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.

Mating Disruption of a Flighted Spongy Moth, Lymantria Dispar Japonica (Motchulsky) in Japan.

Mating disruption of a flighted spongy moth, Lymantria dispar japonica (Motchulsky)(Lepidoptera: Lymantridae), with a synthetic version of its sex pheromone, (+)-disparlure ([7R,8S] -cis-7,8-epoxy-2- methyloctadecane), was tested in the forests in Japan. Pheromone trap catches and the percentage mating of tethered females were measured in the pheromone-treated and untreated control forests. The attraction of male moths to pheromone traps placed at a height of 1.5 m was significantly disrupted when the pheromone dispensers were placed at 1.5 m height, but many moths were captured in control plots. Mating of tethered females placed at 1.5 m was inhibited entirely, while 44% of females were mated in an untreated control forest. We report the first trial of mating disruption against a flighted spongy moth, and these results suggest that mating disruption with the synthetic sex pheromone appears promising for reducing damage caused by L. dispar japonica.

Effect of fluorescent brighteners on the insecticidal activity of Bacillus thuringiensis var. kurstaki and LdMNPV on Lymantria dispar asiatica in Korea.

Stilbene-based fluorescent brighteners (FBs) have been demonstrated to improve the insecticidal activities of entomopathogenic viruses; however, there is limited information regarding their effect on entomopathogenic bacteria. We conducted this study to investigate the effect of two FBs (FB 28 and FB 71) on the insecticidal activities of Bacillus thuringiensis var. kurstaki (Btk) and Lymantria dispar multiple nuclear polyhedrosis virus (LdMNPV) on Lymantria dispar asiatica. FB 28 and Btk combination at low concentration (1.6 × 102 IU/mL) increased the mortality, whereas FB 71 and Btk combination at intermediate and high concentrations (1.6 × 103 and 1.6 × 104 IU/mL) slightly reduced the mortality compared with that with Btk alone. The lethal time was also shorter with combinations of Btk and FB 28 than with FB 71. Both FB 28 and FB 71 increased the mortality in combination with LdMNPV at all concentrations (3 × 102 , 3 × 104 , and 3 × 106 polyhedral occlusion bodies/mL compared with that with LdMNPV alone. Our results suggest that FBs improve the insecticidal activities of Btk and LdMNPV, and their activities depend on their interactions with the midgut structures of the host insect species.

Oviposition behavior of the quasi-gregarious parasitoid, Ooencyrtus kuvanae (Hymenoptera: Encyrtidae).

The parasitoid wasp, Ooencyrtus kuvanae (Howard) (Hymenoptera: Encyrtidae), is a natural enemy of the spongy moth, a significant forest pest in North America. We investigated the oviposition behavior of O. kuvanae females on spongy moth egg masses by (i) presenting female parasitoids with a single spongy moth egg mass that was replaced every day, 2nd day, 4th day, 8th day, or 16th day (which is the total length of the oviposition period) and (ii) presenting female parasitoids with 1, 2, 4, or 8 egg masses at a time. Offspring developmental length ranged from 18 to 24 days. On average, male offspring exhibited faster developmental times, emerging approximately 1 day ahead of females. The amount of time that adult females spent on an egg mass affected the number of parasitized eggs. Specifically, more offspring emerged in the 4-, 8-, and 16-day treatments than in scenarios involving daily or every second-day egg mass replacement. The percentage of male offspring decreased as the number of egg masses presented to females increased. Interestingly, the total number of female offspring remained constant, but the number of male offspring decreased with an increase in the number of egg masses and time spent by the parent within a patch. The observed sexual dimorphism in development time, the influence of resource availability on offspring sex ratios, and flexible oviposition patterns illustrate the adaptability of O. kuvanae in response to varying conditions. These insights have implications for our understanding of parasitoid-host interactions and their potential role in biological control strategies.

Genome-Wide Transcriptomic and Metabolomic Analyses Unveiling the Defence Mechanisms of Populus tremula against Sucking and Chewing Insect Herbivores.

Plants and insects coevolved as an evolutionarily successful and enduring association. The molecular arms race led to evolutionary novelties regarding unique mechanisms of defence and detoxification in plants and insects. While insects adopt mechanisms to conquer host defence, trees develop well-orchestrated and species-specific defence strategies against insect herbivory. However, current knowledge on the molecular underpinnings of fine-tuned tree defence responses against different herbivore insects is still restricted. In the current study, using a multi-omics approach, we unveiled the defence response of Populus tremula against aphids (Chaitophorus populialbae) and spongy moths (Lymantria dispar) herbivory. Comparative differential gene expression (DGE) analyses revealed that around 272 and 1203 transcripts were differentially regulated in P. tremula after moth and aphid herbivory compared to uninfested controls. Interestingly, 5716 transcripts were differentially regulated in P. tremula between aphids and moth infestation. Further investigation showed that defence-related stress hormones and their lipid precursors, transcription factors, and signalling molecules were over-expressed, whereas the growth-related counterparts were suppressed in P. tremula after aphid and moth herbivory. Metabolomics analysis documented that around 37% of all significantly abundant metabolites were associated with biochemical pathways related to tree growth and defence. However, the metabolic profiles of aphid and moth-fed trees were quite distinct, indicating species-specific response optimization. After identifying the suitable reference genes in P. tremula, the omics data were further validated using RT-qPCR. Nevertheless, our findings documented species-specific fine-tuning of the defence response of P. tremula, showing conservation on resource allocation for defence overgrowth under aphid and moth herbivory. Such findings can be exploited to enhance our current understanding of molecular orchestration of tree responses against herbivory and aid in developing insect pest resistance P. tremula varieties.

Integration of satellite remote sensing and MaxEnt modeling for improved detection and management of forest pests.

Forest pests pose a major threat to ecosystem services worldwide, requiring effective monitoring and management strategies. Recently, satellite remote sensing has emerged as a valuable tool to detect defoliation caused by these pests. Lymantria dispar, a major forest pest native to Japan, Siberia, and Europe, as well as introduced regions in North America, is of particular concern. In this study, we used Sentinel-2 satellite imagery to estimate the defoliation area and predict the distribution of L. dispar in Toyama Prefecture, central Japan. The primary aim was to understand the spatial distribution of L. dispar. The normalized difference vegetation index (NDVI) difference analysis estimated a defoliation area of 7.89 km2 in Toyama Prefecture for the year 2022. MaxEnt modeling, using defoliation map as occurrence data, identified the deciduous forests between approximately 35° and 50° at elevations of 400 m and 700 m as highly suitable for L. dispar. This predicted suitability was also high for larval locations but low for egg mass locations, likely due to differences in larval habitats and ovipositing sites. This study is the first attempt to utilize NDVI-based estimates as a proxy for MaxEnt. Our results showed higher prediction accuracy than a previous study based on the occurrence records including larvae, adults, and egg masses, indicating better discrimination of the distribution of L. dispar defoliation. Therefore, our approach to integrating satellite data and species distribution models can potentially enhance the assessment of areas affected by pests for effective forest management.

Effects of Lymantria dispar multiple nucleopolyhedrovirus and Bacillus thuringiensis var. kurstaki on different larval instars of Lymantria dispar asiatica.

Outbreaks of Lymantria dispar asiatica (the Asian spongy moth; Lepidoptera: Erebidae) occur sporadically, causing widespread damage to forest and fruit trees. Owing to the development of pesticide resistance and environmental contamination, biopesticides, including L. dispar multiple nucleopolyhedrovirus (LdMNPV) and Bacillus thuringiensis var. kurstaki (Btk), can significantly contribute to controlling overall larval stage of this species. Although both pathogens are highly effective at the larval stage, their effects on different instar stages have not been investigated. In this study, we analyzed the mortality and lethality in different L. dispar asiatica instars exposed to single or combined pathogen treatments. Treatments with low or medium LdMNPV concentrations induced lower mortality and had higher LT50 values at the 4th and 5th instars compared with other instars, whereas high LdMNPV treatments induced high mortality in all instars, with higher LT50 values at later instars. Treatment with Btk induced a rapid 100% mortality in all instars, with higher LT50 values for the later instars. The combination of LdMNPV and Btk delayed the killing time compared with the effects of single treatments, with the effect being more pronounced in the 1st and 5th instar stage than at other stages at low Btk concentrations. Our findings indicate that the pathogenic effects of LdMNPV and Btk on L. dispar asiatica differ according to larval stage, thereby providing novel insights into enhancing the biological control efficacy of these agents against L. dispar asiatica in the field.

Spatial ensemble modeling for predicting the potential distribution of Lymantria dispar asiatica (Lepidoptera: Erebidae: Lymantriinae) in South Korea.

The spongy moth, Lymantria dispar, is a pest that damages various tree species throughout North America and Eurasia, has recently emerged in South Korea, threatening local forests and landscapes. The establishment of effective countermeasures against this species' outbreak requires predicting its potential distribution with climate change. In this study, we used species distribution models (CLIMEX and MaxEnt) to predict the potential distribution of the spongy moth and identify areas at risk of exposure to a sustained occurrence of the pest by constructing an ensemble map that simultaneously projected the outcomes of the two models. The results showed that the spongy moth could be distributed over the entire country under the current climate, but the number of suitable areas would decrease under a climate change scenario. This study is expected to provide basic data that can predict areas requiring intensive control and monitoring in advance with methodologically improved modeling technique.

Building a reference indicator model using co-kriging interpolation to determine the geographical origin of the flighted spongy moth complex in China.

Using stable isotopes to detect and analyze the geographical origin of insects represents an important traceability technology, which requires a rich isotope database. In this study, we representatively sampled the Chinese provinces where flighted spongy moth complex (FSMC) has been reported and, for the first time, used co-kriging interpolation to predict the distribution patterns of FSMC δ13 C values in the main distribution areas. From 2020 to 2022, we set up 60 traps in 12 provinces and cities in China and collected 795 FSMCs. Then, 6 main climatic factors were obtained by multi-collinearity screening from 21 types of meteorological data collected at the sample plots, and a correlation analysis was carried out by combining longitude, latitude, and altitude data with the δ13 C values of FSMC. Next, we performed a co-kriging interpolation using the 2 climatic factors with the highest correlation (isothermality and altitude) and the δ13 C values of FSMC. A cross-validation was performed to systematically test 11 candidate models and select the best semi-variogram model ("Exponential"), which was then used to build a co-kriging interpolation model. The geographical distribution patterns of the FSMC δ13 C values obtained from the 2 interpolation models (i.e., interpolated with isothermality and altitude, respectively) were almost the same. Moreover, the δ13 C values varied significantly at the regional scale, showing regular changes in spatial distribution. Overall, the reference indicator map of the δ13 C values generated from stable isotopes can be used to greatly improve the efficiency of discrimination analyses on the geographical origin of FSMC.

Assessment of spongy moth infestation impacts on forest productivity and carbon loss using the Sentinel-2 satellite remote sensing and eddy covariance flux data.

Background: Deciduous forests in eastern North America experienced a widespread and intense spongy moth (Lymantria dispar) infestation in 2021. This study quantified the impact of this spongy moth infestation on carbon (C) cycle in forests across the Great Lakes region in Canada, utilizing high-resolution (10 × 10 m2) Sentinel-2 satellite remote sensing images and eddy covariance (EC) flux data. Study results showed a significant reduction in leaf area index (LAI) and gross primary productivity (GPP) values in deciduous and mixed forests in the region in 2021. Results: Remote sensing derived, growing season mean LAI values of deciduous (mixed) forests were 3.66 (3.18), 2.74 (2.64), and 3.53 (2.94) m2 m-2 in 2020, 2021 and 2022, respectively, indicating about 24 (14)% reduction in LAI, as compared to pre- and post-infestation years. Similarly, growing season GPP values in deciduous (mixed) forests were 1338 (1208), 868 (932), and 1367 (1175) g C m-2, respectively in 2020, 2021 and 2022, showing about 35 (22)% reduction in GPP in 2021 as compared to pre- and post-infestation years. This infestation induced reduction in GPP of deciduous and mixed forests, when upscaled to whole study area (178,000 km2), resulted in 21.1 (21.4) Mt of C loss as compared to 2020 (2022), respectively. It shows the large scale of C losses caused by this infestation in Canadian Great Lakes region. Conclusions: The methods developed in this study offer valuable tools to assess and quantify natural disturbance impacts on the regional C balance of forest ecosystems by integrating field observations, high-resolution remote sensing data and models. Study results will also help in developing sustainable forest management practices to achieve net-zero C emission goals through nature-based climate change solutions.

The Impact of a Cypovirus on Parental and Filial Generations of Lymantria dispar L.

Recently, we found that the spongy moth Lymantria dispar L. is susceptible to infection by a Dendrolimus sibiricus cytoplasmic polyhedrosis virus (DsCPV-1). In the present study, we evaluated the pathogenicity of DsCPV-1 against L. dispar larvae and its impact on surviving insects after the infection. Offspring of virally challenged insects were tested for susceptibility to a stress factor (starvation). In addition, we used light microscopy and quantitative polymerase chain reaction (qPCR) to test the ability of DsCPV-1 to be transmitted vertically. We found insect mortality of the L. dispar parents following the infection was positively associated with DsCPV-1 dose. DsCPV-1 was lethal to second-instar L. dispar larvae with a 50% lethal dose (LD50) of 1687 occlusion bodies per larva. No vertical transmission of DsCPV-1 to offspring larvae was detected, while the majority of insect deaths among offspring larvae were caused by microsporidia (Vairimorpha lymantriae), which was harbored by the parents. The offspring of virally challenged parents exhibited a higher number of detected microsporidia compared to the control. Our findings suggest that the application of DsCPV-1 is effective in controlling pests in terms of transgenerational impact following virus exposure.

Lymantria Dispar Iflavirus 1 RNA Comprises a Large Proportion of RNA in Adult L. dispar Moths.

The spongy moth virus Lymantria dispar iflavirus 1 (LdIV1), originally identified from a Lymantria dispar cell line, was detected in 24 RNA samples from female moths of four populations from the USA and China. Genome-length contigs were assembled for each population and compared with the reference genomes of the first reported LdIV1 genome (Ames strain) and two LdIV1 sequences available in GenBank originating from Novosibirsk, the Russian Federation. A whole-genome phylogeny was generated for these sequences, indicating that LdIV1 viruses observed in North American (flightless) and Asian (flighted) spongy moth lineages indeed partition into clades as would be expected per their host's geographic origin and biotype. A comprehensive listing of synonymous and non-synonymous mutations, as well as indels, among the polyprotein coding sequences of these seven LdIV1 variants was compiled and a codon-level phylogram was computed using polyprotein sequences of these, and 50 additional iflaviruses placed LdIV1 in a large clade consisting mostly of iflaviruses from other species of Lepidoptera. Of special note, LdIV1 RNA was present at very high levels in all samples, with LdIV1 reads accounting for a mean average of 36.41% (ranging from 1.84% to 68.75%, with a standard deviation of 20.91) of the total sequenced volume.

Improvement in the Identification Technology for Asian Spongy Moth, Lymantria dispar Linnaeus, 1758 (Lepidoptera: Erebidae) Based on SS-COI.

Lymantria dispar (Linnaeus, 1758), which is commonly known as spongy moth, with two subspecies, is found in Asia: Lymantria dispar asiatica and Lymantria dispar japonica, collectively referred to as the Asian spongy moth (ASM). The subspecies Lymantria dispar dispar occurs in Europe and is commonly known as the European spongy moth (ESM). The ASM is on the quarantine list of many countries because it induces greater economic losses than the ESM. Accurate identification is essential to prevent the invasion of ASM into new areas. Although several techniques for identifying ASMs have been developed, the recent discovery of complex patterns of genetic variation among ASMs in China as well as new subspecies in some areas has necessitated the development of new, improved identification techniques, as previously developed techniques are unable to accurately identify ASMs from all regions in China. Here, we demonstrate the efficacy of an improved technique for the identification of the ASM using ASM-specific primers, which were designed based on cytochrome oxidase I sequences from samples obtained from all sites where ASMs have been documented to occur in China. We show that these primers are effective for identifying a single ASM at all life stages and from all ASM populations in China, and the minimum detectable concentration of genomic DNA was 30 pg. The inclusion of other Lymantria samples in our analysis confirmed the high specificity of the primers. Our improved technique allows the spread of ASMs to be monitored in real time and will help mitigate the spread of ASMs to other areas.

SAFARIS: a spatial analytic framework for pest forecast systems.

Non-native pests and diseases pose a risk of economic and environmental damage to managed and natural U.S. forests and agriculture. The U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) Plant Protection and Quarantine (PPQ) protects the health of U.S. agriculture and natural resources against invasive pests and diseases through efforts to prevent the entry, establishment, and spread of non-native pests and diseases. Because each pest or disease has its own idiosyncratic characteristics, analyzing risk is highly complex. To help PPQ better respond to pest and disease threats, we developed the Spatial Analytic Framework for Advanced Risk Information Systems (SAFARIS), an integrated system designed to provide a seamless environment for producing predictive models. SAFARIS integrates pest biology information, climate and non-climate data drivers, and predictive models to provide users with readily accessible and easily customizable tools to analyze pest and disease risks. The phenology prediction models, spread forecasting models, and other climate-based analytical tools in SAFARIS help users understand which areas are suitable for establishment, when surveys would be most fruitful, and aid in other analyses that inform decision-making, operational efforts, and rapid response. Here we introduce the components of SAFARIS and provide two use cases demonstrating how pest-specific models developed with SAFARIS tools support PPQ in its mission. Although SAFARIS is designed to address the needs of PPQ, the flexible, web-based framework is publicly available, allowing any user to leverage the available data and tools to model pest and disease risks.

Lymantria dispar (L.) (Lepidoptera: Erebidae): Current Status of Biology, Ecology, and Management in Europe with Notes from North America.

The European Spongy moth, Lymantria dispar (L.) (Lepidoptera: Erebidae), is an abundant species found in oak woods in Central and Southern Europe, the Near East, and North Africa and is an important economic pest. It is a voracious eater and can completely defoliate entire trees; repeated severe defoliation can add to other stresses, such as weather extremes or human activities. Lymantria dispar is most destructive in its larval stage (caterpillars), stripping away foliage from a broad variety of trees (>500 species). Caterpillar infestation is an underestimated problem; medical literature reports that established populations of caterpillars may cause health problems to people and animals. Inflammatory reactions may occur in most individuals after exposure to setae, independent of previous exposure. Currently, chemical and mechanical methods, natural predators, and silvicultural practices are included for the control of this species. Various insecticides have been used for its control, often through aerial sprayings, which negatively affect biodiversity, frequently fail, and are inappropriate for urban/recreational areas. However, bioinsecticides based on various microorganisms (e.g., entomopathogenic viruses, bacteria, and fungi) as well as technologies such as mating disruption using sex pheromone traps have replaced insecticides for the management of L. dispar.

Rock Microhabitats Provide Suitable Thermal Conditions for Overwintering Insects: A Case Study of the Spongy Moth (Lymantria dispar L.) Population in the Altai Mountains.

Many insect species overwinter in various rock shelters (cavities and crevices), but the microclimates of rock biotopes remain poorly understood. We investigated the temperature dynamics in rock microhabitats where clusters of egg masses of the wintering spongy moth Lymantria dispar L. (SM) were observed. Our research objective was to find the relation between the ovipositing behaviour of females and the landscape features in different parts of this species' range. Studies of the ecology of the SM are important from a practical point of view, as the moth causes significant economic damage to forests of the Holarctic. We found that the average monthly temperature of rock surfaces in the studied microhabitats was 2-5 °C above the average air temperature. More importantly, the minimum temperatures in these microhabitats were 4-13 °C higher than the minimum air temperature. These results help to reassess the role of the mountain landscape in the spread of insect species. Rock biotopes provided a significant improvement in the conditions for wintering insects. We believe that, when modelling the spread of invasive species (such as the SM), it is necessary to account for the influence of rock biotopes that may facilitate shifts in the northern boundaries of their range.