Dispar Larvae Research Articles

Evaluating the ecotoxicological effects of Pb contamination on the resistance against Lymantria dispar in forest plant, Larix olgensis.

Heavy metal contamination in forest ecosystems has become increasingly severe, and there is an urgent need to better understand the ecotoxicological effects of heavy metals on the whole forest ecosystems, especially their effects on insect resistance of forest plant. In the present study, the resistance against gypsy moth (Lymantria dispar) larvae in Larix olgensis seedlings grown in non-amended or Pb-amended (at 500 and 1500 mg kg-1 ) soil was evaluated. Pb from the treated soil could be transferred and exerted bio-toxicological effects along the food chain consisting of L. olgensis seedlings and gypsy moth larvae, eventually causing significantly reduction in seedlings growth, as well as larval weight, survival rate and antioxidant capacity. With regard to phytochemical defense, the activities or contents of protease inhibitors (trypsin and chymotrypsin inhibitors) and secondary metabolites (condensed tannin and total phenolics) in Pb-treated larch needles presented a tendency of 'low-promotion, high-inhibition' with the increase of Pb exposure concentration. At the same time, Pearson's correlation coefficients showed that the trade-off hypothesis on energy allocation between phytochemical defense and plant growth was not supported by the data from the L. olgensis seedlings that were exposed to Pb stress, and elemental defense might replace the dominant role of phytochemical defense in L. olgensis seedlings under Pb stress against the gypsy moth larvae. These findings emphasize ecotoxicological effects of heavy metal contaminations along the food chains (forest plants and forest defoliators), and provide a new perspective for optimizing forest pest control strategies in the heavy metal polluted regions. © 2020 Society of Chemical Industry.

Silencing Br-C impairs larval development and chitin synthesis in Lymantria dispar larvae

In insects, 20-hydroxyecdysone (20E) mediates developmental transitions and regulates molting processes through activation of a series of transcription factors. Broad-Complex (Br-C), a vital gene in the 20E signalling pathway, plays crucial roles during insect growth processes. However, whether Br-C affects chitin synthesis in insects remains unclear. In the present study, the Br-C gene from Lymantria dispar, a notorious defoliator of forestry, was identified based on transcriptome data, and subjected to bioinformatic analysis. The regulatory functions of LdBr-C in chitin synthesis and metabolism in L. dispar larvae were analysed by RNA interference (RNAi). The full-length LdBr-C gene (1431 bp) encodes a 477 amino acid (aa) polypeptide containing a common BRcore region (391 aa) at the N-terminus and a C-terminal Zinc finger domain (56 aa) harbouring two characteristic C2H2 motifs (CXXC and HXXXXH). Phylogenetic analyses showed that LdBr-C shares highest homology and identity with Br-C isoform 7 (83.12%) of Helicoverpa armigera. Expression profiles indicate that LdBr-C was expressed throughout larval and pupal stages, and highly expressed in prepupal and pupal stages. Furthermore, LdBr-C expression was strongly induced by exogenous 20E, and suppressed dramatically after application of dsLdBr-C. Bioassay results showed that knockdown of LdBr-C caused larval developmental deformity, significant weight loss, and a mortality rate of 67.18%. Knockdown of LdBr-C significantly down-regulated transcription levels of eight critical genes (LdTre1, LdTre2, LdG6PI, LdUAP, LdCHS1, LdCHS2, LdTPS and LdCHT) related to chitin synthesis and metabolism, thereby lowering the chitin content in the midgut and epidermis. Our findings demonstrate that Br-C knockdown impairs larval development and chitin synthesis in L. dispar.

Avermectin stress varied structure and function of gut microbial community in Lymantria dispar asiatica (Lepidoptera: Lymantriidae) larvae

Lymantria dispar asiatica is a globally distributed herbivorous pest. Avermectin is a highly effective, broad-spectrum insecticide. In this study, fourth instar L. dispar asiatica larvae were exposed to a LC30 dose of avermectin. The structure and function of larval gut microbial community was analyzed to examine how gut microbiota in L. dispar asiatica larvae responded to avermectin stress. Results showed that the structure and function of gut microbial community in L. dispar asiatica larvae were varied by avermectin stress. To be precise, more than half quantity of the observed Optical Taxonomic Units (OTUs) showed significantly different abundances under avermectin stress. Linear discriminant analysis effect size (LEfSe) suggested nine bacterial genera and 12 fungal genera contributed to the different gut microbial community structure in L. dispar asiatica larvae. Gut microbial function classification (PICRUSt and FUNGuild) suggested that three bacterial function categories and a fungal function guild were significantly increased, and two fungal function guilds were significantly decreased by avermectin stress. This study furthers our understanding of the physiology of L. dispar asiatica larvae under avermectin stress, and is an essential step towards future development of potential pesticide targets.

Variation in the pH of experimental diets affects the performance ofLymantria dispar asiaticalarvae and its gut microbiota

To study dietary pH effects on Lymantria dispar asiatica larvae and provide a theoretical basis for its control in different forests, phosphate buffers (PBs) of pH 6, 7, and 8 were used to prepare experimental diets. The diet prepared with pH 6 PB was named as DPB6, with pH 8 PB as DPB8, and with pH 7 PB as DPB7 (control). The dietary pH was 5.00 in DPB6, 6.05 in control, and 6.50 in DPB8. After feeding on the diets with different pH values for 84 hr, fourth-instar caterpillars were randomly collected. Growth and various physiological traits were determined and 16S recombinant DNA sequencing was performed using the intestinal microflora of surviving larvae. Results showed that the mortality was 30% in DPB6, and 10% in DPB8, while no mortality was observed in control. The partial least squares discriminant analyses suggested that diets prepared with PB of different pH resulted in different food intake, amount of produced feces, weight gain, digestive enzyme activities, and antioxidant enzyme activities in larvae. Interestingly, both the highest weight gain and the lowest total antioxidant capacities were seen in control larvae. Results also showed that the larval gut microbiota community structure was significantly affected by dietary pH. Moreover, linear discriminant analysis effect size suggested that the family Acetobacteraceae in control, genus Prevotella in DPB8, and genus Lactococcus, family Flavobacteriaceae, family Mitochondria, and family Burkholderiaceae in DPB6 contributed to the diversity of the larval gut microbial community.

The improved resistance against gypsy moth in Larix olgensis seedlings exposed to Cd stress association with elemental and chemical defenses.

Cadmium (Cd), as an environmental pollutant, can endanger various biological and chemical characteristics of plants in multiple aspects. In this study, the effects of Cd contamination or exposure for 30 and 60 days at 1, 2, 4 mg kg-1 concentrations on the resistance of Larix olgensis seedlings to the gypsy moth (Lymantria dispar) larvae were investigated. Our results showed that Cd stress did not significantly affect the growth and biomass parameters of the larch seedlings, which might be attributed to the scavenging mechanism of reactive oxygen species (e.g. superoxide dismutase and peroxidase). Regarding the phytochemical defense, we found that Cd stress significantly changed the contents or activities of protease inhibitors (such as trypsin and chymotrypsin inhibitors) and secondary metabolites (tannins and phenolic acids) in L. olgensis seedling needles; however, their response trends varied with Cd exposure concentrations with a significant increase at low concentrations and a significant decrease at high concentrations. Moreover, both chemical and elemental defenses contributed to the resistance of L. olgensis seedlings to the gypsy moth larvae, and their synergistic effects (between toxic elements and organic metabolites) could provide an overall improved defense of L. olgensis seedlings even at low concentrations of single components, resulting in a detrimental effect on the growth of gypsy moth larvae. These findings call for an urgent need to adjust and optimize pest control strategies in heavy metal polluted areas based on the effects of heavy metal stress on woody plant resistance to pest insects. © 2019 Society of Chemical Industry.

Fenoxycarb and methoxyfenozide (RH-2485) affected development and chitin synthesis through disturbing glycometabolism in Lymantria dispar larvae

Fenoxycarb as a juvenile hormone analogue and methoxyfenozide (RH-2485) as a 20-hydroxyecdysone (20E) agonist are two main insect growth regulators (IGRs) used for pest control, whose insecticidal mechanisms had been widely reported in past decades. However, there were few studies focused on their effects on the carbohydrate metabolism of insects. Here, we reported that two IGRs (fenoxycarb and RH-2485) significantly affected growth and development of L. dispar larvae and caused larval lethality. Furthermore, both contens of three sugars (glycogen, threhalose, glucose) in four tissues (fat body, midgut, hemolymph and epidermis) and trehalase activity in three tissues (fat body, midgut and hemolymph) of L. dispar larvae were markedly affected by these two IGRs. Moreover, we found that mRNA expression levels of LdTPS, LdTre1 and LdTre2 in L. dispar larvae were dramatically suppressed by two IGRs. Additionally, chitin content in both midgut and epidermis decreased significantly after L. dispar larvae treated with fenoxycarb or RH-2485. Summarily, these results indicated that these two IGRs disturbed glycometabolism in L. dispar larvae, resulting in impeding chitin synthesis, generating new epidermis failure, disrupting molting and larval lethality in the end.

Effects of poplar secondary metabolites on performance and detoxification enzyme activity of Lymantria dispar

To identify the effects of poplar secondary metabolites on Lymantria dispar, six poplar secondary metabolites (i.e., caffeic acid, salicin, rutin, quercetin, flavone, and catechol) and three mixtures containing characteristic secondary metabolites in poplar were selected. Mixture 1 contained flavone and salicin, mixture 2 contained salicin, caffeic acid, and catechol, and mixture 3 contained flavone, catechol, and caffeic acid. Mixtures were added to artificial diets used to feed 2nd instar L. dispar larvae. The effects of different secondary metabolites on larval growth and development, antifeedant activity, nutrient utilization, and detoxifying enzymatic activity were investigated. Results revealed that there were different influences on L. dispar larvae. The maximum antifeedant rate of flavone was 87.58%. Larvae treated with mixture 2 had a significantly longer development time of 5.61 d with a survival rate of 38.75% for 15 d, which is lower than a single secondary metabolite. No L. dispar larvae survived on feeding diets containing flavone for 7 d. An increase in GST and P450 activities in larvae was significantly induced during the 72 h feeding on artificial diets containing experimental secondary metabolites. After treatment containing salicin and flavone for 24–72 h, P450 activity increased at first then decreased. These results provide a foundation for further investigation on the host selection and underlying adaptation mechanisms in L. dispar.

Ambient temperature-mediated enzymic activities and intestinal microflora in Lymantria dispar larvae.

To understand how ambient temperature affect the gypsy moth larvae, and provide a theoretical basis for pest control in different environments. Fourth instar gypsy moth larvae were incubating for 3 hr at 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃, respectively. Afterward, digestive and antioxidant enzyme activities, total antioxidant capacity, and intestinal microflora community were analyzed to reveal how the caterpillars respond to ambient temperature stress. Results showed that both digestive and antioxidant enzymes were regulated by the ambient temperature. The optimum incubation temperatures of protease, amylase, trehalase, and lipase in gypsy moth larvae were 30℃, 25℃, and 20℃, respectively. When the incubation temperature was deviated optimum temperatures, digestive enzyme activities would be downregulated depending on the extent of temperature stress. In addition, glutathione S-transferase, peroxidase, catalase, and polyphenol oxidase would be activated under a sufferable temperature stress, but superoxide dismutase and carboxylesterase (CarE) would be inhibited. In addition, results showed that the top two abundant phyla were Proteobacteria and Firmicutes. The phylum Firmicutes abundance was decreased and phylum Proteobacteria abundance was increased by ambient temperature stress. Moreover, it suggested that gypsy moth caterpillars at different ambient temperature mainly differed from each other by Escherichia-Shigella and Bifidobacterium in control, Acinetobacter in T15, and Lactobacillus in T40, respectively.

Effect of fluoranthene on antioxidative defense in different tissues of Lymantria dispar and Euproctis chrysorrhoea larvae

This study examined the effect of long-term exposure to environmentally relevant concentrations of dietary fluoranthene (6.7 and 67 ng / g dry food weight) on defense mechanisms of the polyphagous forest insects Lymantria dispar L. and Euproctis chrysorrhoea L. The activities and expression of isoforms of superoxide dismutase (SOD) and catalase (CAT), the activities of glutathione S-transferase (GST) and glutathione reductase (GR), and total glutathione content (GSH) were determined in the whole midgut and midgut tissue, while SOD and CAT activities were assessed in hemolymph of the larvae. The results showed significant changes of enzyme activities, with more pronounced responses in larval midgut tissues, and between-species differences in patterns of response. Significantly increased activity of SOD was recorded in the whole midgut and midgut tissue of L. dispar larvae, as well as in midgut tissue of E. chrysorrhoea larvae. Fluoranthene increased CAT activity in midgut tissue of L. dispar larvae, and in the whole midgut and midgut tissue of E. chrysorrhoea larvae. Different expression patterns were detected for enzyme isoforms in tissues of larvae exposed to dietary fluoranthene. Total GSH content and GST activity increased in E. chrysorrhoea larval midgut tissue. Significantly decreased SOD activity in hemolymph of L. dispar larvae, and opposite changes in CAT activity were recorded in the hemolymph of larvae of two insect species. The tissue-specific responses of enzymes to dietary fluoranthene, recorded in each species, enabled the larvae to overcome the pollutant induced oxidative stress, and suggest further assessment of their possible use as early-warning signals of environmental pollution.

DNA insecticide developed from the Lymantria dispar 5.8S ribosomal RNA gene provides a novel biotechnology for plant protection

Having observed how botanicals and other natural compounds are used by nature to control pests in the environment, we began investigating natural polymers, DNA and RNA, as promising tools for insect pest management. Over the last decade, unmodified short antisense DNA oligonucleotides have shown a clear potential for use as insecticides. Our research has concentrated mainly on Lymantria dispar larvae using an antisense oligoRING sequence from its inhibitor-of-apoptosis gene. In this article, we propose a novel biotechnology to protect plants from insect pests using DNA insecticide with improved insecticidal activity based on a new antisense oligoRIBO-11 sequence from the 5.8S ribosomal RNA gene. This investigational oligoRIBO-11 insecticide causes higher mortality among both L. dispar larvae grown in the lab and those collected from the forest; in addition, it is more affordable and faster acting, which makes it a prospective candidate for use in the development of a ready-to-use preparation.

Effects of Temperature on First Instar Lymantria (Lepidoptera: Erebidae) Survival and Development With and Without Food.

Lymantria dispar L. and Lymantria monacha (L.) are Eurasian pests that have the potential for accidental introduction via trade into other world areas. Establishment of first instars of Lymantria depends on larvae surviving long enough to disperse and finding suitable hosts. The survival and development of newly hatched Lymantria larvae from nine geographic populations at seven temperatures (1-30°C) held without food, with summer foliage of a preferred or conifer host was determined. There was considerable variation both within and among the Lymantria populations in the survival of larvae at different temperatures when held with and without food. Without food survival declined from about a month at 5°C to a few days at 30°C, following a typical enzymatic kinetic rate function. At 1°C larval survival was less than at 5°C likely because the larvae were susceptible to freezing. Larvae from the one L. monacha population fed and gained weight on the summer foliage, particularly on the conifer, at 10-15°C but < 20% survived for 14 d at 20-30°C. The newly hatched L. dispar larvae from all eight populations fed (at 10-30°C) and developed (at 15-30°C) on the summer foliage of one or both of the hosts. This suggests that they may be able to find adequate food for establishment even if hatch is not synchronous with bud break in the invaded habitat. Survival on the conifer was highest for one Chinese and two European populations of L. dispar, suggesting the ability to utilize conifers is population and not subspecies specific.

Extraction optimization of insecticidal compounds from Lysurus mokusin by response surface methodology

We optimized the extraction process of Lysurus mokusin (L.) Fr. insecticidal compound (LMIC) using response surface methodology with ultrasonic extraction (UE). The surface morphologies of maceration extract (ME) and UE residues were compared by scanning electron microscopy, and then the activity of LMIC on Lymantria dispar (Asian gypsy moths) larvae was determined. Results showed that the optimal condition of UE was followed by 80 min of extraction time, 60% of ethanol concentration and 80 mL g−1 of liquid–solid ratio. The surface morphologies of UE residue were looser and rougher than ME residue. The corrected mortalities of LMIC on L. dispar larvae was 59.77%. This result indicated that L. mokusin was a good candidate as pesticide for pest management.

Cu/ZnSOD always responded stronger and rapider than MnSOD in Lymantria dispar larvae under the avermectin stress

Metalloenzyme SODs play important roles in insects dealing with environmental stress. Here, we cloned the Cu/ZnSOD (LdCZS) and MnSOD (LdMS) mRNA of Lymantria dispar by rapid amplification of cDNA ends (RACE). Afterwards their expression patterns were detected by quantitative real-time polymerase chain reaction (qPCR) after bioinformatic analysis. We found that both LdCZS and LdMS were widely detected in all gypsy moth larvae and all five tissues that we analyzed, and both of them were up-regulated after larvae were fed with avermectin of sublethal concentration and LC10. The LdCZS expression value are always higher than LdMS after treating with avermectin of sublethal concentrations. In addition, temporal expression profile in avermectin treated larvae showed that LdCZS expressed highest at 2nd hour, and LdMS expressed highest at 6th hour. The cuticulas transcribed LdCZS and LdMS significantly higher than heads, fat bodies, Malpighian tubes, and midguts after spraying avermectin of sublethal concentration. These results suggested that both Cu/ZnSOD and MnSOD are important antioxidant enzymes in L. dispar defensing against pesticide stress, and LdCZS always responded rapider and stronger than LdMS.

Benzo[a]pyrene-induced changes in carboxylesterase, acetylcholinesterase and heat shock protein 70 of Lymantria dispar (Lepidoptera: Lymantriidae) from unpolluted and polluted forests

Plant vegetation accumulates polycyclic aromatic hydrocarbons (PAHs) among which benzo[a]pyrene (B[a]P) is recognized as being very toxic, including cancerogenic. Lymantria dispar L. larvae are sensitive to changes in the environment, providing potential signs of pollutant presence. We examined the chronic effects of two concentrations of B[a] P on the activity of carboxylesterase (CaE), acetylcholinesterase (AChE) and heat shock protein 70 (Hsp70) levels in the brain tissue of two populations of L. dispar larvae, originating from unpolluted and polluted habitats. We found that the relative growth rate was significantly lower in both populations and that only larvae from polluted forests were sensitive to low B[a]P concentrations, exhibiting a significant increase in brain tissue CaE activity and Hsp70 concentration. AChE activity showed no changes in response to B[a]P exposure in either population. Examined biochemical parameters indicate that their sensitivity to chronic treatment with B[a]P was highly dependent on the pre-exposure history of L. dispar larvae, suggesting that they could be promising biomarkers of B[a]P and PAH pollution in forest ecosystems.

Phototransduction and circadian entrainment are the key pathways in the signaling mechanism for the baculovirus induced tree-top disease in the lepidopteran larvae

The tree-top disease is an altered behavioral state, displayed by baculovirus-infected lepidopteran larvae, and characterized by climbing to an elevated position before death. The detailed molecular mechanism underlying this phenomenal behavior change has not been reported yet. Our study focused on the transcriptomic changes in the host larvae due to baculovirus infection from pre-symptomatic to tree-top disease stage. Enrichment map visualization of the gene sets grouped based on the functional annotation similarity revealed 34 enriched pathways in signaling mechanism cluster during LdMNPV induced tree-top disease in third instar Lymantria dispar asiatica larvae. Directed light bioassay demonstrated the positively phototactic larvae during tree-top disease and the gene expression analysis showed altered rhythmicity of the host’s core circadian genes (per and tim) during the course of infection emphasizing the role of Circadian entrainment and Phototransduction pathways in the process, which also showed maximum interactions (>50% shared genes with 24 and 23 pathways respectively) among other signaling pathways in the enrichment map. Our study provided valuable insights into different pathways and genes, their coordinated response and molecular regulation during baculovirus infection and also improved our understanding regarding signaling mechanisms in LdMNPV induced tree-top disease.

Sanguinarine in Chelidonium majus induced antifeeding and larval lethality by suppressing food intake and digestive enzymes in Lymantria dispar

Our previous studies had identified that both crude extracts and total alkaloid from Chelidonium majus exerted a significant antifeeding and larval lethality on Lymantria dispar. Moreover, sanguinarine, chelidonine, berberine hydrochloride and coptisine were the main alkaloid in C. majus exerting toxicity to L. dispar. In this paper, we evaluated the insecticidal and antifeeding activities of each alkaloid on the 3rd instar L. dispar larvae by bioassay. Meanwhile, the effects of alkaloids from C. majus on the activities and mRNA levels of three main digestive enzymes in L. dispar larvae were investigated. The results indicated that sanguinarine possessed the strongest insecticidal activity with a LD50 value of 4.963 μg/larva, and the coptisine showed little lethality to 3 rd instar L. dispar larvae among four alkaloids from C. majus. The insecticidal capacity of four alkaloids on 3rd instar L. dispar larvae was in the following decreasing order of sanguinarine > chelidonine > berberine hydrochloride > coptisine. Similarly, except coptisine, the other three alkaloids significantly reduced food intakes of third instar L. dispar larvae and suppressed activities of three digestive enzymes (α-amylase, lipase and total protease) simultaneously. Finally, qRT-PCR analysis revealed that the transcriptions of α-amylase, lipase and serine protease were affected by sanguinarine. Especially, at 48 h after treatment, the mRNA expressions of those digestive enzymes were significantly suppressed by sanguinarine. In conclusion, we suggested that alkaloids from C. majus induced antifeeding and larval lethality on L. dispar larvae by suppressing food intake and digestive enzymes in L. dispar. Our findings provide a novel insight into evaluating the antifeeding and insecticidal properties of C. majus, which afford a new strategy for integrated pest management programs as well.

Heavy Metal Accumulation/Excretion in and Food Utilization of Lymantria dispar Larvae Fed With Zn- or Pb-Stressed Populus alba berolinensis Leaves.

Heavy metal contaminations have attracted increasing concern worldwide due to their potential damages to the whole ecosystem. This study investigated the heavy metal-accumulation and excretion in, and food utilization of the gypsy moth (Lymantria dispar) larvae that were fed with leaves plucked from poplar seedlings (Populus alba berolinensis) grown in either noncontaminated soil (control), Zn-contaminated soil (500mg/kg), or Pb-contaminated soil (500mg/kg). Our results showed that excretion of heavy metals via insect feces and exuvia is an effective approach to reduce the internal Zn and Pb concentrations, and result in the decrease of Zn and Pb concentrations in the gypsy moth larvae with the increased larval age. In addition, the gypsy moth larvae seemed to have a strong homeostatic adjustment mechanism [between approximate digestibility (AD) and efficiency of conversion of digested food (ECD)] that maintains a stable level of "efficiency of conversion of ingested food (ECI)" regardless of heavy metal (Zn or Pb) contaminations or not, except the fifth instar larvae in which the increase in AD was insufficient to compensate for the decrease of ECD. These results suggest that heavy metal excretions could help the gypsy moth larvae cope with Zn or Pb stress, and the increased digestion of food could meet their energy requirements for both detoxification and growth. However, further increase in Zn or Pb exposure time seemed to inhibit the larval food utilization.

Food utilization and excretion mechanism of Cd in gypsy moth larvae fed on Cd-enriched Populus albaXP. berolinensis leaves

To investigate the eco-physiological responses of phytophagous insects to host plants exposed to heavy metal stress, the gypsy moth (Lymantria dispar) larvae were reared with leaves of Populus albaXP. berolinensis grown in Cd-contaminated soil to analyze their food utilization and mechanisms underlying Cd excretion. Our results showed that after feeding on Cd-stressed poplar leaves, Cd concentrations and contents in the 3rd-6th instar larvae were significantly higher than those in control. The Cd concentrations significantly decreased with the larval age, whereas it was reverse for Cd contents. Cd concentrations in the larval faces and exuvia were significantly higher than those in control. Food digestion/consumption rate and conversion rate of the 3rd-5th instar larvae in the Cd-treatment group were significantly higher and lower than those in the control, respectively. Food utilization rate of the Cd-stressed 3rd-4th instar larvae were not significantly different from that of the control larvae with the same age. In contrast, the Cd-stressed 5th instar larvae showed a significantly lower food utilization rate than the control. These results indicated that gypsy moth larvae could remove some Cd from their bodies through effective excretion (e.g. feces and exuvia), with the mature larvae being more effective than the juveniles. The increases of larval body mass seemed to have a "dilution effect" on its Cd concentration. Under Cd stress, the gypsy moth larvae might maintain a certain level of food utilization rate for growth and development via a modulation of food consumption/conversion rates, but Cd-stress would still reduce the larval food utilization rate when it was beyond a certain threshold.

Characterisation of three Alphabaculovirus isolates from the gypsy moth, Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

ABSTRACTIn this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170 kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 103 and 106 OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC50 and LC95 values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC50 value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.

Molecular Alliance of Lymantria dispar Multiple Nucleopolyhedrovirus and a Short Unmodified Antisense Oligonucleotide of Its Anti-Apoptotic IAP-3 Gene: A Novel Approach for Gypsy Moth Control.

Baculovirus IAP (inhibitor-of-apoptosis) genes originated by capture of host genes. Unmodified short antisense DNA oligonucleotides (oligoDNAs) from baculovirus IAP genes can down-regulate specific gene expression profiles in both baculovirus-free and baculovirus-infected insects. In this study, gypsy moth (Lymantria dispar) larvae infected with multiple nucleopolyhedrovirus (LdMNPV), and LdMNPV-free larvae, were treated with oligoDNA antisense to the RING (really interesting new gene) domain of the LdMNPV IAP-3 gene. The results with respect to insect mortality, biomass accumulation, histological studies, RT-PCR, and analysis of DNA apoptotic fragmentation suggest that oligoRING induced increased apoptotic processes in both LdMNPV-free and LdMNPV-infected insect cells, but were more pronounced in the latter. These data open up possibilities for promising new routes of insect pest control using antisense phosphodiester DNA oligonucleotides.