Genetics of flight in spongy moths (Lymantria dispar ssp.): functionally integrated profiling of a complex invasive trait.

BACKGROUND: Flight can drastically enhance dispersal capacity and is a key trait defining the potential of exotic insect species to spread and invade new habitats. The phytophagous European spongy moths (ESM, Lymantria dispar dispar) and Asian spongy moths (ASM; a multi-species group represented here by L. d. asiatica and L. d. japonica), are globally invasive species that vary in adult female flight capability-female ASM are typically flight capable, whereas female ESM are typically flightless. Genetic markers of flight capability would supply a powerful tool for flight profiling of these species at any intercepted life stage. To assess the functional complexity of spongy moth flight and to identify potential markers of flight capability, we used multiple genetic approaches aimed at capturing complementary signals of putative flight-relevant genetic divergence between ESM and ASM: reduced representation genome-wide association studies, whole genome sequence comparisons, and developmental transcriptomics. We then judged the candidacy of flight-associated genes through functional analyses aimed at addressing the proximate demands of flight and salient features of the ecological context of spongy moth flight evolution. RESULTS: Candidate gene sets were typically non-overlapping across different genetic approaches, with only nine gene annotations shared between any pair of approaches. We detected an array of flight-relevant functional themes across gene sets that collectively suggest divergence in flight capability between European and Asian spongy moth lineages has coincided with evolutionary differentiation in multiple aspects of flight development, execution, and surrounding life history. Overall, our results indicate that spongy moth flight evolution has shaped or been influenced by a large and functionally broad network of traits. CONCLUSIONS: Our study identified a suite of flight-associated genes in spongy moths suited to exploration of the genetic architecture and evolution of flight, or validation for flight profiling purposes. This work illustrates how complementary genetic approaches combined with phenotypically targeted functional analyses can help to characterize genetically complex traits.

A TaqMan Assay for the Detection and Monitoring of Potentially Invasive Lasiocampids, With Particular Attention to the Siberian Silk Moth, Dendrolimus sibiricus (Lepidoptera: Lasiocampidae).

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.

Novel antimicrobial anionic cecropins from the spruce budworm feature a poly-L-aspartic acid C-terminus.

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.

Landscape-scale population connectivity in two parasitoid species associated with the spruce budworm: Testing the birdfeeder effect using genetic data.

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.

Convergent herbivory on conifers by Choristoneura moths after boreal forest formation.

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 Palearctic species 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.

Distinct sources of gene flow produce contrasting population genetic dynamics at different range boundaries of a Choristoneura budworm.

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 1,195 single nucleotide polymorphisms, we documented genomewide, locus-specific patterns of introgression between Choristoneura occidentalis biennis spruce budworms and two ecologically divergent relatives, C. o. occidentalis and Choristoneura fumiferana, that it interacts with at alternate boundaries of its range. We observe contrasting hybrid indexes between the two 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 genomewide 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.

Genomic and Proteomic Analyses Indicate that Banchine and Campoplegine Polydnaviruses Have Similar, if Not Identical, Viral Ancestors.

UNLABELLED: 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.

Characterization of the polydnaviral 'T. rostrale virus' (TrV) gene family: TrV1 expression inhibits in vitro cell proliferation.

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.

Ultrastructural and genomic characterization of a second banchine polydnavirus confirms the existence of shared features within this ichnovirus lineage.

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 <5 kb, an impressively high degree of genome segmentation and a very similar gene content (same gene families in both viruses). Altogether, the data presented here confirm the existence of shared characteristics within this banchine IV lineage.

Steps in the biosynthesis of fuscumol in the longhorn beetles Tetropium fuscum (F.) and Tetropium cinnamopterum Kirby.

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.

Range-wide population genomics of the spongy moth, Lymantria dispar (Erebidae): Implications for biosurveillance, subspecies classification and phylogeography of a destructive moth.

The spongy moth, Lymantria dispar, is an irruptive forest pest native to Eurasia where its range extends from coast to coast and overspills into northern Africa. Accidentally introduced from Europe in Massachusetts in 1868-1869, it is now established in North America where it is considered a highly destructive invasive pest. A fine-scale characterization of its population genetic structure would facilitate identification of source populations for specimens intercepted during ship inspections in North America and would enable mapping of introduction pathways to help prevent future incursions into novel environments. In addition, detailed knowledge of L. dispar's global population structure would provide new insight into the adequacy of its current subspecies classification system and its phylogeographic history. To address these issues, we generated >2000 genotyping-by-sequencing-derived SNPs from 1445 contemporary specimens sampled at 65 locations in 25 countries/3 continents. Using multiple analytical approaches, we identified eight subpopulations that could be further partitioned into 28 groups, achieving unprecedented resolution for this species' population structure. Although reconciliation between these groupings and the three currently recognized subspecies proved to be challenging, our genetic data confirmed circumscription of the japonica subspecies to Japan. However, the genetic cline observed across continental Eurasia, from L. dispar asiatica in East Asia to L. d. dispar in Western Europe, points to the absence of a sharp geographical boundary (e.g., the Ural Mountains) between these two subspecies, as suggested earlier. Importantly, moths from North America and the Caucasus/Middle East displayed high enough genetic distances from other populations to warrant their consideration as separate subspecies of L. dispar. Finally, in contrast with earlier mtDNA-based investigations that identified the Caucasus as L. dispar's place of origin, our analyses suggest continental East Asia as its evolutionary cradle, from where it spread to Central Asia and Europe, and to Japan through Korea.

Phenological Features of the Spongy Moth, Lymantria dispar (L.) (Lepidoptera: Erebidae), in the Northernmost Portions of Its Eurasian Range.

The spongy moth, Lymatria dispar, is a classic example of an invasive pest accidentally introduced from Europe to North America, where it has become one of the most serious forest defoliators, as in its native range. The present study was aimed at (i) identifying the current northern limit of L. dispar's Eurasian range and exploring its northward expansion in Canada using pheromone trap data, and (ii) comparing northern Eurasian populations with those from central and southern regions with respect to male flight phenology, the sums of effective temperatures (SETs) above the 7 °C threshold necessary for development to the adult stage, and heat availability. We show that the range of L. dispar in Eurasia now reaches the 61st parallel, and comparisons with historical data identify the average speed of spread as 50 km/year. We also document the northern progression of L. dispar in southern Canada, where the actual northern boundary of its range remains to be identified. We show that the median date of male flight does not vary greatly between northern and southern regions of the spongy moth range in Eurasia despite climate differences. Synchronization of flight at different latitudes of the range is associated with an acceleration of larval development in northern Eurasian populations. Similar changes in developmental rate along a latitudinal gradient have not been documented for North American populations. Thus, we argue that this feature of spongy moths from northern Eurasia poses a significant invasive threat to North America in terms of enhanced risks for rapid northward range expansion.

Analysis of virion structural components reveals vestiges of the ancestral ichnovirus genome.

Many thousands of endoparasitic wasp species are known to inject polydnavirus (PDV) particles into their caterpillar host during oviposition, causing immune and developmental dysfunctions that benefit the wasp larva. PDVs associated with braconid and ichneumonid wasps, bracoviruses and ichnoviruses respectively, both deliver multiple circular dsDNA molecules to the caterpillar. These molecules contain virulence genes but lack core genes typically involved in particle production. This is not completely unexpected given that no PDV replication takes place in the caterpillar. Particle production is confined to the wasp ovary where viral DNAs are generated from proviral copies maintained within the wasp genome. We recently showed that the genes involved in bracovirus particle production reside within the wasp genome and are related to nudiviruses. In the present work we characterized genes involved in ichnovirus particle production by analyzing the components of purified Hyposoter didymator Ichnovirus particles by LC-MS/MS and studying their organization in the wasp genome. Their products are conserved among ichnovirus-associated wasps and constitute a specific set of proteins in the virosphere. Strikingly, these genes are clustered in specialized regions of the wasp genome which are amplified along with proviral DNA during virus particle replication, but are not packaged in the particles. Clearly our results show that ichnoviruses and bracoviruses particles originated from different viral entities, thus providing an example of convergent evolution where two groups of wasps have independently domesticated viruses to deliver genes into their hosts.

Development of a qPCR-based method for counting overwintering spruce budworm (Choristoneura fumiferana) larvae collected during fall surveys and for assessing their natural enemy load: a proof-of-concept study.

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. © 2021 Her Majesty the Queen in Right of Canada. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. Reproduced with the permission of the Minister of Natural Resources Canada.

The Spruce Budworm Genome: Reconstructing the Evolutionary History of Antifreeze Proteins.

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.

Rational design of Lepidoptera-specific insecticidal inhibitors targeting farnesyl diphosphate synthase, a key enzyme of the juvenile hormone biosynthetic pathway.

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.

Sequencing, assembly and annotation of the whole-insect genome of Lymantria dispar dispar, the European gypsy moth.

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.

New insights into short-chain prenyltransferases: structural features, evolutionary history and potential for selective inhibition.

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 Unconventional Viruses of Ichneumonid Parasitoid Wasps.

To ensure their own immature development as parasites, ichneumonid parasitoid wasps use endogenous viruses that they acquired through ancient events of viral genome integration. Thousands of species from the campoplegine and banchine wasp subfamilies rely, for their survival, on their association with these viruses, hijacked from a yet undetermined viral taxon. Here, we give an update of recent findings on the nature of the viral genes retained from the progenitor viruses and how they are organized in the wasp genome.

In Situ Processing and Efficient Environmental Detection (iSPEED) of tree pests and pathogens using point-of-use real-time PCR.

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.