Pathology and genome sequence of a Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) isolate from Heilongjiang, China.

The pathogenicity and genome sequence of isolate LdMNPV-HrB of the gypsy moth alphabaculovirus, Lymantria dispar multiple nucleopolyhedrovirus from Harbin, Heilongjiang, China, were determined. A stock of this virus from one passage through the gypsy moth New Jersey Standard Strain (LdMNPV-HrB-NJSS) exhibited 6.2- to 11.9-fold greater pathogenicity against larvae from a Harbin colony of L. dispar asiatica than both Gypchek and a Massachusetts, USA LdMNPV isolate (LdMNPV-Ab-a624). Sequence determination and phylogenetic analysis of LdMNPV-HrB and LdMNPV-HrB-NJSS revealed that these isolates were most similar to other east Asian LdMNPV isolates with 98.8% genome sequence identity and formed a group with the east Asian LdMNPV isolates which was separate from groups of isolates from Russia, Europe, and USA.

Geographic isolates of Lymantria dispar multiple nucleopolyhedrovirus: Genome sequence analysis and pathogenicity against European and Asian gypsy moth strains.

Isolates of the baculovirus species Lymantria dispar multiple nucleopolyhedrovirus have been formulated and applied to suppress outbreaks of the gypsy moth, L. dispar. To evaluate the genetic diversity in this species at the genomic level, the genomes of three isolates from Massachusetts, USA (LdMNPV-Ab-a624), Spain (LdMNPV-3054), and Japan (LdMNPV-3041) were sequenced and compared with four previously determined LdMNPV genome sequences. The LdMNPV genome sequences were collinear and contained the same homologous repeats (hrs) and clusters of baculovirus repeat orf (bro) gene family members in the same relative positions in their genomes, although sequence identities in these regions were low. Of 146 non-bro ORFs annotated in the genome of the representative isolate LdMNPV 5-6, 135 ORFs were found in every other LdMNPV genome, including the 37 core genes of Baculoviridae and other genes conserved in genus Alphabaculovirus. Phylogenetic inference with an alignment of the core gene nucleotide sequences grouped isolates 3041 (Japan) and 2161 (Korea) separately from a cluster containing isolates from Europe, North America, and Russia. To examine phenotypic diversity, bioassays were carried out with a selection of isolates against neonate larvae from three European gypsy moth (Lymantria dispar dispar) and three Asian gypsy moth (Lymantria dispar asiatica and Lymantria dispar japonica) colonies. LdMNPV isolates 2161 (Korea), 3029 (Russia), and 3041 (Japan) exhibited a greater degree of pathogenicity against all L. dispar strains than LdMNPV from a sample of Gypchek. This study provides additional information on the genetic diversity of LdMNPV isolates and their activity against the Asian gypsy moth, a potential invasive pest of North American trees and forests.

Role of ocular albinism type 1 (OA1) GPCR in Asian gypsy moth development and transcriptional expression of heat-shock protein genes.

The ocular albinism type 1 gene, named OA1, is a coding pigment cell-specific G protein-coupled receptor exclusively localized in intracellular organelles. However, the function of OA1 in insects remains generally unknown. In the present study, we explore for the first time the function of LdOA1 in the Asian gypsy moth, Lymantria dispar. To identify the function of LdOA1 gene in the development and growth of the Asian gypsy moth, the LdOA1 gene in third instar larvae was knocked down by RNAi. Compared with the controls, the knockdown of LdOA1 increased larval mortality but did not significantly affect their utilization of nutrition. Moreover, LdOA1 was stably transformed into the third chromosome of Drosophila melanogaster. The LdOA1 gene in the transformed D. melanogaster modulated the expression of heat-shock protein (hsp) and increased the expression of hsp genes under deltamethrin stress, which indicates that LdOA1 is involved in the regulation of hsp gene expression. These results deepen our understanding of the molecular function of OA1 in insects.

Characterization of the transcriptome of the Asian gypsy moth Lymantria dispar identifies numerous transcripts associated with insecticide resistance.

Although the Asian gypsy moth Lymantria dispar causes extensive forest damage worldwide, little is known regarding the genes involved in its development or response to insecticides. Accordingly, characterization of the transcriptome of L. dispar larvae would promote the development of toxicological methods for its control. RNA-seq analysis of L. dispar larvae messenger RNA (mRNA) generated 62,063 unigenes with N50 of 993 bp, from which 23,975 unique sequences (E-value < 10(-5)) were identified using a BLASTx search of the NCBI non-redundant (nr) database. Using functional classification in the Gene Ontology (GO) and Clusters of Orthologous Groups (COG) databases, 7,309 indentified sequences were categorized into 51 functional groups and 8,079 sequences were categorized into 25 functional groups, respectively. Moreover, we identified a large number of transcripts encoding known insecticide targets, or proteins involved in the metabolism of insecticides. Reads per kilobase of unigene length per million mapped reads (RPKM) analysis identified 39 high abundance transcripts, of which 27 exhibited significantly altered expression patterns across the egg, larvae, pupae, male and female adult stages. Our study provides the most comprehensive transcriptomic sequence resource for L. dispar, which will form the basis for future identification of candidate insecticide resistance genes in L. dispar.

A Multiplex Real-Time PCR Assay for Screening Gypsy Moths (Lepidoptera: Erebidae) in the United States for Evidence of an Asian Genotype.

European gypsy moth populations (Lymantria dispar L.) are well established and a proven destructive force in hardwood trees throughout the United States and Canada. Introduction of the exotic Asian gypsy moth into North America would be even more impactful, as Asian gypsy moth populations have wider host ranges, and are capable of naturally dispersing more rapidly due to female flight ability. To support early detection and exclusion of Asian gypsy moth, the U.S. Department of Agriculture (USDA) uses molecular techniques to screen moths trapped in North America for evidence of common Asian genotype. In order to strengthen U.S. domestic capacity to screen moths quickly and efficiently, we report a real-time PCR assay for this pest. A probe system using TaqMan 5' nuclease chemistry is reported for detection of an allele associated with common Asian gypsy moth genotypes. The targeted allele is located at the nuclear FS1 locus currently used by the USDA in conventional PCR tests to screen for evidence of Asian gypsy moth introductions or introgression. The diagnostic probe is successfully multiplexed with a conserved 18S probe system to detect reaction failure due to poor sample quality or quantity. The specificity, sensitivity, and repeatability of the FS1-18S multiplex real-time PCR assay were tested on laboratory-reared and field-collected moths to demonstrate diagnostic utility. Implications of the new assay as a screening tool for evidence of Asian gypsy moth introgression and introduction are discussed.

Transcription profiling of 12 asian gypsy moth (Lymantria dispar) cytochrome P450 genes in response to insecticides.

As the main group of detoxification enzymes, cytochrome P450 monoxygenases (P450s) catalyse an extremely diverse range of reactions that play an important role in the detoxification of foreign compounds. Transcription profiling of 12 Lymantria dispar P450 genes from the CYP6 subfamily believed to be involved in insecticide metabolism was performed in this study. Life-stage transcription profiling of CYP6 genes revealed significant variations between eggs, larvae, pupae, and adult males and females. Exposure of larvae to sublethal doses of deltamethrin, omethoate, and carbaryl enhanced the transcription of most of the CYP6 P450 genes, with induction peaking between 24 and 72 h after exposure. Transcription profiles were dependent on the levels of insecticide exposure and the various developmental stages.

RNAi-based functional analysis of bursicon genes related to wing expansion in gypsy moths.

Bursicon is a heterodimeric neuropeptide composed of Burs-α and Burs-ß subunits that plays an important role in cuticle tanning and wing expansion in insects. In this study, full-length cDNAs of Burs-α (LdBurs-α) and Burs-ß (LdBurs-ß) genes were identified in gypsy moth (Lymantria dispar) and cloned. The 480 bp and 420 bp open reading frames (ORFs) encode 159 and 129 amino acid polypeptides, respectively. LdBurs-α and LdBurs-ß have 11 conserved cysteine residues, and LdBurs-α and LdBurs-ß genes were expressed during all developmental stages according to quantitative reverse transcription PCR (qRT-PCR), with highest expression in the egg stage. High expression levels were also detected in the haemolymph, cuticle and head. To explore the physiological functions of LdBurs-α and LdBurs-ß, the genes were knocked down in larvae and pupae using RNA interference (RNAi), and expression levels of LdBurs-α and LdBurs-ß were decreased by 42.26-80.09%. Wing defects were observed in L. dispar pupae following Ldbursion silencing, with a phenotypic percentage ranging from 10.17% to 15.00%. RNAi-mediated knockdown of Ldbursicon prevented the expansion of male and female L. dispar adult wings, with malformation rates ranging from 6.38% and 30.00% to 57.69% and 69.23%, but no cuticle tanning defects were observed in pupae or adults. The results indicate that bursicon plays a key role in wing expansion in L. dispar adults, making it a potentially novel molecular target for insecticide-based control of this pest species.

A Phenology Model for Asian Gypsy Moth Egg Hatch.

Phenology models are useful tools in pest management interventions, biosecurity operations targeting alien invaders, and answering questions regarding the potential for range expansion/shift. The Gypsy Moth Life Stage model (GLS) has been used to predict the invasive range of the North American gypsy moth (Lymantria dispar dispar Linnaeus [Lepidoptera: Erebidae]) in North America and New Zealand. It has been used to examine the role of supra-optimal temperatures in range expansion/stasis/retraction. However, GLS has also been used where the target organism is the Asian subspecies L. d. asiatica Vnukovskij, despite observed differences between the predominant phenotypes of the two subspecies in the temperature requirements for egg hatch and the absence of egg phenology model parameters specific to the Asian phenotype. Here we describe the results of temperature and exposure duration on the timing of Asian gypsy moth egg hatch, and we present phenology model parameters for the Asian phenotype. Sum of squared differences (observed minus predicted day of median egg hatch) was reduced from 7,818 d2 (North American parameters) to 178 d2. Days of simulated median egg hatch differed from the observed days by 0-7 d (x¯=0.2; SD=3.1). The pattern of simulated egg hatch closely mimicked the irregular pattern of observed egg hatch from the temperature regimes of our experiment. Egg hatch is arguably the most important life cycle event in gypsy moth population suppression/eradication interventions and in estimating their potential invasive range. The model parameters described here produce accurate predictions of Asian gypsy moth egg hatch.

Ocular Albinism Type 1 Regulates Deltamethrin Tolerance in Lymantria dispar and Drosophila melanogaster.

The ocular albinism type 1 (OA1), a pigment cell-specific integral membrane glycoprotein, is a member of the G-protein-coupled receptor (GPCR) superfamily that binds to heterotrimeric G proteins in mammalian cells. We aimed to characterize the physiological functions an insect OA1 from Lymantria dispar (LdOA1) employs in the regulation of insecticide tolerance. In the present study, we investigated the roles of LdOA1 in response to deltamethrin exposure in both L. dispar and Drosophila melanogaster. LdOA1 was expressed at the lowest level during the 4th instar stage, while LdOA1 was significantly upregulated in the 5th instar and male stages. Knockdown of LdOA1 by injecting dsRNA of LdOA1 into gypsy moth larvae caused a 4.80-fold higher mortality than in control larvae microinjected with dsRNA of GFP under deltamethrin stress. Nine out of 11 L. dispar CYP genes were significantly downregulated under deltamethrin stress in LdOA1 silenced larvae as compared to control larvae. Moreover, the LdOA1 gene was successfully overexpressed in D. melanogaster using transgenic technique. The deltamethrin contact assay showed that the LdOA1 overexpression in flies significantly enhanced the tolerance to deltamethrin compared to the control flies. Furthermore, the downstream Drosophila CYP genes were upregulated in the LdOA1 overexpression flies, suggesting LdOA1 may play a master switch role in P450-mediated metabolic detoxification. This study is the first report of an insect OA1 gene regulating insecticide tolerance and potentially playing a role in the regulation of downstream cytochrome P450 expression. These results contribute to the future development of novel insecticides targeting insect GPCRs.

Effects of Temperature on Development of Lymantria dispar asiatica and Lymantria dispar japonica (Lepidoptera: Erebidae).

Periodic introductions of the Asian subspecies of gypsy moth, Lymantria dispar asiatica Vnukovskij and Lymantria dispar japonica Motschulsky, in North America are threatening forests and interrupting foreign trade. Although Asian gypsy moth has similar morphology to that of European and North American gypsy moth, it has several traits that make it a greater threat, the most important being the flight capability of females. Asian gypsy moth is not yet established in North America; however, infestations have been detected multiple times in Canada and the United States. To facilitate detection and eradication efforts, we evaluated the effect of a range of temperatures on development time, survivorship, and fertility of eight populations of Asian gypsy moth. There were significant impacts of temperature and population on these life history characteristics. The larval developmental rate increased with temperature until it reached an optimum at 29 °C. Larvae experienced significant molting problems at the highest and lowest temperatures tested (10 °C and 30 °C). At 30 °C, female fitness was markedly compromised, as evidenced by reduced fecundity and fertility. This suggests that development and survival of Asian gypsy moth may be limited by summer temperature extremes in the Southern United States. We also determined the degree-day requirements for two critical life stages and two populations of Asian gypsy moth, which represent the extremes in latitude, to predict the timing for biopesticide application and adult trap deployment. Our data will benefit pest managers in developing management strategies, pest risk assessments, and timing for implementation of management tactics.

Evaluation of the Effects of Light Intensity and Time Interval After the Start of Scotophase on the Female Flight Propensity of Asian Gypsy Moth (Lepidoptera: Erebidae).

Asian gypsy moth, Lymantria dispar L. (Lepidoptera: Erebidae), females are capable of flight, but little is known about what causes the variation in flight propensity that has been observed. The female flight propensity and capability of Asian gypsy moth from seven geographic populations (three from China, two from Russia, one from Japan, and one from Korea) were compared under all combinations of three light intensities (0.05, 0.10, and 0.40 lux) and during three time intervals after the start of scotophase. A total of 567 females were flight tested. Female flight propensity, time to initiate walking, fanning, and flying, and duration of fanning differed significantly among geographic populations. Females were less likely to voluntarily fly during the 0-1-h time interval after the start of scotophase than during the later time intervals (1-2 and 2-3 h), suggesting that the light intensity cue has to occur at the correct time after the expected start of scotophase for flight initiation. Light intensity did not significantly affect the proportion of females that voluntarily flew, but did impact the timing of the walking and fanning preflight behaviors. The interaction between light intensity and time interval after the start of scotophase had a significant effect on the proportion of females that fanned. The proportion of females with sustained flight capability varied among the populations evaluated. These results may aid in determining the risk of Asian gypsy moth dispersal, but further work is needed to assess other factors that play a role in flight propensity.

Sequencing and de novo assembly of the Asian gypsy moth transcriptome using the Illumina platform

Abstract The Asian gypsy moth (Lymantria dispar) is a serious pest of forest and shade trees in many Asian and some European countries. However, there have been few studies of L. dispar genetic information and comprehensive genetic analyses of this species are needed in order to understand its genetic and metabolic sensitivities, such as the molting mechanism during larval development. In this study, high-throughput sequencing technology was used to sequence the transcriptome of the Asian subspecies of the gyspy moth, after which a comprehensive analysis of chitin metabolism was undertaken. We generated 37,750,380 high-quality reads and assembled them into contigs. A total of 37,098 unigenes were identified, of which 15,901 were annotated in the NCBI non-redundant protein database and 9,613 were annotated in the Swiss-Prot database. We mapped 4,329 unigenes onto 317 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. Chitin metabolism unigenes were found in the transcriptome and the data indicated that a variety of enzymes was involved in chitin catabolic and biosynthetic pathways.