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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Continent-wide population genomic structure and phylogeography of North America's most destructive conifer defoliator, the spruce budworm (Choristoneura fumiferana).

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.

Temporal variation in spatial genetic structure during population outbreaks: Distinguishing among different potential drivers of spatial synchrony.

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.

Expansion of LINEs and species-specific DNA repeats drives genome expansion in Asian Gypsy Moths.

Two subspecies of Asian gypsy moth (AGM), Lymantria dispar asiatica and L. dispar japonica, pose a serious alien invasive threat to North American forests. Despite decades of research on the ecology and biology of this pest, limited AGM-specific genomic resources are currently available. Here, we report on the genome sequences and functional content of these AGM subspecies. The genomes of L.d. asiatica and L.d. japonica are the largest lepidopteran genomes sequenced to date, totaling 921 and 999 megabases, respectively. Large genome size in these subspecies is driven by the accumulation of specific classes of repeats. Genome-wide metabolic pathway reconstructions suggest strong genomic signatures of energy-related pathways in both subspecies, dominated by metabolic functions related to thermogenesis. The genome sequences reported here will provide tools for probing the molecular mechanisms underlying phenotypic traits that are thought to enhance AGM invasiveness.

The dual life of ichnoviruses.

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.

Insights into the Structure of the Spruce Budworm (Choristoneura fumiferana) Genome, as Revealed by Molecular Cytogenetic Analyses and a High-Density Linkage Map.

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.

Unexpected invasion of miniature inverted-repeat transposable elements in viral genomes.

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.

Impact of rearing temperature on encapsulation and the accumulation of transcripts putatively involved in capsule formation in a parasitized lepidopteran host.

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 ß1, 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.