Gypsy Moth Larvae Research Articles

LdAMPKα2 knockdown accelerated the growth but depressed the chitin biosynthesis in Lymantria dispar larvae

AMPK (AMP-activated protein kinase) is a crucial cellular energy sensor across all eukaryotic species. Its multiple roles in maintaining energy homeostasis, regulating cellular metabolic processes have been widely investigated in mammals. In contrast, the function of AMPK in insects has been less reported. Here, we successfully identified three AMPK subunits from Lymantria dispar (L. dispar), a Lepidoptera pest in forestry. Based on that, in particular, the role of AMPK signaling in regulating larval development, as well as chitin biosynthesis was investigated by the application of RNAi-mediated LdAMPKα2 knockdown. The results indicated that knockdown of LdAMPKα2 significantly increased the body weight of L. dispar larvae, and dramatically upregulated the expression of LdmTOR, LdS6K and LdSREBP1, the key genes in mTOR (mammalian target of rapamycin) signaling pathway. While, it significantly reduced the expression of Ld4EBP, a critical repressor of mTOR pathway. Besides, the glucose level was increased and trehalose level was decreased in L. dispar after LdAMPKα2 silencing. Furthermore, we found that the chitin content in the epidermis, as well as the expressions of four key genes in the chitin biosynthesis pathway, LdGFAT, LdPAGM, LdUAP and LdCHSA, were significantly decreased after LdAMPKα2 knockdown. Taken together, these results revealed that AMPK signaling played a pivotal role in regulating the growth and development, as well as carbohydrate metabolism and chitin biosynthesis in L. dispar larvae. The findings expand our understanding of the comprehensive regulatory role of AMPK signaling in insects.

The impact of carvacrol on the larval gut bacterial structure and function of Lymantria dispar.

The gut bacteria of insects play an important role in regulating their metabolism, immune system and metabolizing pesticides. Our previous results indicate that carvacrol has certain gastric toxic activity on Lymantria dispar larvae and affects their detoxification metabolism at the mRNA level. However, the impact of carvacrol on the gut bacteria of L. dispar larvae has been unclear. In this study, the 16S rRNA sequencing technology was used to sequence and analyze the gut bacteria of the larvae which were exposed with sublethal concentration (0.297 mg/mL) and median lethal concentration (1.120 mg/mL), respectively. A total of 10 phyla, 16 classes, 47 orders, 72 families, 103 genera, and 135 species were obtained by using a 97% similarity cutoff level. The dominant bacterial phyla in the gut of the L. dispar larvae are Firmicutes and Proteobacteria. The treatment with carvacrol can significantly affect the structure of gut bacteria in the larvae of the L. dispar. At both doses, carvacrol can shift the dominant gut bacteria of the larvae from Proteobacteria to Firmicutes. At the genus level, two doses of carvacrol can significantly enhance the relative abundance of probiotic Lactobacillus in the gut of L. dispar larvae (p ≤ 0.01). Additionally, significant differences were observed among the five bacterial genera Burkholderia-Caballeronia-Paraburkholderia, Anoxybacillus, Pelomonas, Mesorhizobium (p ≤ 0.05). The analysis of α-diversity and β-diversity indicates that the treatment with carvacrol at two doses significantly affect the bacterial richness and diversity in the larvae. However, the results of functional classification prediction (PICRUSt) indicate that carvacrol significantly down-regulate 7 functions, including Energy metabolism, Cell growth and death, and up-regulate 2 functions, including Carbohydrate metabolism and Membrane transport. The network analysis indicates that the correlation between gut bacteria also has been changed. In addition, the insecticidal activity results of carvacrol against L. dispar larvae with gut bacteria elimination showed that gut bacteria can reduce the insecticidal activity of carvacrol against L. dispar larvae. This study provides a theoretical foundation for understanding the role of gut bacteria in detoxifying plant toxins and conferring pesticide resistance.

Biological activity of essential oils of Calocedrus decurrens and Cupressus arizonica on Lymantria dispar larvae and Phytophthora root pathogens

Composition of Calocedrus decurrens and Cupressus arizonica essential oils (CDEO and CAEO, respectively), their antifeedant activity on spongy moth (Lymantria dispar) larvae, as well as their antimicrobial activity on three Phytophthora species were investigated. In leaves of these two conifers from the Cupressaceae family, monoterpenes strongly dominated (90.5 and 85.0%, respectively), but their terpene profiles were different. The dominant compound of CDEO was δ-3-carene (49.5%), followed by myrcene (9.4%), terpinolene (8.6%) and α-pinene (7.0%). In CAEO, the most dominant compounds were limonene (23.3%) and α-pinene (20.5%), followed by umbellulone (12.0%) and terpinen-4-ol (9.5%). CDEO showed a strong antifeedant activity, while leaf discs treated with CAEO had phagostimulatory effect on spongy moth larvae. CAEO had no significant effect on relative consumption and larval growth rate, while CDEO reduced consumption and stimulated growth at the lowest EO concentration (0.05%). Both EOs significantly affected the growth of colonies of the Phytophthora species tested. At concentrations of 0.5% and 1%, a 100% inhibition was observed in almost all experimental groups, with the exception of P. plurivora on 0.5% CDEO. At a concentration of 0.1% inhibition rate varied from 15% in P. ×cambivora treated with CDEO to 90% in P. quercina treated with CAEO. These findings indicate that these two EOs could be used to control tree pests.

A new insight into Cd exposure-induced hemocyte reduction in Lymantria dispar larvae: Involvement of the ROS-ATF6-ER stress-apoptosis pathway

Hemocytes are important targets for heavy metal-induced immunotoxicity in insects. This study aimed to investigate the mechanism by which cadmium (Cd) exposure affects the hemocyte count in Lymantria dispar larvae. The results showed that the number of larval hemocytes was significantly decreased under Cd exposure, accompanied by a significant increase in the apoptosis rate and the expression of Caspase-3. The endoplasmic reticulum (ER) of hemocytes in the Cd-treated group showed irregular swelling. Expression levels of ER stress indicator genes (CHOP, Bip1, Bip2, Bip3, and Bip4) were significantly higher in the Cd-treated group. Among the three pathways that potentially mediate ER stress, only the key genes in the ATF6 pathway (ATF6, S1P-1, S1P-2, and WFS1) exhibited differential responses to Cd exposure. Cd exposure significantly increased the levels of reactive oxygen species (ROS) and the expression of oxidative stress-related genes (CNCC, P38, and ATF2) in hemocytes. Studies using inhibitors confirmed that apoptosis mediated the decrease in hemocyte count, ER stress mediated apoptosis, ATF6 pathway mediated ER stress, and ROS or oxidative stress mediated ER stress through the activation of the ATF6 pathway. Taken together, the ROS-ATF6-ER stress-apoptosis pathway is responsible for the reduction in the hemocyte count of Cd-treated L. dispar larvae.

Isolation characterization and pathogenicity of Helicoverpa armigera single nucleopolyhedrovirus isolate from Türkiye

The cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a major pest of agricultural crops, such as cotton, horticultural plants, a range of vegetable crops and forest trees. Many dead and live larvae and pupa samples of H. armigera, an extremely polyphagous pest, were collected from Adana in Türkiye. It was observed that cadaveric integuments were fragile, and their body tissues were also liquefied. Studies with phase-contrast microscopy revealed a very intense baculovirus infection in cadavers. Transmission electron microscopy exhibited that the new baculovirus had one nucleocapsid in an envelope in the occlusion bodies. Therefore, the isolate was named Helicoverpa armigera single nucleopolyhedrovirus-D (HearSNPV-D). The phylogenetic analysis according to the polyhedrin and late expression factor-8 genes appeared that the new isolate was in close relationship with HearSNPV China and Türkiye isolate. The new isolate was tested on six different insects including its own host Helicoverpa armigera, Hyphantria cunea, Malacosoma neustria, Lymantria dispar, Thaumetopea pityocampa and Spodoptera exigua larvae. Bioassay studies showed that HearSNPV-D was virulent to H. armigera and had a narrow host range. The LC50 value was determined as 0.7 × 105 and 1.9 × 107 OBs/ml for H. armigera and S. exigua, respectively. Non-significant mortality was observed in non-host species by HearSNPV-D, which may reveal the highly specific nature of HearSNPV-D. Therefore, HearSNPV-D can be used as a specific, environmentally friendly control agent for H. armigera.

Genome Sequencing and Organization of Three Geographically Different Isolates of Nucleopolyhedrovirus from the Gypsy Moth Reveal Significant Genomic Differences.

The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of trees and forests. Lymantria dispar nucleopolyhedrovirus (LdMNPV) belongs to the Baculoviridae family and is an insect virus specific to gypsy moth larvae. In this study, we describe the complete genome sequences of three geographically diverse isolates, H2 (China), J2 (Japan), and T3 (Turkey), of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). The genomes of isolates H2, J2, and T3 were subjected to shotgun pyrosequencing using Roche 454 FLX and assembled using Roche GS De Novo Assembler. Comparative analysis of all isolates was performed using bioinformatics methods. The genomes of LdMNPV-H2, J2, and T3 were 164,746, 162,249, and 162,614 bp in size, had GC content of 57.25%, 57.30%, and 57.46%, and contained 162, 165, and 164 putative open reading frames (ORFs ≥ 150 nt), respectively. Comparison between the reference genome LdMNPV-5/6 (AF081810) and the genomes of LdMNPV-H2, J2, and T3 revealed differences in gene content. Compared with LdMNPV-5/6, ORF5, 6, 8, 10, 31, and 67 were absent in LdMNPV-H2, ORF5, 13, and 66 were absent in LdMNPV-J2, and ORF10, 13, 31, and 67 were absent in LdMNPV-T3. In addition, the gene encoding the mucin-like protein (ORF4) was split into two parts in isolates H2 and T3 and designated ORF4a and ORF4b. Phylogenetic analysis grouped isolates H2 and J2 in a different cluster than isolate T3, which is more closely related to the Turkish and Polish isolates. In addition, H2 was found to be closely related to a South Korean LdMNPV isolate. This study provided a more detailed overview of the relationships between different geographic LdMNPV isolates. The results showed remarkable differences between groups at the genome level.

The Influence of Dietary Cadmium on Changes in the Midgut Mass Related to the Mass of Gypsy Moth Larvae

The Influence of Dietary Cadmium on Changes in the Midgut Mass Related to the Mass of Gypsy Moth Larvae

The effects of temperature stress and population origin on the thermal sensitivity of Lymantria dispar L. (Lepidoptera: Erebidae) larvae

Increased environmental temperature is one of the most frequent stresses effecting metabolic rate in herbivorous insect species. Our goal was to compare the influence of increased environmental temperature and induced thermotolerance on the activity of midgut phosphatases and brain tissue hsp70 concentration in 5th instar Lymantria dispar larvae originating from an unpolluted and polluted forest. Induced thermotolerance (larval pre-treatment at high, sub-lethal temperature) increases the species ability to overcome the negative effects of thermal stress, therefore we monitored the effect of this regime in larvae originating from both forests. Thermal regimes in this experiment predominantly influenced the alkaline phosphatases activity and it was affected by temperature, population origin, and their combined effect. Total acid phosphatases activity was changed only by the joint effect of temperature and population origin. Brain hsp70 concentration was under a significant individual and joint effect of temperature and population. In both populations, brain tissue hsp70 concentration and alkaline phosphatases activity should be taken under consideration as a battery with biomarker potential for thermal stress in L. dispar larvae as a bioindicator species.

Antifeeding, Toxic, and Growth-Reducing Activity of trans-Anethole and S-(+)-Carvone against Larvae of the Gypsy Moth Lymantria dispar (L.)

Botanicals, such as essential oils (EO) and their compounds, are considered a viable eco-friendly alternative to synthetic insecticides, which threaten human health and ecosystem functioning. In the present study, we explored the potential use of two EO compounds, trans-anethole (phenylpropanoid) and S-(+)-carvone (monoterpene ketone), against gypsy moth larvae (GML), a serious pest of deciduous forests and orchards. GML feeding, survival, molting, and nutritional physiology were assessed at different compound concentrations and compared with the effects of the commercial botanical product NeemAzal®-T/S (neem). The impact of botanicals on GML feeding was assessed by the leaf-dipping method and showed the highest antifeeding activity of neem in the no-choice assay. GML that were offered a choice were deterred by anethole and attracted by low concentrations of carvone and neem. Ingestion of botanicals was more effective in inducing mortality and reducing molting than residual contact exposure. Anethole and carvone were better toxicants but worse growth regulators than neem. Assessing nutritional indices revealed reduced growth, consumption, and food utilization in larvae fed on botanical-supplemented diets. The highest metabolic cost of food processing was recorded in carvone-fed larvae, which exhibited a negative growth rate. The results suggest that anethole and carvone might be used as control agents against GML.

Effects of Arbuscular Mycorrhizal Fungi-Colonized Populus alba × P. berolinensis Seedlings on the Microbial and Metabolic Status of Gypsy Moth Larvae.

Arbuscular mycorrhizal fungi (AMF) are considered as important biological factors that can affect insect resistance of plants. Herein, we used AMF-poplar seedlings that could either increase or decrease the resistance to gypsy moth larvae, to elucidate the mechanism of mycorrhizal-induced insect resistance/susceptibility at the larval microbial and metabolic levels. Our results found that larval plant consumption and growth were significantly inhibited in the Glomus mossae (GM)-colonized seedlings, whereas they were enhanced in the Glomus intraradices (GI)-colonized seedlings. GM inoculation reduced the beneficial bacteria abundance in the larval gut and inhibited the detoxification and metabolic functions of gut microbiota. However, GI inoculation improved the larval gut environment by decreasing the pathogenic bacteria and activating specific metabolic pathways. Furthermore, GM inoculation triggers a metabolic disorder in the larval fat body, accompanied by the suppression of detoxification and energy production pathways. The levels of differentially accumulated metabolites related to amino acid synthesis and metabolism and exogenous toxin metabolism pathways were significantly increased in the GI group. Taken together, the disadaptation of gypsy moth larvae to leaves of GM-colonized seedlings led to the GM-induced insect resistance in poplar, and to the GI-induced insect susceptibility involved in the improvement of larval gut environment and fat body energy metabolism.

Suitability of Turkey Oak, European Beech, and Hornbeam to Gypsy Moth Feeding

Pest resistance of trees should be taken into account in future forest strategy planning and predicting risks of defoliation. The gypsy moth (GM) (Lymantria dispar L.) is a serious forest pest with outbreaking population dynamics. To estimate defoliation risk of the most widely distributed tree species in Balkan Peninsula and Europe (Turkey oak Quercus cerris L., European beech Fagus sylvatica L. and hornbeam Carpinus betulus L.), we carried out laboratory feeding trials and investigated their acceptability and suitability for GM development. We determined morphological and chemical attributes of these hosts as well as larval host preference, growth and nutritional indices. Preference, growth, and efficiency of food conversion into biomass were ranked in the order: Turkey oak ˃ European beech ˃ hornbeam. Hornbeam was the most avoided and showed the lowest conversion efficiency although, comparing to optimal oak host, its leaves were less tough, contained more water and exhibited similar values of nitrogen (index of protein content) and C/N ratio (index of investment into carbon based plant defense). We suggest that hornbeam and beech leaf chemical profiles should be further studied to reveal specific compounds that impose high metabolic cost to GM larvae. Moreover, additional research are needed to understand how intermediate hosts in natural populations affect GM outbreaks.

Unraveling arbuscular mycorrhizal fungi-induced resistance of purple branch rose (Rosa rugosa ‘Zizhi’) to Lymantria dispar based on metabolomics

Currently, people primarily rely on chemical pesticides to prevent and control insect pests, which not only introduce problems with environmental pollution but also produce chemical residues that will make it difficult to guarantee the safety of edible plants. Arbuscular mycorrhizal fungi (AMF) are soil microorganisms that form a symbiotic relationship with plants, which improves plant resistance. This natural phenomenon provides new ideas for eco-friendly biological control. The present study assessed the effects of AMF (Funneliformis mosseae, Rhizophagus intraradices and their combination) inoculation on rose growth and its resistance to gypsy moth larvae (Lymantria dispar) by investigating the growth performance and metabolomics of rose plants and the growth and physiological parameters of gypsy moth larvae. AMF inoculation improved the growth of both the aboveground and belowground parts of the rose plants. The developmental duration of gypsy moth larvae feeding on mycorrhizal rose leaves was prolonged by 4.8–5.8 days. Moreover, feeding on mycorrhizal rose leaves significantly reduced the body weight of the gypsy moth larvae by 9–24%, body length by 4–6%, and the width of head sheath by 11–15%. The superoxide dismutase (SOD) and carboxylesterase (CarE) enzymes in the larvae that fed on the leaves of mycorrhizal rose were significantly higher than those in the control, while the acid phosphatase (ACP) was inhibited (P < 0.05). Peroxidase (POD) and alkaline phosphatase (AKP) in larvae that fed on mycorrhizal rose leaves were significantly higher than those in the control at 5 days. Glutathione S transferases (GSTs) were significantly higher than the control at 3 and 5 days. Metabolomics analyses showed that inoculation with AMF promoted the accumulation of jasmonic acid (JA) and abscisic acid (ABA) in rose leaves. The treatment groups were 1.6- and 3.4-fold higher than those of the control, respectively. The AMF significantly regulated 1,589 metabolites in rose leaves. The AMF promoted the accumulation of flavonoids and inhibited the accumulation of glycosides in rose leaves. A total of 19 metabolites that significantly correlated with the growth and development of gypsy moths were screened out. Among ABA, glycerophosphocholine, 1-(sn-glycero-3-phospho)-1D-myo-inositol, (-)-catechin, N-acetyl-D-glucosamine 6-phosphate, 3-(4-hydroxyphenyl)-1-propanol and 3-trans-caffeoyltormentic acid were significantly different from those of the control. These compounds may play a role in the resistance of rose to gypsy moth induced by AMF. In conclusion, AMF inoculation improved the resistance of R. rugosa to L. dispar through modifications on the metabolism and physiology. These findings highlight the interaction of “mycorrhizal-plant-insect”. It provides a reference for the application of AMF to control plant pest.

Cd exposure-triggered susceptibility to Bacillus thuringiensis in Lymantria dispar involves in gut microbiota dysbiosis and hemolymph metabolic disorder

The immunotoxicity induced by heavy metals on herbivorous insects reflect the alterations of the susceptibility to entomopathogenic agents in herbivorous insects exposed to heavy metal. In the present study, the susceptibility of gypsy moth larvae to Bacillus thuringiensis under Cd treatment at low and high dosages was investigated, and the gut microbiome-hemolymph metabolome responses that affected larval disease susceptibility caused by Cd exposure were examined. Our results showed that mortality of gypsy moth larvae caused by B. thuringiensis was significantly higher in larvae pre-exposed to Cd stress, and there was a synergistic effect between Cd pre-exposure and bacterial infection. Exposure to Cd significantly decreased the abundance of several probiotics (e.g., Serratia for the low Cd dosage and Weissella, Aeroonas, and Serratia for the high Cd dosage) and increased the abundances of several pathogenic bacteria (e.g., Stenotrophomonas, Gardnerella, and Cutibacterium for the low Cd dosage and Pluralibacter and Tsukamurella for the high Cd dosage) compared to the controls. Moreover, metabolomics analysis indicated that amino acid biosynthesis and metabolism were significantly perturbed in larval hemolymph under Cd exposure at both the low and high dosages. Correlation analysis demonstrated that several altered metabolites in larval hemolymph were significantly correlated with changes in the gut microbial community. The results demonstrate that prior exposure to Cd increases the susceptibility of gypsy moth larvae to B. thuringiensis in a synergistic fashion due to gut microbiota dysbiosis and hemolymph metabolic disorder, and thus microbial-based biological control may be the best pest control strategy in heavy metal-polluted areas.

The immunotoxicity of Cd exposure to gypsy moth larvae: An integrated analysis of cellular immunity and humoral immunity

Heavy metal exposure-triggered growth retardation and physiology disorder in phytophagous insects have been widely understood, but only a few studies have investigated its immunomodulatory effects on herbivorous insects. Here, the innate immunity of gypsy moth (Lymantria dispar) larvae under Cd stress was evaluated by integrating cellular and humoral immunity, and the immunomodulation mechanism of Cd stress was further understood by the proteomics analysis of larval hemolymph. Our results showed that the total hemocyte count, as well as phagocytic, encapsulation and bacteriostatic activity, of hemolymph in gypsy moth larvae exposed to Cd stress was significantly lower than that in un-treated larvae. Further proteomic analysis revealed that Cd exposure may reduce the total hemocyte count in larval hemolymph by inducing endoplasmic reticulum pathway-mediated hemocyte apoptosis, thereby causing the collapse of cellular immunity in gypsy moth larvae. In addition, the transcriptional level of signal transduction genes (IMD, Toll, Relish, JAK and STAT) and antimicrobial peptide genes (cecropin and lebocin), as well as the protein abundance of pattern recognition receptors (PGRP and GNBP3) in the Toll, IMD and JAK/STAT signaling pathways was significantly decreased in Cd-treated larvae, clearly implying an immunosuppresive effect of Cd stress on pathogen recognition, signal transduction and effector synthesis of humoral immunity in gypsy moth larvae. Taken together, these results suggest that Cd exposure decreases both cellular immunity and humoral immunity of gypsy moth larvae, and provides a new entry point for systematically and comprehensively unraveling the heavy metal pollutants-caused immunotoxicity.

Cd exposure-triggered metabolic disruption increases the susceptibility of Lymantria dispar (Lepidoptera: Erebidae) larvae to Mamestra brassicae nuclear polyhedrosis virus: A multi-omics study

Biological control is an environmentally friendly and effective pest control strategy, but it is often affected by a variety of abiotic factors in the pest control area. Here, the susceptibility of gypsy moth larvae to Mamestra brassicae nuclear polyhedrosis virus (MbNPV) under Cd treatment at the low and high dosages was investigated, and the mechanism of Cd stress affecting virus susceptibility of gypsy moth larvae was analyzed from a metabolic perspective by combining transcriptome and metabolome of the larval fat body. Our results showed that the mortality of MBNPV infection on gypsy moth larvae pre-exposed to Cd was significantly higher than that of larvae without Cd pre-exposure, and the joint effects of Cd exposure and virus infection on larval mortality were demonstrated to be synergistic. Transcriptome analysis revealed that amino acid and carbohydrate transport and metabolism accounted for most of the differently expressed genes in the low Cd and high Cd treatment groups. Consistent with the transcriptome results, metabolome analysis also showed that most metabolites affected by Cd exposure were involved in amino acid and carbohydrate metabolism. Function analysis showed that the contents of several amino acids (e.g. tryptophan and tyrosine) with antioxidant properties were significantly increased in Cd-treated gypsy moth larvae. Taken together, Cd exposure as an environmental factor, promotes the susceptibility of gypsy moth larvae to MbNPV, and metabolic disruption, especially amino acids and carbohydrates-related metabolism, is responsible for the increased susceptibility of gypsy moth larvae to virus under Cd stress.

The PI3K/AKT Pathway and PTEN Gene Are Involved in "Tree-Top Disease" of Lymantria dispar.

Nucleopolyhedrovirus (NPV) can alter its host behaviour such that infected larvae hang at the top of trees before their death. This phenomenon was firstly described by Hofmann in 1891 and named as “tree-top disease”. Subsequent studies have described effects during the infection proceedings as NPVs manipulate the host to avoid the immune response, cross defensive barriers and regulate hormones. In this study, we demonstrate that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway is involved in host manipulation by Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Particularly at the late stage of infection, a multifunctional dephosphorylase in the PI3K/AKT signaling pathway is dynamically upregulated, namely, the phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) gene. The biological assays of PTEN gene knockdown showed that an increase in PTEN gene expression was necessary for the infected Lymantria dispar larvae’s terminal climbing behavior, death postponement and virion production. The results imply that the PI3K/AKT signaling pathway and PTEN gene might play an essential role in “tree-top disease” induced by LdMNPV.

Bioactivity of Chamaecyparis lawsoniana (A. Murray) Parl. and Thuja plicata Donn ex D. Don essential oils on Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae) larvae and Phytophthora de Bary 1876 root pathogens

We examined essential oils (EOs) of Chamaecyparis lawsoniana and Thuja plicata (Cupressaceae), their antifeedant activity on Lymanthria dispar larvae and their antimicrobial activity. Studies of EOs showed that these two conifer species differed both in content of terpene classes and the major compounds. Total monoterpenes strongly dominated in T. plicata (96.4%), while C. lawsoniana was rich in both mono- and sesquiterpenes (40.8% and 30.3%, respectively) as well as in diterpenes (19.1%). The most dominant compounds of C. lawsoniana EO were: limonene (16.7%), oplopanonyl acetate (14.5%), beyerene (10.1%), and 13-epi-dolabradiene (6.7%). The dominant compound of T. plicata EOs was α-thujone (76.9%), followed by relatively small amounts of β- thujone (5.3%), sabinene (4.5%) and terpinene-4-ol (3.2%). The difference in EO compositions of the conifers was reflected on Lymantria dispar larvae performance. Larvae fed on the leaf discs treated by C. lawsoniana EO had a slight phagostimulatory effect at lower concentration shown by higher relative rate of food consumption and relative growth rate than the larvae in the control group. Contrastingly, leaf discs treated with EO of T. plicata EO had an antifeedant effect and lower relative consumption rate (RCR) and relative growth rate RGR than the larvae in the control group. Both tested EOs influenced substantially the colony growth of the subjected Phytophthora plurivora and P. quercina. Namely, 100% inhibitory effect was recorded at concentration of 0.1% in the case of C. lawsoniana EO, whereas of T. plicata colonies did not grow at 0.5% concentration. The implications of these findings and possibility of using the tested EOs in further experiments in vitro and in vivo are discussed.

Transcriptomic analysis of interactions between Lymantria dispar larvae and carvacrol

Due to its biological activity, carvacrol (CAR) is widely used in medicine, agriculture, and forestry. Our previous studies showed that in Lymantria dispar larvae, CAR treatment can induce the production of antifeedants and lead to growth inhibition and death of larvae. However, the effect CAR exerts on RNA levels in L. dispar larvae remains unclear. In this study, the Illumina HiSeq4000 sequencing platform was used to sequence the total RNA of L. dispar larvae. A total of six cDNA libraries (three treatments and three controls) were established and 39,807 genes were generated. Compared with the control group, 296 differentially expressed genes (DEGs) (142 up-regulated and 154 down-regulated) were identified after CAR treatment. GO and KEGG enrichment analyses showed that these DEGs mainly clustered in the metabolism of xenobiotics, carbohydrates, and lipids. Furthermore, 12 DEGs were found to be involved in detoxification, including six cytochrome P450s, two esterases, one glutathione peroxidase, one UDP-glycosyltransferase gene, and two genes encoding heat shock proteins. The expression levels of detoxification genes changed under CAR treatment (especially P450s), which further yielded candidate genes for explorations of the insecticidal mechanism of CAR. The reliability of transcriptome data was verified by qRT-PCR. The enzyme activities of CYP450 and acid phosphatase significantly increased (by 38.52 U/mg·prot and 0.12 μmol/min·mg, respectively) 72 h after CAR treatment. However, the activity of alkaline phosphatase did not change significantly. These changes in enzyme activity corroborated the reliability of the transcriptome data at the protein level. The results of GO enrichment analysis of DEGs indicated that CAR influenced the oxidation-reduction process in L. dispar larvae. Furthermore, CAR can cause oxidative stress in L. dispar larvae, identified through the determination of peroxidase and polyphenol oxidase activities, total antioxidant capacity, and hydrogen peroxide content. This study provides useful insight into the insecticidal mechanism of CAR.

Defense responses of arbuscular mycorrhizal fungus-colonized poplar seedlings against gypsy moth larvae: a multiomics study

Arbuscular mycorrhizal (AM) fungi may help protect plants against herbivores; however, their use for the pest control of woody plants requires further study. Here, we investigated the effect of Glomus mosseae colonization on the interactions between gypsy moth larvae and Populus alba × P. berolinensis seedlings and deciphered the regulatory mechanisms underlying the mycorrhizal-induced resistance in the leaves of mycorrhizal poplar using RNA-seq and nontargeted metabolomics. The resistance assay showed that AM fungus inoculation protected poplar seedlings against gypsy moth larvae, as evidenced by the decreased larval growth and reduced larval survival. A transcriptome analysis revealed that differentially expressed genes (DEGs) were involved in jasmonic acid biosynthesis (lipoxygenase, hydroperoxide dehydratase, and allene oxide cyclase) and signal transduction (jasmonate-ZIM domain and transcription factor MYC2) and identified the genes that were upregulated in mycorrhizal seedlings. Except for chalcone synthase and anthocyanidin synthase, which were downregulated in mycorrhizal seedlings, all DEGs related to flavonoid biosynthesis were upregulated, including 4-coumarate-CoA ligase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, and leucoanthocyanidin reductase. The metabolome analysis showed that several metabolites with insecticidal properties, including coumarin, stachydrine, artocarpin, norizalpinin, abietic acid, 6-formylumbelliferone, and vanillic acid, were significantly accumulated in the mycorrhizal seedlings. These findings suggest the potential of mycorrhiza-induced resistance for use in pest management of woody plants and demonstrate that the priming of JA-dependent responses in poplar seedlings contributes to mycorrhiza-induced resistance to insect pests.

Antifeeding and Insecticidal Activity of Ailanthus altissima and Morus alba Extracts Against Gipsy Moth (Lymantria dispar (L.), Lepidoptera, Lymantridae) Larvae Under Laboratory Conditions

Gypsy moth (Lymantria dispar L.) is one of most important defoliating pests of deciduous trees. Due to increased environmental demands, the use of plant-based preparations is gaining in importance as a control option for this pest in forestry, agriculture and horticulture. The aim of this study was to evaluate antifeeding and insecticidal activity of 0.5, 1 and 2% extracts of Ailanthus altissima bark and leaves, and Morus alba leaves, against L. dispar larvae under laboratory conditions. Antioxidant capacity of plant extracts was determined, as well as the content of phenolic compounds by&nbsp;spectrophotometric and HPLC-DAD methods. Antifeeding and insecticidal effects were tested in a “no-choice” test. The highest content of all bioactive phenolic compounds was in A. altissima bark and M. alba leaf extracts. The lowest leaf consumption after 24 and 48 h was in A. аltissima bark (5.03, 9.48%, respectively) and M. alba leaf (1.44, 2.97%, respectively) extracts. A. altissima bark and M. alba leaf extracts expressed strong antifeeding activity. After 24 h, all extracts expressed slight insecticidal effect (1.25-17.50% of mortality). The mortality increased after 48 h in treatments with A. altissima bark extract, at all applied concentrations (40.0-58.9%) and M. alba leaves at 1 and 2% concentrations (30.0-58.9%). Our results indicate that extracts of A. altissima bark and M. alba leaves may act as effective low-cost natural protectants able to control the presence of gypsy moth in ecosystems.&nbsp;Extracts of A. altissima bark and M. alba leaves expressed strong antifeeding activity and significant insecticidal effect on gypsy moth larvae, at all applied concentrations.