or rates of locus-specific introgression), and variable linkage disequilibrium among those outliers. At the same time, correlated genome-wide rates of introgression between zones suggest the presence of processes common to both boundaries. These findings highlight the contrasting population genetic dynamics that can occur at separate frontiers of a single population, while also suggesting that shared patterns may frequently accompany cases of divergence-with-gene-flow that involve a lineage in common. Our results point to potentially complex evolutionary outcomes for populations experiencing multiple sources of gene flow. This article is protected by copyright. All rights reserved.

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.

Ichnoviruses (IVs) are mutualistic, double-stranded DNA viruses playing a key role in the successful parasitism of thousands of endoparasitoid wasp species. IV particles are produced exclusively in the female wasp reproductive tract. They are co-injected along with the parasitoid egg into caterpillar hosts upon parasitization. The expression of viral genes by infected host cells leads to an immunosuppressive state and delayed development of the host, two pathologies that are critical to the successful development of the wasp egg and larva. Ichnovirus is one of the two recognized genera within the family Polydnaviridae (polydnaviruses or PDVs), the other genus being Bracovirus (BV), associated with braconid wasps. IVs are associated with ichneumonid wasps belonging to the subfamilies Campopleginae and Banchinae; attempts to identify IV particles in other ichneumonid subfamilies have so far been unsuccessful. Functional studies targeting IV genes expressed in parasitized hosts, along with investigations of the molecular mechanisms responsible for viral morphogenesis in the female wasp, have resulted in a better understanding of the biology of these atypical viruses.

Species delimitation requires an assessment of varied traits that can contribute to reproductive isolation, as well as of the permanence of evolutionary differentiation among closely related lineages. Integrative taxonomy, including the combination of genome-wide molecular data with ecological data, offers an effective approach to this issue. We use genotyping-by-sequencing together with a review of ecological divergence to assess the traditionally recognized species status of three closely related members of the spruce budworm species complex, Choristoneura fumiferana (Clemens), C. occidentalis Freeman (=C. freemani Razowski) and C. biennis Freeman, each of which is a major defoliator of conifer forests. We sampled a broad region of overlap between these three taxa in Alberta and British Columbia (Canada) where potential for gene flow provides a strong test of the durability of divergence among lineages. A total of 2218 single nucleotide polymorphisms (SNPs) were assayed, and patterns of differentiation were evaluated under the biological, ecological, genotypic cluster and phylogenetic species concepts. Choristoneura fumiferana was genetically distinct with substantial barriers to genetic exchange with C. occidentalis and C. biennis. Conversely, divergence between C. occidentalis and C. biennis was limited to a small subset of outlier loci and was within the range observed within any one of the taxa. Considering both population genetic and ecological patterns of divergence, C. fumiferana should continue to be recognized as a distinct species, and C. biennis (syn.n.) should be treated as a subspecies (C. occidentalis biennis Freeman, 1967) of C. occidentalis, thereby automatically establishing the nominate name C. occidentalis occidentalis Freeman, 1967 for univoltine populations of this species.

Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C 5 ), with the allylic diphosphate primer dimethylallyl diphosphate (DMAPP; C 5 ) to generate the C 15 prenyl chain (FPP) used for protein prenylation as well as sterol and terpene biosynthesis. Here, we designed and prepared a series of pyridinium bisphosphonate (PyrBP) compounds, with the aim of selectively inhibiting FPPS of the lepidopteran insect order. FPPSs of Drosophila melanogaster and the spruce budworm, Choristoneura fumiferana , were inhibited by several PyrBPs, and as hypothesized, larger bisphosphonates were more selective for the lepidopteran protein and completely inactive towards dipteran and vertebrate FPPSs. Cell growth of a D. melanogaster cell line was adversely affected by exposure to PyrPBs that were strongly inhibitory to insect FPPS, although their effect was less pronounced than that observed upon exposure to the electron transport disrupter, chlorfenapyr. To assess the impact of PyrBPs on lepidopteran insect growth and development, we performed feeding and topical studies, using the tobacco hornworm, Manduca sexta , as our insect model. The free acid form of a PyrBP and a known bisphosphonate inhibitor of vertebrate FPPS, alendronate, had little to no effect on larval M. sexta ; however, the topical application of more lipophilic ester PyrBPs caused decreased growth, incomplete larval molting, cuticle darkening at the site of application, and for those insects that survived, the formation of larval–pupal hybrids. To gain a better understanding of the structural differences that produce selective lepidopteran FPPS inhibition, homology models of C. fumiferana and D. melanogaster FPPS ( Cf FPPS2, and Dm FPPS) were prepared. Docking of substrates and PyrBPs demonstrates that differences at the −3 and −4 positions relative to the first aspartate rich motif (FARM) are important factors in the ability of the lepidopteran enzyme to produce homologous isoprenoid structure and to be selectively inhibited by larger PyrBPs.

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.

Fuscumol [(2S,5E)-6,10-dimethyl-5,9-undecadien-2-ol] was recently identified as the male-produced aggregation pheromone of the brown spruce longhorn beetle, Tetropium fuscum (F.), and the eastern larch borer, Tetropium cinnamopterum Kirby. Several other species use this homoterpenoid alcohol motif, its ketone, or its acetate as part of their pheromone system. Investigation of the biosynthesis of this compound in these two Tetropium species demonstrated that geranylacetone [(5E)-6,10-dimethyl-5,9-undecadien-2-one] and farnesol [(2E,6E)-3,7,11-trimethyl-2,6,10-dodecatrien-1-ol] are both intermediates in this process. This was accomplished by applying deuterium-labeled geranylacetone and deuterium-labeled farnesol in separate experiments to the abdominal sterna of live T. fuscum and T. cinnamopterum and analyzing the deuterium labeling in the fuscumol and geranylacetone emitted by the insects with solid-phase microextraction (SPME) and GC/MS analysis. Deuterium labeling studies also showed that nerolidol[(3S,6E)-3-hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene] and 2,3-epoxyfarnesol are not intermediates in fuscumol or geranylacetone synthesis in T. fuscum or T. cinnamopterum. Tissue-specific expression of T. fuscum farnesyl diphosphate synthase (TfFPPS), an enzyme expected to provide a key fuscumol precursor, was measured. TfFPPS transcripts were relatively abundant in male midguts, but were also present at significant levels in other tissues.

Endoparasitoids face the challenge of overcoming the immune reaction of their hosts, which typically consists of encapsulation and melanisation of parasitoid eggs or larvae. Some endoparasitic wasps such as the solitary Tranosema rostrale (Hymenoptera: Ichneumonidae) that lay their eggs in larvae of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), have evolved a symbiotic relationship with a polydnavirus (PDV), which in turn helps them suppress the host’s immune response. We observed an increase in mortality of immature T. rostrale with increasing temperature, and we tested two hypotheses about the mechanisms involved: high temperatures (1) hamper the expression of T. rostrale PDV genes and (2) enhance the expression of spruce budworm immunity-related genes. Dissections of parasitized spruce budworm larvae reared at 30 °C revealed that most parasitoid eggs or larvae had died as a result of encapsulation and melanisation by the host. A qPCR analysis of T. rostrale PDV (TrIV) gene expression showed that the transcription of several TrIV genes in host larvae was downregulated at high temperature. On the other hand, encapsulation, but not melanisation, of foreign bodies in spruce budworm larvae was enhanced at high temperatures, as shown by the injection of Sephadex™ beads into larvae. However, at the molecular level, the transcription of genes related to spruce budworm’s melanisation process (prophenoloxidase 1 and 2) was upregulated. Our results support the hypothesis that a temperature-dependent increase of encapsulation response is due to the combined effects of reduced expression of TrIV genes and enhanced expression of host immune genes.

High throughput sequencing technologies have revolutionized the potential to reconcile incongruence between gene and species trees, and numerous approaches have been developed to take advantage of these advances. Genotyping-by-sequencing is becoming a regular tool for gathering phylogenetic data, yet comprehensive evaluations of phylogenetic methods using these data are sparse. Here we use multiple phylogenetic and population genetic methods for genotyping-by-sequencing data to assess species relationships in a group of forest insect pests, the spruce budworm (Choristoneura fumiferana) species complex. With few exceptions, all methods agree on the same relationships, most notably placing C. pinus as basal to the remainder of the group, rather than C. fumiferana as previously suggested. We found strong support for the monophyly of C. pinus, C. fumiferana, and C. retiniana, but more ambiguous relationships and signatures of introgression in a clade of western lineages, including C. carnana, C. lambertiana, C. occidentalis occidentalis, C. occidentalis biennis, and C. orae. This represents the most taxonomically comprehensive genomic treatment of the spruce budworm species group, which is further supported by the broad agreement among multiple methodologies.

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.

Isopentenyl diphosphate isomerase (IPPI) of the spruce budworm, Choristoneura fumiferana, and of the tobacco hornworm, Manduca sexta, was cloned and its catalytic properties assessed. In the presence of Mg(2+) or Mn(2+), the recombinant protein from C. fumiferana (CfIPPI) efficiently isomerized IPP to dimethylallyl diphosphate (DMAPP). While C. fumiferana IPPI transcript levels were evenly distributed in a wide variety of tissues, they were highly abundant in the corpora allata. Because IPPI plays an alternate role in lepidopteran juvenile hormone (JH) biosynthesis by catalyzing the isomerization of the homologous substrate, homoisopentenyl diphosphate (HIPP), the ability of CfIPPI to convert HIPP to homodimethylallyl diphosphate (HDMAPP) was also studied. As expected, HIPP isomerization was efficient and the formation of HDMAPP occurred, but the regiospecificity of the reaction was lower than previously found in M. sexta corpora allata homogenates and with purified Bombyx mori IPPI. Differences in inhibitory potency for several alkylated ammonium diphosphates and higher homologs of DMAPP were noted between CfIPPI and a vertebrate IPPI, suggesting that the lepidopteran enzyme has a larger active site cavity. To determine the structural factors responsible for homologous substrate coupling, site directed mutagenesis of several residues identified through sequence alignment and homology modeling analysis was performed. The results suggest that unlike other IPPIs, W216 (C. fumiferana numbering) works in concert with a tyrosine residue (Y105) to allow binding of larger substrates and to stabilize the high-energy intermediate formed during substrate isomerization.

The effects of a methanolic extract of the plant Haplophyllum tuberculatum (ME-Ht) and of teflubenzuron (TFB) were compared on several reproductive variables and ecdysteroid titers in the females of Locusta migratoria. The test products were administered orally to newly emerged females at doses of 1500 (ME-Ht) and 10 μg/female (TFB). The methanolic extract and TFB had comparable effects on several of the variables examined. Both significantly delayed the first oviposition and reduced fecundity and fertility. ME-Ht and TFB also displayed similar effects on ovarian growth, vitellogenesis and ecdysteroid titers. Both treatments induced a drop in hemolymph protein levels as well as a reduction in vitellogenin uptake by oocytes. This delay in oogenesis was accompanied by a resorption of terminal oocytes. However, whereas TFB completely blocked egg hatch, ME-Ht only had a modest inhibitory effect on this variable. Hemolymph and ovarian ecdysteroid titers, as measured by radioimmunoassay, were similar and low in both control and treated females, except for a peak observed only in control females at the end of vitellogenesis. We discuss the functional significance of the observed effects in the context of the putative modes of action of the methanolic plant extract and TFB.

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.

Proteins that are capable of binding chitin play essential roles in the synthesis and structural integrity of the insect cuticle and peritrophic matrix. In the course of developing expressed sequence tag (EST) libraries for the eastern spruce budworm, Choristoneura fumiferana, we identified an abundant cDNA encoding a homolog of the Drosophila “gasp” gene (Gene Analogous to Small Peritrophins). For the present work, we undertook the characterization of this new homolog, CfGasp, in an effort to identify its role during larval development. As shown for DmGasp, the C. fumiferana homolog was found to contain three type-2 chitin-binding domains (CBDs), which were also found in Gasp orthologs retrieved from GenBank. In a phylogenetic analysis, these Gasp proteins formed a tight cluster, distinct from the midgut-specific peritrophins with which they share the cysteine-containing CBDs so far considered absent from cuticular proteins. However, unlike what has been shown for peritrophins, CfGasp transcript levels were low in larval midguts and most abundant in epidermis, while they were low in trachea and ovaries. Transcript levels increased during larval molts in a pattern similar to that observed for exocuticular proteins in other insects. In addition, the recombinant protein was shown to be capable of binding chitin. Altogether, these results suggest a structural role for CfGasp in exocuticle formation.

A trypsin-like molting-related serine protease cDNA (CfMRSP) was cloned from the spruce budworm, Choristoneura fumiferana. The full-length CfMRSP comple- mentary DNA (cDNA) encoded a 43 kDa protein that contained a trypsin-like serine protease catalytic domain, but no clip domain. The C-terminal extension contained five cystein residues, which may allow the protein to form a homodimer through interchain disulfide bonds and regulate the activity of CfMRSP. Phylogenetic tree analysis showed that CfMRSP clusters with lepidopteran homologues such as serine protease 1 of Lonomia obliqua, hemolymph proteinase 20 (HP20), pattern recognition serine proteinase precursor (ProHP14) and a trypsin-like protein of Manduca sexta. Northern blot analysis of devel- opmental expression of CfMRSP indicated that its transcripts were found primarily in the epidermis and were produced during all of the tested stadia, from 4th instar larvae to pupae, but increased levels of CfMRSP transcripts were always found after each molt. A high level of the protein was found in the epidermis by immunohistochemistry analysis. Altogether these data suggest that CfMRSP plays a role in the epidermis during molting and metamorphosis.

The ichnovirus TrIV, transmitted by the endoparasitic wasp Tranosema rostrale to its lepidopteran host during oviposition, replicates asymptomatically in wasp ovaries and causes physiological dysfunctions in parasitized caterpillars. The need to identify ichnoviral genes responsible for disturbances induced in lepidopteran hosts has provided the impetus for the sequencing and annotation of ichnovirus genomes, including that of TrIV. In the latter, 86 putative genes were identified, including 35 that could be assigned to recognized ichnoviral gene families. With the aim of assessing the relative importance of each TrIV gene, as inferred from its level of expression, and evaluating the accuracy of the gene predictions made during genome annotation, the present study builds on an earlier qPCR quantification of transcript abundance of TrIV rep ORFs, in both lepidopteran and wasp hosts, extending it to other gene families as well as to a sample of unassigned ORFs. We show that the majority (91%) of putative ORFs assigned to known gene families are expressed in infected larvae, while this proportion is lower (67%) for a sample taken among the remaining ORFs. Selected members of the TrV and rep gene families are shown to be transcribed in infected larvae at much higher levels than genes from any other TrIV gene family, pointing to their likely involvement in host subjugation. In wasp ovaries, the transcriptional profile is dominated by a rep gene and a member of a newly described gene family encoding secreted proteins displaying a novel cysteine motif, which we identified among previously unassigned ORFs.

Immune challenge in arthropods is frequently accompanied by melanization of the hemolymph, a reaction triggered by the activa- tion of prophenoloxidase (PPO). Because their immature stages are spent inside the hemocoel of insect larvae, endoparasitoids have evolved strategies to escape or counter melanin formation. Very little molecular information is available on these endoparasitoid counterstrategies. We have sought to shed light on the inhibition of melanization in the spruce budworm, Choristoneura fumiferana, by the parasitic wasp Tranosema rostrale, by cloning two host PPO homologs and studying their transcriptional regulation after parasitization. The two polypeptides are encoded by transcripts of ~3.3 kb (for Cf PPO1) and 3.0 kb (for Cf PPO2) and possess structural features typical of other insect PPOs. While there appears to be a single Cf PPO2 gene in the C. fumiferana genome, we detected three Cf PPO1 mRNA variants displaying insertions/deletions in the 3' untranslated region, suggesting that there may be more than one Cf PPO1 gene copy. Both Cf PPO1 and Cf PPO2 were expressed at high levels in C. fumiferana 6th instars, and parasitization by T. rostrale had no apparent impact on the level of their transcripts. Injection of a large dose (0.5 female- equivalent) of polydnavirus-laden calyx fluid extracted from T. rostrale, which is known to inhibit melanization in C. fumiferana, only caused a transient decrease in Cf PPO1 and Cf PPO2 transcript accumulation at 2-3 d post injection. It thus appears that transcriptional downregulation of C. fumiferana PPO by T. rostrale plays a minor role in the inhibition of hemolymph melanization in this host-parasitoid system. Arch. Insect Biochem. Physiol. 67:188-201, 2008. Published 2008 Wiley-Liss, Inc. †

Unlike most viruses, the mature ichnovirus particle possesses two unit membrane envelopes. Following loss of the outer membranein vivo, nucleocapsids are believed to gain entry into the cytosol via a membrane fusion event involving the inner membrane and the plasma membrane of susceptible host cells; accordingly, experimentally induced damage to the outer membrane might be expected to increase infectivity. Here, in an attempt to develop anin vitromodel system for studying ichnovirus infection, we show that digitonin-induced disruption of the virion outer membrane not only increases infectivity, but also uncovers an activity not previously associated with any polydnavirus: fusion from without.

Polydnaviruses form a group of unconventional double-stranded DNA (dsDNA) viruses transmitted by endoparasitic wasps during egg laying into caterpillar hosts, where viral gene expression is essential to immature wasp survival. A copy of the viral genome is present in wasp chromosomes, thus ensuring vertical transmission. Polydnaviruses comprise two taxa, Bracovirus and Ichnovirus , shown to have distinct viral ancestors whose genomes were “captured” by ancestral wasps. While evidence indicates that bracoviruses derive from a nudivirus ancestor, the identity of the ichnovirus progenitor remains unknown. In addition, ichnoviruses are found in two ichneumonid wasp subfamilies, Campopleginae and Banchinae, where they constitute morphologically and genomically different virus types. To address the question of whether these two ichnovirus subgroups have distinct ancestors, we used genomic, proteomic, and transcriptomic analyses to characterize particle proteins of the banchine Glypta fumiferanae ichnovirus and the genes encoding them. Several proteins were found to be homologous to those identified earlier for campoplegine ichnoviruses while the corresponding genes were located in clusters of the wasp genome similar to those observed previously in a campoplegine wasp. However, for the first time in a polydnavirus system, these clusters also revealed sequences encoding enzymes presumed to form the replicative machinery of the progenitor virus and observed to be overexpressed in the virogenic tissue. Homology searches pointed to nucleocytoplasmic large DNA viruses as the likely source of these genes. These data, along with an analysis of the chromosomal form of five viral genome segments, provide clear evidence for the relatedness of the banchine and campoplegine ichnovirus ancestors. IMPORTANCE Recent work indicates that the two recognized polydnavirus taxa, Bracovirus and Ichnovirus , are derived from distinct viruses whose genomes integrated into the genomes of ancestral wasps. However, the identity of the ichnovirus ancestor is unknown, and questions remain regarding the possibility that the two described ichnovirus subgroups, banchine and campoplegine ichnoviruses, have distinct origins. Our study provides unequivocal evidence that these two ichnovirus types are derived from related viral progenitors. This suggests that morphological and genomic differences observed between the ichnovirus lineages, including features unique to banchine ichnovirus genome segments, result from evolutionary divergence either before or after their endogenization. Strikingly, analysis of selected wasp genomic regions revealed genes presumed to be part of the replicative machinery of the progenitor virus, shedding new light on the likely identity of this virus. Finally, these genes could well play a role in ichnovirus replication as they were overexpressed in the virogenic tissue.

The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota.

In 1998, Palli et al. found that in the eastern spruce budworm, Choristoneura fumiferana, two hexameric proteins termed CfDAP1 and CfDAP2 are expressed abundantly before and during diapause. To determine whether the accumulation of these proteins in early instars is a direct consequence of the diapause phenomenon, we assessed the abundance of CfDAP-like proteins in first (L1), second (L2), and third (L3) instars of a diapause-free strain of the closely related western spruce budworm, Choristoneura occidentalis. Using sodium dodecyl sulfate – polyacrylamide gel electrophoresis and Western-blot analyses of L2 whole-body proteins, a CfDAP-specific antiserum reacted with two C. occidentalis proteins displaying apparent molecular masses identical with those of CfDAP1 and CfDAP2. Among the three larval stadia examined, CfDAP-like proteins accumulated to significant levels only in the first two, with enhanced accumulation observed in L2s starved from the time of hatching. Accumulation of CfDAP1 and CfDAP2 in post-diapause C. fumiferana L2s was affected similarly by food availability. Thus, the data presented here, combined with those of Palli et al., suggest that expression of CfDAP and CfDAP-like proteins in early larval life is more or less limited to L1 and L2, and is associated with starvation stress imposed either experimentally or by elements of the life cycle (i.e., diapause).

The locust, Locusta migratoria , has the capacity to develop a behavioural fever which reduces fungal infection by Metarhizium anisopliae var acridum . We investigated hemocyte and blastospore kinetics in infected insects under conditions that did or did not allow thermoregulation. Hemocyte concentrations were severely reduced in inoculated insects that did not thermoregulate but remained similar to those of controls in inoculated insects that were allowed to thermoregulate. Reductions in hemocyte counts were accompanied by an increase in the concentration of blastospores. In non-thermoregulating insects, circulating blastospores were first observed two days post-inoculation and had heavily colonized the hemolymph by day 5; in contrast, no blastospores were recovered from hemolymph of inoculated-thermoregulating insects. We used fluorescein isothiocyanate (FITC)-labelled silica beads to examine in vivo phagocytosis in thermoregulating and non-thermoregulating locusts. In the absence of fungus, a greater proportion of beads were engulfed by hemocytes in thermoregulating than in non-thermoregulating locusts early (4 and 24 h) after bead injection, but the proportions were similar thereafter. In infected locusts, phagocytosis in non-thermoregulating insects was progressively impaired; such impairment, however, was not observed in challenged, thermoregulating insects. Our results suggest that thermoregulation helped keep fungal growth in check, apparently through the maintenance of hemocyte population levels and the direct inhibition of blastospore propagation by elevated temperatures.

Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10(-6)M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.

Tranosema rostrale (Brishke) (Hymenoptera, Ichneumonidae) and Actia interrupta Curran (Hymenoptera: Tachinidae) are the two endoparasitoids most frequently encountered in low-density populations of the spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera, Tortricidae), in the Quebec City region. Monitoring of attack rates of implanted C. fumiferana larvae at two different study sites suggested the possible existence of competition between the two parasitoids, with A. interrupta seemingly displacing T. rostrale. Here, we show that multiparasitism involving these two species does occur in the field, but at a frequency too low to explain the seasonal pattern of decline in apparent parasitism by T. rostrale that accompanies the rise of A. interrupta attack rates. We also provide preliminary evidence, from laboratory experiments, that A. interrupta has a competitive advantage over T. rostrale and that the success of parasitism by A. interrupta may be enhanced by prior parasitism by T. rostrale under certain conditions, possibly due to the presence of the latter species' polydnavirus. In addition, we describe a PCR-based method that we developed to help detect the presence of T. rostrale eggs which often escape detection by simple visual examination of the dissected host larvae; DNA sequences specific to the polydnavirus injected by the female wasp at the time of oviposition can be readily amplified from whole host larvae.

Ichnoviruses (IVs), unique symbiotic viruses carried by ichneumonid campoplegine wasps, derive from integration of a paleo-ichnovirus into an ancestral wasp genome. The modern ‘genome’ is composed of both regions that are amplified, circularized and encapsidated into viral particles and non-encapsidated viral genomic regions involved in particle morphogenesis. Packaged genomes include multiple circular dsDNAs encoding many genes mostly organized in gene families. Virus particles are assembled in specialized ovarian cells from which they exit into the oviduct lumen; mature virions are injected during oviposition into the insect host. Expression of viral proteins in infected cells correlates with physiological alterations of the host enabling success of parasitism.

We investigated interactions between Bacillus thuringiensis Berliner var. kurstaki and parasitoids that attack late instars of the eastern spruce budworm, Choristoneura fumiferana (Clemens). In a petri-dish arena, females of Tranosema rostrale rostrale (Brishke) (Hymenoptera: Ichneumonidae) were able to discriminate between untreated fourth instars and fourth instars that had been given a known dose of a commercial product (Foray 48B). When the choice tests were conducted before host mortality due to B. thuringiensis had occurred among treated larvae (24 h post ingestion), the parasitoid attacked untreated larvae more readily. When females were given a choice between control larvae and treated larvae that were still alive 72 h post ingestion, they were able to discriminate between the two only when the larvae had been treated with at least a 50% lethal dose. Under laboratory conditions, female T. r. rostrale were thus able to detect and avoid treated larvae that exhibited a lethal response to the pathogen, and to a lesser extent larvae that had survived pathogen exposure. The ability of the latter was not apparent under field conditions. When treated and untreated larvae were exposed for 1 week to a complex of indigenous parasitoids in the field, there was no difference between treatments in the rates of parasitism by either T. r. rostrale or Actia interrupta Curran (Diptera: Tachinidae). Parasitism averaged 91% for larvae in the control treatment compared with 92% for larvae treated with Foray 48B. The field data suggest that spruce budworm larvae that survive exposure to B. thuringiensis are just as likely to be parasitized as unexposed, healthy larvae. This means that prolonged development of late-instar spruce budworm larvae after treatment with B. thuringiensis could possibly result in increased attack rates by parasitoids.

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides (M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only approximately 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent ( approximately 100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.