Introduction

The spongy moth (formerly the gypsy moth) Lymantria dispar L. (Lepidoptera: Erebidae) is one of the species that causes significant economic damage to forests1,2. Traditionally, three subspecies have been recognized based on morphology, the geographic range, and female flight capability3. Particularly, the European spongy moth, L. dispar dispar L. is native to Europe, and its adult females are flightless4, L. dispar asiatica Vnukovskij is found in eastern Russia, China, and Korea, and L. dispar japonica Motschulsky occurs on the main islands of Japan. The latter two subspecies are known as the Asian spongy moth, in which adult females are capable of flight. It should be noted that the name "Asian spongy moth" also encompasses several closely related Lymantria species, one of which, L. umbrosa, is found in eastern Hokkaido, Japan5,6. In the eastern United States and Canada, L. dispar dispar has become established since its introduction to Massachusetts from Western Europe in the late 1860s and has been classified as a quarantine pest7. Larvae of L. dispar are known to defoliate a wide array of plant species1 and are responsible for regular outbreaks throughout the Holarctic region6,8,9,10. Recent genetic studies suggest that the spongy moth’s population structure may be more complex than the three traditionally recognized subspecies11 and that a Caucasian subspecies may be identified as a distinct clade in future studies12,13.

Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), a representative of the family Baculoviridae, causes epizootics in L. dispar populations. LdMNPV is a highly pathogenic host-specific baculovirus that has been used as the basis of biological insecticides for spongy moth control1,14. Viruses of this family possess a double-stranded circular DNA genome ranging from 80 to 180 kbp in size and harboring 90 to 180 open reading frames (ORFs)15. Two virion phenotypes occur in the replication cycle of this virus: budded viruses (BVs) and occlusion-derived viruses (ODVs). The latter are embedded in a proteinaceous occlusion body (OB), which contains multiple virions. Primary peroral infection of insect larvae is initiated in midgut epithelial cells by ODVs, whereas the spread of the infection to other larval tissues is mediated by BVs16.

It has been shown that there are differences among LdMNPV isolates in the virulence toward L. dispar larvae. There are some possible explanations for the observed differences in LdMNPVs’ virulence. The first potential explanation involves the presence of viral enhancing factor genes (vef-1 and vef-2) in certain LdMNPV strains17. Protein products of these genes are integral components of the ODV envelope18, probably responsible for ODV binding to an insect midgut cell during primary infection19. A second explanation deals with evolutionary coadaptation of L. dispar populations and baculoviruses in geographically isolated regions. Prolonged coexistence in localized environments may lead to enhanced viral efficacy against native host populations compared to geographically distant hosts20,21,22. Later, Harrison et. al23 suggested that LdMNPV isolates originating from the Asian spongy moth may have greater efficacy against both the European spongy moth and invasive spongy moth populations in North America. Finally, the third explanation is that the response of L. dispar populations to baculoviruses is dependent on a host plant’s metabolites, whose set differs among dominant host plants of corresponding localities and may affect the insect’s immune function and physical barriers to the pathogen’s entry24,25. This variation may significantly affect the host–pathogen interaction, thereby contributing to the observed differences in virulence26.

Prior laboratory studies involving spongy moth artificial diets have revealed that LdMNPV virulence is subject to variation that is not contingent upon prior host–baculovirus coexistence or the source of an isolate23,27. Nonetheless, field applications of the virus require a more realistic examination of pest–plant interactions. It can be inferred that LdMNPV virulence is influenced by both genetic characteristics of the virus, such as the presence of vef genes, and the variation of preferences for host plant species among local L. dispar populations28,29.

In this study, we evaluated the virulence of six LdMNPV isolates toward five geographically distinct L. dispar populations and one L. umbrosa population across their geographic ranges. Extensive bioassays were conducted to determine whether the susceptibility of L. dispar populations to LdMNPV infection depends on the origin of a viral isolate. These L. dispar populations are associated with different environmental conditions and host plant species. Additionally, we sequenced complete genomes of novel variants of LdMNPV (designated as LdMNPV-KR, -KG, and -BibJ) isolated from spongy moths located in Krasnodar (Russia), Kyrgyzstan (Jalal-Abad region), and Bibai (Hokkaido, Japan), respectively. These isolates were selected as the most distant from each other for a comparison with previously determined genome sequences of LdMNPV isolates available in GenBank. The results will expand our knowledge about the susceptibility of L. dispar from various localities (i.e., different populations) to different LdMNPV isolates for selecting a promising isolate for the development of novel biological pesticides that are effective regardless of a baculovirus source and of a spongy moth subspecies.

Results

Bioassay results

We estimated LD50 values of four LdMNPV isolates to compare their virulence against three different L. dispar populations (Table 1). The LD50 values of LdMNPV-27/0, -KR, and -BibJ obtained from Novosibirsk, Krasnodar and Bibai, respectively, were not significantly different when assayed on L. dispar larvae from Novosibirsk. Meanwhile, when the larvae from Krasnodar were assayed, LdMNPV-27/0 exhibited greater efficacy than the original LdMNPV-KR (Table 1).

Table 1 Dose–response mortality (LD50s expressed in ln of OBs/larva, with 95% fiducial limits) of 2d instar Lymantria dispar larvae infected with LdMNPV isolates.
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Although no significant differences in the LD50 values were observed between LdMNPV-KG and -BibJ, the Krasnodar L. dispar population required a significantly higher amount of OBs to induce 50% insect mortality with LdMNPV-KG compared to other isolates. Moreover, LdMNPV-KG had no significant effect on the survival rate of the Novosibirsk larvae (χ2 = 1.8, df = 1, p = 0.177), whereas infection with the same isolate resulted in a reduction of survival rate in the Krasnodar larvae (Table 1). Toward L. dispar from Khabarovsk, LD50 of LdMNPV-BibJ was significantly lower than that of spatially distant isolate LdMNPV-KR.

To assess the virulence of different LdMNPV isolates on various L. dispar populations, including those from Japan, survival curves were compared. Significant differences were observed between insect populations for most of the LdMNPV isolates (Fig. 1a,b,c,d,e), with the exception of LdMNPV-KG (Fig. 1f). LdMNPV-KuzJ was found to act faster in the Nara (native host) and Sapporo populations (χ2 = 8.3, df = 2, p = 0.02, Fig. 1a). LdMNPV-ShinJ exhibited rapid mortality in larvae from its native host (Teshikaga) and the Sapporo L. dispar population (χ2 = 19.6, df = 2, p < 0.0001, Fig. 1c). LdMNPV-BibJ showed significantly higher virulence toward larvae from Nara and Sapporo compared to populations from Novosibirsk and Teshikaga (χ2 = 40.5, df = 5, p < 0.0001, Fig. 1b). Similarly, isolate LdMNPV-27/0 was highly effective against most tested L. dispar populations, although notably less virulent against its native host population from Novosibirsk (χ2 = 30.6, df = 4, p < 0.0001, Fig. 1d). In general, treatment of the Novosibirsk population with the viral isolates resulted in higher insect survival as compared to the other populations. Overall, the LD50 values and survival curves obtained in this study did not indicate a clear relation between a moth popualtion’s susceptibility and its specific viral isolate. These results mean that the LdMNPV isolates possess various degrees of virulence against different L. dispar populations.

Fig. 1
figure 1

Cumulative survival probability of L. dispar populations orally exposed to different LdMNPV isolates at a dose of 50 OBs per larva. Curves are labeled with different letters if a p-value denotes significance (log-rank test with Holm adjustment, p < 0.05).

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Assembly and annotation of LdMNPV-KR, -KG, and -BibJ genomes

Complete genome sequences of the three new LdMNPV isolates from geographically distant locations were determined. Genome sizes of LdMNPV-KR, -KG, and -BibJ were 153,804, 164,499 and 162,228 bp with a GC content of 57.8%, 57.7%, and 57.2% respectively (Table 2). The isolates were found to have the number of open reading frames (ORFs), homologous repeat (hr) regions, and baculovirus repeat ORF (bro) family members.

Table 2 The main characteristics of LdMNPV-KR, -KG and -BibJ genomes.
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A comparative analysis of the three newly LdMNPV genomes revealed a range of 97.0% to 98.3% identity with the LdMNPV-27/0 sequence. Whole-genome alignments with other LdMNPV sequences revealed extensive collinearity. The circular genome maps illustrate the genomic organization of these isolates (Fig. 2). The degree of identity that was observed between annotated genes and known genes of nucleopolyhedroviruses showed variation, e.g., LdMNPV-KR ORFs manifested higher sequence identity in the blastp search and a greater number of conserved amino acid sequences, ranging from 77.3% (mucin-like protein) to 100% for 33 proteins (polh, chtB1, odv-e27, me-53, orf-28 protein, 39 K, ADPRase, p47, helicase-1, ac52, ac53, vp105, ac57, gp37, ac75, ac76, gp41, p33, odv-e25, odv-e28, p40, p12, orf-96 protein, pkip-1, orf-102 protein, orf-107 protein, lef-1, orf-114 protein, orf-124 protein, pep, orf-134 protein, orf-148 protein, and orf-152 protein). LdMNPV-KG ORFs encode amino acid sequences with identities ranging from 60.6% (iap-2) to 100% for 19 proteins (chtB1, odv-e27, odv-e18, orf-26 protein, lef-6, ADPRase, ac53, vp1054, lef-9, orf-71 protein, ac75, ac76, gp41, p18, odv-e28, p40, p12, lef-1, and orf-132 protein). LdMNPV-BibJ was found to have the least conserved ORFs: from 68.2% (chaB1) to 100% for five proteins (polh, chtB1, odv-e18, ac75, and p12).

Fig. 2
figure 2

Circular genome maps illustrating genomic organization of the LdMNPV isolates (LdMNPV-KR, -KG, and -BibJ). Genome coordinates (in kbp) are marked on the innermost circle. Annotated genes are indicated around the genome.

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A subsequent analysis of homologous sequences using the blastp algorithm revealed 85, 95, and 91 functionally annotated ORFs for LdMNPV-KR, -KG, and -BibJ, respectively. For the remaining sequences, a search for protein domains was conducted using InterProScan. Consequently, the number of annotated ORFs homologous to the available nucleotide sequences increased to 116, 122, and 123 for LdMNPV-KR, -KG, and -BibJ, respectively. The analysis primarily identified Autographa californica nucleopolyhedrovirus open reading frames (ORFs) homologous to those previously characterized (e.g., AC29, AC18, AC106)30. Furthermore, the analysis revealed the presence of proteins exhibiting chitin-binding and RNA-binding domains, as well as ChaB-like, DNase I-like, and telokin-like proteins. Additionally, the study identified ADP-ribose pyrophosphatase31, the per os infectivity factor 632, and the poxin33. Consequently, the proportion of ORFs with a known biological function or a homologue was found to be nearly equivalent for all isolates, at approximately 75%.

Multiple sequence alignment analysis of the vef-1 gene of three new isolates showed that deletion of this gene was detected in isolate LdMNPV-BibJ. The sequences of LdMNPV-KR and LdMNPV-KG isolates showed no deletion compared to the reference sequence (AF081810) (Fig. 3).

Fig. 3
figure 3

Multiple alignment visualization of amino acid sequences of the vef-1 gene. Gray shading indicates conserved residues (matches), blue shading highlights mismatches, and red shading represents gaps. Isolates LdMNPV-304123, -27/027, -RR0141, and -BibJ (this study) possess a deletion of the vef-1 gene and are therefore not displayed in this alignment.

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Phylogenetic analysis

Phylogenetic relationships among 23 selected of baculoviruses were deduced from whole-genome nucleotide sequence alignments by ML analysis (Fig. 4). The three new isolates LdMNPV-KR, -KG, and -BibJ were found to formed distinct lineages. LdMNPV-KR is grouped into a cluster containing isolates from Turkey, the USA, and Poland. LdMNPV-KG ended up in a cluster with isolates from Russia. LdMNPV-BibJ did not cluster with other isolates, but in terms of the evolutionary transitions, it is closest to other Japanese isolates.

Fig. 4
figure 4

Phylogeny of 23 selected baculoviruses on the basis of the whole-genome nucleotide sequence alignments. The phylogeny was reconstructed by the ML analysis with 500 replicates. The numbers at the nodes indicate bootstrap support; new isolates LdMNPV-KR, -KG, and -BibJ are marked with a red color; the scale bar indicates a phylogenetic distance expressed as the number of nucleotide substitutions in the entire sequence.

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Discussion

The bioassay data on relative virulence of the six LdMNPV isolates in this study cover five distinct L. dispar populations (belonging to subspecies L. dispar dispar, L. dispar asiatica, and L. dispar japonica) and one population of L. umbrosa. Our primary comparisons were based on virulence measures (LD50 and survival curves) and genetic characteristics of isolates. LD50 values of four LdMNPV isolates in this study were determined toward three geographically distant L. dispar populations. The maximal distance between the compared insect population habitats is over 6000 km (Krasnodar vs Khabarovsk) and > 3000 km for the closest locations (Krasnodar vs Novosibirsk or Novosibirsk vs Khabarovsk). The larvae from these populations continued to consume their respective host plant species after the treatment with LdMNPV, and the OBs stocks used in the bioassays were derived from the native host larvae.

Outcomes of the LdMNPV infection were confirmed to be dose-dependent as expected, with higher virus doses resulting in greater insect mortality. A significant variation of LD50 values was observed among the isolates (Table 1). L. dispar dispar from Krasnodar exhibited greater susceptibility to LdMNPV-27/0, sourced from a geographically distant location, compared to the local isolate LdMNPV-KR. Interestingly, despite consuming the same host plant species (Betula pendula), the Novosibirsk and Krasnodar populations differed markedly in their responses to LdMNPV-27/0. Recent studies indicate that an L. dispar population from Novosibirsk, located in Western Siberia, likely belongs to the subspecies L. dispar dispar according to microsatellite genotyping34 and mitochondrial-DNA analyses13,35,36,37. Nevertheless, the boundary between the European and Asian subspecies remains unresolved in Eurasia11. One of the key criteria for the division of L. dispar into subspecies is the dimorphism in female flight capabilities. Females of the subspecies L. dispar dispar are unable to engage in strong directed flights4. According to our observations, L. dispar females in Novosibirsk are indeed unable to fly, suggesting that they belong to the subspecies L. dispar dispar.

Survival curve analyses revealed additional complexity in isolate virulence, indicating high variability among isolates toward different host populations. LdMNPV-27/0 exhibited higher virulence against various populations of L. dispar, including L. dispar japonica and L. umbrosa (Fig. 1d). Japanese isolates (LdMNPV-KuzJ, -BibJ, and -ShinJ) also showed significant variation of survival curves toward both their native and distant host populations within the Japanese islands (Fig. 1a,b,c). Both LD50 values and survival analysis indicated heightened susceptibility of the Krasnodar population to most isolates, including LdMNPV-KG. This outcome underscores the notion that mechanisms of L. dispar’s resistance to LdMNPV may play a major role in these differences38,39. The findings of the present study are consistent with previous ones23,27,40. Outcomes of such an infection are determined primarily by characteristics of an isolate rather than by a coevolutionary history between baculoviruses and their respective hosts. The present study indicates that an insect population’s origin may determine the level of susceptibility of larvae to LdMNPV, regardless of the source of the baculovirus.

The observed differences in genome sizes among the isolates in this study can largely be attributed to variations in the number and presence of bros genes. These genes frequently involved in deletions or duplications might contribute significantly to isolate-specific differences in virulence. These results are consistent with those of a previous study23, which has identified a correlation between genome size and multiplication of bro genes in LdMNPV. However, the precise biological functions of bro genes remain to be elucidated.

The absence of the vef-1 gene in LdMNPV-BibJ previously reported also for LdMNPV-304123, -27/027 and -RR0141—challenges existing assumptions regarding its role in virulence. Although vef genes (vef-1 and vef-2) have been associated with the enhanced virulence of LdMNPV42, our bioassay results indicated significantly lower LD50 values for isolates lacking the vef-1 gene such as LdMNPV-27/0 and -BibJ compared to LdMNPV-KR, which retains this gene (Table 1, Fig. 3). Moreover, isolate LdMNPV-3041, also originating from Japan, previously demonstrated high virulence despite lacking vef-1 23. Thus, the phenotypic consequences of the vef-1 deletion appear to be more complex than previously assumed and its recurrent absence in various isolates, including LdMNPV-27/0, -3041, -RR01 and -BibJ suggests that this gene might not be under strong positive selection pressure.

Phylogenetic analysis based on complete genome sequences supports affiliation of the new baculovirus isolates (LdMNPV-KR, -KG, and -BibJ) with three separate lineages (Fig. 4). The phylogenetic relationships detected in this study for LdMNPV-KR, -KG, and -BibJ are in agreement with previous phylogenies40,43. LdMNPV-BibJ from the Asian L. dispar japonica population was placed in a clade with other Japanese isolates which is consistent with its geographic origin. This divergence highlights genetic origin of LdMNPV-BibJ, which may be a result of long isolation and adaptation to local populations of L. dispar japonica in Japan6,13. Isolate LdMNPV-KR formed a clade with an isolate from Turkey, the United States, and Poland, thereby giving a well-supported clade (bootstrap support = 1). This grouping points to a common evolutionary host history despite the substantial geographic distances among these locations. Such relationships may reflect historical migration of L. dispar subspecies13 and global dispersal of baculoviruses. LdMNPV-KG isolate from the L. dispar asiatica population was grouped together with isolates from Russia. This clustering corroborates the findings of Martemyanov et al., (2017) that the LdMNPV-3029 strain (utilized in practice to make a viral agent called Virin ENsh) was initially isolated from an insect population in the Tien Shan mountain region (Jalal-Abad, Kyrgyzstan)44. This is despite Russia’s being shown as the origin in the GenBank entry; this discrepancy may be attributed to the production of Virin ENsh in Moscow. The present study describes a new LdMNPV-KG isolate that exhibits divergence from LdMNPV-3029 strain. LdMNPV-KG and -3029 strain have markedly disparate phenotypes. LdMNPV-KG, originating from native Kyrgyzstan larvae, maintained vef-1 gene (Fig. 3), yet demonstrated relatively low virulence tested host populations, particularly larvae from Novosibirsk (Table 1). This observation further underscores the complexity of the phenotypic role of the vef-1 gene, indicating that its presence is not necessarily correlate with enhanced virulence. Consequently, it is likely that other genetic loci or regulatory mechanisms significantly influence isolate-specific differences in LdMNPV virulence, warranting further investigations.

This study lends further credence to the hypothesis that the efficacy of LdMNPV is predominantly contingent on viral characteristics rather than host–pathogen coevolution. However, variations in local host populations, potentially influenced by environmental conditions and host plant metabolites, also play crucial roles independently of coevolutionary interactions. LdMNPV possesses considerable potential as a safe and environmentally sustainable method for the control of L. dispar populations. The extensive bioassay and genomic data presented in this study are expected to facilitate further advances in the research on (and applications of) this nucleopolyhedrovirus, thereby contributing to a more profound understanding of baculovirus genetics in particular and molecular biology in general. The most potent strain used for the production of biologicals may be employed for application in any area, regardless of a host plant species and spongy moth subspecies.

Material and methods

L. dispar populations

The L. dispar populations used in this study included three insect populations from Russia (from Novosibirsk, Krasnodar and Khabarovsk) and three insect populations from Japan (from Honshu island (Nara) and from Hokkaido island (Sapporo and Teshikaga)) (Table 3). The L. dispar population from Novosibirsk was maintained in the Laboratory of Ecological Physiology at the Institute of Systematics and Ecology of Animals (Novosibirsk, Russia), as previously described in detail45. Briefly, L. dispar larvae were maintained in ventilated plastic containers (approximately 100 larvae in a 20 l container). The branches were inserted into 50 ml tubes with water and sealed with paraflm to prevent water loss. The insects were reared under laboratory conditions at a constant temperature (23 °C) and with a natural daylight regime. The L. dispar populations from Krasnodar (Slavic Experimental Station affiliated with the All-Russian Research Institute of Plant Protection, St. Petersburg, Russia) and Khabarovsk (Laboratory of Animal Ecology, Institute of Water and Environmental Problems FEB RAS) were maintained under similar conditions and fed with the host plant corresponding to the pest’s habitat (Table 3). The L. dispar populations from Japan (Tokyo University of Agriculture and Technology) were maintained under similar conditions on a spongy-moth artificial diet (Frontier Scientific Services, Newark, USA).

Table 3 Lymantria dispar subspecies, host plant species, and sampling sites of the insects subjected to LdMNPV infection.
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LdMNPV isolates

LdMNPV isolates used in bioassays were isolated from cadavers of L. dispar larvae collected in Krasnodar region of Russia; Jalal-Abad region of Kyrgyzstan; and Kuzumaki (Hoshu), Bibai (Hokkaido), and Shintotsukawa (Hokkaido) islands of Japan (Table 4). In particular, 3d or 4th instar L. dispar larval cadavers were homogenized in sterile water and filtered through three layers of cheesecloth to remove larval debris. The OBs were collected by centrifugation for 10 min at 10,000 × g and 4 °C. The OBs were washed by resuspension in 50 ml of sterile water and re-pelleted by centrifugation, then washed again in sterile water and collected again by centrifugation. The purified OBs of LdMNPV isolates were resuspended in sterile water. Purified LdMNPV-BibJ, collected by ultracentrifugation for 45 min at 35,000 g and 4 C (Beckman Coulter, CA, USA) in a Percoll solution (pH 7.0) (GE Healthcare UK Ltd., Buckinghamshire, UK), was acquired from Tokyo University of Agriculture and Technology. The obtained OBs were then used for genomic sequencing and bioassays. The LdMNPV-27/0 used in bioassays was preliminarily amplified in a single passage through 4th instar L. dispar larvae from Novosibirsk. Specifically, the Novosibirsk larvae were infected orally with an OB suspension (108 OBs/ml) and reared until death. The OBs of LdMNPV-27/0 from virus-killed larvae were then isolated and purified as described above. NPV infections are usually obvious as virtually the infected larvae become flaccid and showed sign of liquefaction. The OB concentration was determined using a hemocytometer under phase contrast microscopy (Carl Zeiss Axioscope 40, Germany) at 400 × and stored at 4 °C.

Table 4 Sources of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) isolates used for insect infection.
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Virus extraction and genome sequencing

Three LdMNPV isolates were selected as those of the most distant origins from each other for genome sequencing, namely LdMNPV-KR, -KG, and -BibJ. Viral DNA was extracted from the original OB sample isolated from virus-killed L. dispar larvae from Krasnodar (Russia), Kyrgyzstan, and Bibai (Hokkaido, Japan), respectively (Table 4). Before DNA extraction, the samples of purified OBs were incubated in carbonate buffer (0.1 M Na2CO3, 0.15 M NaCl, 0.01 M EDTA, pH 10.8) at room temperature for 30 min to dissolve OBs. DNA libraries were prepared with the NEBNext Ultra DNA Library Preparation Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions and analyzed on a MiSeq genome sequencer (Illumina, USA).

Genome assembly and annotation

The full-length genome was assembled de novo in Unicycler v0.5.046 and SPAdes v3.15.547 software. Three novel LdMNPV genomes were deposited in GenBank under accession numbers PQ586371 (LdMNPV-KR), PQ586372.2 (-KG), and PQ586373 (-BibJ) (Table S2). Genome size and GC content were calculated by means of QUAST v5.2.048. LdMNPV genomes’ annotation was performed by the Viral Genome Annotation System49. The criterion for ORF identification was a minimum length of 150 bp, and ORF-encoded amino acid sequences were used in blastp queries (GenBank: all nonredundant protein sequences, nr) to identify homologous ORFs. An additional search for functional protein domains was also carried out for ORFs with unclear functions. We employed InterProScan v5.67–99.050 for searches in available databases (InterPro, PANTHER, Pfam, PIRSF, and SUPERFAMILY) for further validation. The whole-genome sequences of the three new isolates of LdMNPV were aligned with the reference isolate LdMNPV-27/0 (KY249580) using Multiple Alignment Fast Fourier Transform (MAFFT) v7.11051. Genomic maps were generated using GCview.ca platform (https://gcview.ca) for visualization of genome organization.

Phylogenetic analysis

For the whole-genome phylogenetic analysis, the nucleotide sequences of the three newly sequenced LdMNPV genomes, (designated as LdMNPV-KR, -KG, and -BibJ) and 20 available LdMNPV genome sequences (Table S2) were aligned in MAFFT. A phylogenetic tree was inferred by the maximum likelihood (ML) method using HKY + G(4) + I evolutionary model with 500 bootstrap replicates. The sequences of Lymantria xylina MNPV-5 were used as an outgroup (not shown)52. The analysis was carried out using R53 packages “ape” v5.854 and “phangorn” v2.11.155. We performed an additional analysis of the vef-1 gene, where amino acid of vef-1 from the GenBank-available sequences (Table S2) were aligned using MAFFT. Visualization of the alignment was performed in R package “seqvisr” v0.2.756.

Bioassay

Infection of L. dispar larvae with LdMNPV isolates was carried out by the droplet feeding method57. Newly molted 2d instar larvae were offered a droplet (0.5 µl) of an aqueous suspension consisting of 100 mg/ml sucrose, 0.01 mg/ml red dye, and OBs. The LdMNPV doses were 5, 50, 150, 500, and 5000 OBs/larva. These dose ranges of baculovirus led to larval mortalities between 5 and 95%. Larvae from Japanese populations were offered the droplet with only one LdMNPV dose: 50 OBs/larva. The larvae that ingested the whole droplet were placed in a 350 ml ventilated plastic container with either the host plant (corresponded to Table 3 for L. dispar populations from Russia) or artificial diet (corresponded to Table 3 for L. dispar populations from Japan). The larvae that failed to consume the whole droplet were excluded from the bioassay. Four to three replicates (containers) were used with 10 larvae per container for each LdMNPV dose (40 or 30 larvae/dose). Larval mortality was monitored daily for 16 days after infection.

Statistical analysis

Statistical analysis was performed in the R software (v4.1.3)53 with the RStudio graphical user interface. Insect mortality in control of L. dispar populations were less than 9%, and no corrections were required. To estimate LD50 values of LdMNPV-27/0, -KR, -KG, and -BibJ, doses of these isolates were log transformed. The transformed doses and the proportion of dead larvae were analyzed using a generalized linear model with a binomial distribution and a logit link function. LD50 and slope values were calculated using the “drc” package (v3.0.1)58. The compParm function in the “drc” package was run to compare LD50 values among isolates and to test whether the slopes were parallel. To evaluate the susceptibility of L. dispar larvae, including the Japanese ones, we performed survival curves using the survfit function from the “survival” package (v3.5.7)59. Survival curve pairwise multiple comparisons were analyzed using the pairwise_survdiff function from the same package (log-rank test with Holm adjustment).