Molecular characterization and enzyme inhibition studies of NADP+- farnesol dehydrogenase from diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

Juvenile hormone III (JH III) plays an important role in insect reproduction, development, and behavior. The second branch of JH III production includes oxidation of farnesol to farnesal by farnesol dehydrogenase. This study reported the identification and characterization of Plutella xylostella farnesol dehydrogenase (PxFoLDH). Our results showed that PxFoLDH belongs to the short-chain dehydrogenase/reductase superfamily, consisting of a single domain with a structurally conserved Rossman fold, an NAD(P) (H)-binding region and a structurally diverse C-terminal region. The purified enzyme displayed maximum activity at 55$\ $°C with pH 9.5 and was stable in the temperature below 70$\ ^\circ $C. PxFoLDH was determined to be a monomer with a relative molecular weight of 27 kDa and highly specific for trans, trans-farnesol, and NADP+. Among analog inhibitors tested, farnesyl acetate was the most effective inhibitor with the lowest Ki value of 0.02 µm. Our findings showed this purified enzyme may represent as NADP+-farnesol dehydrogenase.

Horizontal Gene Transfer and Gene Duplication of ß-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits.

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect ß-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of ß-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of ß-fructofuranosidase were found in Coleoptera and Hymenoptera. ß-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran ß-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of ß-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of ß-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.

Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

Consequences of 'no-choice, fixed time' reciprocal host plant switches on nutrition and gut serine protease gene expression in Pieris brassicae L. (Lepidoptera: Pieridae).

Rapid adaptive responses were evident from reciprocal host-plant switches on performance, digestive physiology and relative gene expression of gut serine proteases in larvae of crucifer pest P. brassicae transferred from cauliflower (CF, Brassica oleracea var. botrytis, family Brassicaceae) to an alternate host, garden nasturtium, (GN, Tropaeolum majus L., family Tropaeolaceae) and vice-versa under laboratory conditions. Estimation of nutritional indices indicated that larvae of all instars tested consumed the least food and gained less weight on CF-GN diet (significant at p≤0.05) as compared to larvae feeding on CF-CF, GN-GN and GN-CF diets suggesting that the switch to GN was nutritionally less favorable for larval growth. Nevertheless, these larvae, especially fourth instars, were adroit in utilizing and digesting GN as a new host plant type. In vitro protease assays conducted to understand associated physiological responses within twelve hours indicated that levels and properties of gut proteases were significantly influenced by type of natal host-plant consumed, change in diet as well as larval age. Activities of gut trypsins and chymotrypsins in larvae feeding on CF-GN and GN-CF diets were distinct, and represented shifts toward profiles observed in larvae feeding continuously on GN-GN and CF-CF diets respectively. Results with diagnostic protease inhibitors like TLCK, STI and SBBI in these assays and gelatinolytic zymograms indicated complex and contrasting trends in gut serine protease activities in different instars from CF-GN diet versus GN-CF diet, likely due to ingestion of plant protease inhibitors present in the new diet. Cloning and sequencing of serine protease gene fragments expressed in gut tissues of fourth instar P. brassicae revealed diverse transcripts encoding putative trypsins and chymotrypsins belonging to at least ten lineages. Sequences of members of each lineage closely resembled lepidopteran serine protease orthologs including uncharacterized transcripts from Pieris rapae. Differential regulation of serine protease genes (Pbr1-Pbr5) was observed in larval guts of P. brassicae from CF-CF and GN-GN diets while expression of transcripts encoding two putative trypsins (Pbr3 and Pbr5) were significantly different in larvae from CF-GN and GN-CF diets. These results suggested that some gut serine proteases that were differentially expressed in larvae feeding on different species of host plants were also involved in rapid adaptations to dietary switches. A gene encoding nitrile-specifier protein (nsp) likely involved in detoxification of toxic products from interactions of ingested host plant glucosinolates with myrosinases was expressed to similar levels in these larvae. Taken together, these snapshots reflected contrasts in physiological and developmental plasticity of P. brassicae larvae to nutritional challenges from wide dietary switches in the short term and the prominent role of gut serine proteases in rapid dietary adaptations. This study may be useful in designing novel management strategies targeting candidate gut serine proteases of P. brassicae using RNA interference, gene editing or crops with transgenes encoding protease inhibitors from taxonomically-distant host plants.

Genome mining and functional analysis of cytochrome P450 genes involved in insecticide resistance in Leucinodes orbonalis (Lepidoptera: Crambidae).

Genome-wide analysis of cytochrome P450 monooxygenase (CYP) genes from the advanced genome project of the Leucinodes orbonalis and the expression analysis provided significant information about the metabolism-mediated insecticide resistance. A total of 72 putative CYP genes were identified from the genome and transcriptome of L. orbonalis. The genes were classified under 30 families and 46 subfamilies based on the standard nomenclature. In the present study, a novel CYP gene, CYP324F1, was identified and it has not been reported from any other living system so far. Biochemical assays showed enhanced titers (5.81-18.5-fold) of O-demethylase of CYP in five field-collected populations. We selected 34 homologous CYP gene sequences, seemed to be involved in insecticide resistance for primer design and quantitative real-time PCR studies. Among the many overexpressed genes (>10 fold), the expression levels of CYP324F1 and CYP306A1 were prominent across all the field populations as compared with the susceptible iso-female line. Oral delivery of ds-CYP324F1 and ds-CYP306A1 directed against CYP324F1 and CYP306A1 to the larvae of one of the insecticide resistance populations caused reduced expression of these two transcripts in a dose-dependent manner (53.4%-85.0%). It appears that the increased titer of O-demethylase is the result of increased transcription level of CYP genes in resistant populations. The data provide insight for identifying the novel resistance management strategies against L. orbonalis.

Inhibition kinetics of digestive proteases for Anticarsia gemmatalis.

Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.

Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera.

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

Evaluation of silica nanoparticle mediated delivery of protease inhibitor in tomato plants and its effect on insect pest Helicoverpa armigera.

The inert and surface tunable nature of silica nanoparticles (SiNPs) makes them suitable for different applications. We have evaluated the potential of SiNPs for delivering proteins in tomato (Lycopersicon esculentum) plants. SiNPs of 20 and 100 nm (Si20 and Si100) were functionalized with (3-aminopropyl) triethoxysilane (APTES) to obtain Si20APT and Si100APT, respectively, that were non-toxic toward plants. The functionalized nanoparticles were taken up by plants through roots as well as leaf surfaces. They were seen to be localized near the vasculature, particularly around the xylem. Si20APT and Si100APT nanoparticles were conjugated with soybean trypsin inhibitor (STI) to yield Si20APT-STI and Si100APT-STI, respectively. Based on the trypsin inhibitory activity of loaded nanoparticles, optimum loading was obtained for 0.4 mg of STI per 0.8 mg of NPs. Si20APT nanoparticles retained higher contents of STI than Si100APT. Exposure of STI-conjugated nanoparticles to 25°C or pH 8.0 aided release of the inhibitor. The particle bound STI inhibited bovine trypsin by 80% and Helicoverpa armigera gut proteinase (HGP) activity by 50%. Second instar H. armigera larvae ingesting STI-loaded particles (incorporated in artificial diet or leaves) showed significant retardation in growth. In choice assays, Si20APT-STI applied leaf discs were strikingly avoided by insect larvae. On the basis of the results obtained in this investigation, we recommend the use of Si20 nanoparticles for developing plant delivery vehicles in the future.

A Series of Compounds Bearing a Dipyrido-Pyrimidine Scaffold Acting as Novel Human and Insect Pest Chitinase Inhibitors.

Chitinases not only play vital roles in the human innate immune system but are also essential for the development of pathogenic fungi and pests. Chitinase inhibitors are efficient tools to investigate the elusive role of human chitinases and to control pathogens and pests. Via hierarchical virtual screening, we have discovered a series of chitinase inhibitors with a novel scaffold that have high inhibitory activities and selectivities against human and insect chitinases. The most potent human chitotriosidase inhibitor, compound 40, exhibited a Ki of 49 nM, and the most potent inhibitor of the insect pest chitinase OfChi-h, compound 53, exhibited a Ki of 9 nM. The binding of these two most potent inhibitors was confirmed by X-ray crystallography. In a murine model of bleomycin-induced pulmonary fibrosis, compound 40 was found to suppress the chitotriosidase activity by 60%, leading to a significant increase in inflammatory cells and suggesting that chitotriosidase played a protective role.

Inhibition of dicer activity in lepidopteran and dipteran cells by baculovirus-mediated expression of Flock House virus B2.

Prior studies have suggested that insect DNA viruses are negatively affected by dicer-2-mediated RNA interference (RNAi). To examine this further, we utilized an in vitro assay to measure dicer activity in lepidopteran and dipteran cells, combined with baculoviruses expressing the RNAi suppressor B2 from Flock House virus or Aedes aegypti dicer-2 (Aedicer-2) using a constitutive heat shock promoter. Addition of cell lysates containing baculovirus-expressed B2 to lysates from dipteran (S2, Aag2) or lepidopteran (Sf9) cells inhibited endogenous dicer activity in a dose-dependent manner, while expression of Aedicer-2 restored siRNA production in Ae. albopictus C6/36 cells, which are dicer-2 defective. However, B2 expression from the constitutive heat shock promoter had no impact on baculovirus replication or virulence in cell lines or larvae that were either highly permissive (Trichoplusia ni) or less susceptible (Spodoptera frugiperda) to infection. We determined that this constitutive level of B2 expression had little to no ability to suppress dicer activity in cell lysates, but higher expression of B2, following heat shock treatment, inhibited dicer activity in all cells tested. Thus, we cannot rule out the possibility that optimized expression of B2 or other RNAi suppressors may increase baculovirus replication and expression of heterologous proteins by baculoviruses.

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.

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.

In Silico Study of the Resistance to Organophosphorus Pesticides Associated with Point Mutations in Acetylcholinesterase of Lepidoptera: B. mandarina, B. mori, C. auricilius, C. suppressalis, C. pomonella, H. armígera, P. xylostella, S. frugiperda, and S. litura.

An in silico analysis of the interaction between the complex-ligands of nine acetylcholinesterase (AChE) structures of Lepidopteran organisms and 43 organophosphorus (OPs) pesticides with previous resistance reports was carried out. To predict the potential resistance by structural modifications in Lepidoptera insects, due to proposed point mutations in AChE, a broad analysis was performed using computational tools, such as homology modeling and molecular docking. Two relevant findings were revealed: (1) Docking results give a configuration of the most probable spatial orientation of two interacting molecules (AChE enzyme and OP pesticide) and (2) a predicted ΔGb. The mutations evaluated in the form 1 acetylcholinesterase (AChE-1) and form 2 acetylcholinesterase (AChE-2) structures of enzymes do not affect in any way (there is no regularity of change or significant deviations) the values of the binding energy (ΔGb) recorded in the AChE-OPs complexes. However, the mutations analyzed in AChE are associated with a structural modification that causes an inadequate interaction to complete the phosphorylation of the enzyme.

An optimized cocktail of chitinolytic enzymes to produce N,N'-diacetylchitobiose and N-acetyl-d-glucosamine from defatted krill by-products.

Defatted krill powder (DKP), the byproduct of krill oil industry, is a resource of biological macromolecules. Here, one bacterial protease, three bacterial chitinases and one insect N-acetyl-d-hexosaminidase were integratively used to produce peptide, N,N'-diacetylchitobiose [(GlcNAc)2] and N-acetyl-d-glucosamine (GlcNAc) from DKP. First, alkaline protease was found to outperform neutral protease in deproteinizing DKP and the resultant krill peptides were rich in essential amino acids (41.4%). Second, the mutant of chitinase A from Serratia marcescens [SmChiA-F232W/F396W (SmChiA-M)] was found to be 32% faster than wild-type SmChiA in hydrolyzing the deproteinized DKP (DDKP) and showed significant synergy with chitinase B from S. marcescens (SmChiB) and chitinase C from S. marcescens (SmChiC). Then two SmChiA-M-based enzyme combinations [SmChiA-M + SmChiB + SmChiC and SmChiA-M + SmChiB + SmChiC + OfHex1 (an insect N-acetyl-d-hexosaminidase from Ostrinia furnacalis)] were designed to produce (GlcNAc)2 and GlcNAc, respectively, from DDKP. A yield of 2.04 g/L (GlcNAc)2 or 2.71 g/L GlcNAc (each with 95% purity) could be obtained from 20 g/L DDKP in 24 h.

Computational Study for the Unbinding Routes of ß-N-Acetyl-d-Hexosaminidase Inhibitor: Insight from Steered Molecular Dynamics Simulations.

ß-N-Acetyl-d-hexosaminidase from Ostrinia furnacalis (OfHex1) is a new target for the design of insecticides. Although some of its inhibitors have been found, there is still no commercial drug available at present. The residence time of the ligand may be important for its pharmacodynamic effect. However, the unbinding routes of ligands from OfHex1 still remain largely unexplored. In the present study, we first simulated the six dissociation routes of N,N,N-trimethyl-d-glucosamine-chitotriomycin (TMG-chitotriomycin, a highly selective inhibitor of OfHex1) from the active pocket of OfHex1 by steered molecular dynamics simulations. By comparing the potential of mean forces (PMFs) of six routes, Route 1 was considered as the most possible route with the lowest energy barrier. Furthermore, the structures of six different states for Route 1 were snapshotted, and the key amino acid residues affecting the dissociated time were analyzed in the unbinding pathway. Moreover, we also analyzed the "open⁻close" mechanism of Glu368 and Trp448 and found that their conformational changes directly affected the dissociation of TMG-chitotriomycin. Our findings would be helpful to understanding and identifying novel inhibitors against OfHex1 from virtual screening or lead-optimization.

Molecular Modeling Investigation of the Interaction between Humicola insolens Cutinase and SDS Surfactant Suggests a Mechanism for Enzyme Inactivation.

One of the largest commercial applications of enzymes and surfactants is as main components in modern detergents. The high concentration of surfactant compounds usually present in detergents can, however, negatively affect the enzymatic activity. To remedy this drawback, it is of great importance to characterize the interaction between the enzyme and the surfactant molecules at an atomistic resolution. The protein enzyme cutinase from the thermophilic and saprophytic fungus called Humicola insolens (HiC) is a promising candidate for use in detergents thanks to its hydrolase activity targeting mostly biopolyesters (e.g., cutin). HiC is, however, inhibited by low concentrations of sodium dodecyl sulfate (SDS), an ubiquitous surfactant. In this work, we investigate the interaction between HiC and SDS using molecular dynamics simulations. Simulations of HiC dissolved in different aqueous concentrations of SDS show the interaction between HiC and SDS monomers, as well as the formation and dynamics of SDS micelles on the surface of the enzyme. These results suggest a mechanism of cutinase inhibition by SDS, which involves the nucleation of aggregates of SDS molecules on hydrophobic patches on the cutinase surface. Notably, a primary binding site for monomeric SDS is identified near the active site of HiC constituting a possible nucleation point for micelles and leading to the blockage of the entrance to the enzymatic site. Detailed analysis of the simulations allow us to suggest a set of residues from the SDS binding site on HiC to probe as engineered mutations aimed at reducing SDS binding to HiC, thereby decreasing SDS inhibition of HiC.

Susceptibility to entomopathogens and modulation of basal immunity in two insect models at different temperatures.

In this work, we analysed the efficacy of different commercial bio-insecticides (Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora and Bacillus thuringiensis) by valuating the mortality induced on two insect models, Galleria mellonella (Lepidoptera) and Sarcophaga africa (Diptera) after exposure to different temperatures (10, 20 and 30 °C). Moreover, we investigated the effects of temperature on the basal humoral immunity of the two target insects; particularly, phenoloxidase (PO) and lysozyme activity. Our results show that G. mellonella is susceptible to all bio-insecticides at all the examined temperatures, except when infected at 10 °C with S. carpocapsae and at 30 °C with S. feltiae and B. thuringiensis. S. africa is more susceptible at 30 °C to all bioinsecticides; whereas, when infected at 10 and 20 °C, H. bacteriophora is the most efficient. Temperature modulates PO activity of both G. mellonella and S. africa, otherwise variations in lysozyme activity is observed only in G. mellonella. Except for a possible correlation between the increased lysozyme activity and the delayed Bt efficacy recorded on G. mellonella at 30 °C, a different resistance to bio-insecticides at different temperatures does not seem to be associated to variations of the host basal immunity, probably due to immunoevasive and immunodepressive strategies of these entomopathogens.

Parameters of oxidative stress, cholinesterase activity, Cd bioaccumulation in the brain and midgut of Lymantria dispar (Lepidoptera: Lymantriidae) caterpillars from unpolluted and polluted forests.

Cadmium (Cd) can display a variety of different effects on living organisms. The objectives of the present study were to investigate Cd bioaccumulation and differences in parameters of oxidative stress (activities of the enzymes: superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase, glutathione S-transferase, and amounts of non-enzymatic free sulfhydryl groups and total glutathione) and cholinesterase activity in larval brain and midgut tissues of the polyphagous forest insect Lymantria dispar collected from unpolluted and polluted oak forests. Fourth instar L. dispar caterpillars from the unpolluted forest had higher body mass but accumulated more Cd in comparison to caterpillars from the polluted forest. In both populations the midgut was more sensitive than the brain to the prooxidative effects of Cd. Enzyme activities and amounts of non-enzymatic parameters tended to be greater in midgut tissues than in the brain, except for cholinesterase activity. Parameters of oxidative stress had higher values in caterpillar tissues from the polluted than from the unpolluted oak forest. The observed differences between the two natural populations point to the importance of knowing the history of population exposure to environmental pollution when monitoring forest ecosystems.

Identification and characterization of the catalase gene involved in resistance to thermal stress in Heortia vitessoides using RNA interference.

To elucidate the role of catalase (CAT) in Heortia vitessoides Moore, which is one of the most destructive defoliating pests in Aquilaria sinensis (Loureiro) Sprenger forests, a CAT gene (HvCAT) was identified in the transcriptome of adult H. vitessoides. Sequence analyses indicated that HvCAT encodes a protein containing 507 amino acids, including a proximal active site sequence (FXRERIPERVVHAKGXGA), heme-ligand sequence (RLFSYNDTX), heme-binding residues (H73, S112, N146, F151, F159, R352, and Y356), and NADPH-binding residues (P149, H192, Y196, G199, R201, N211, H233, K235, I300, W301, P302, H303, Q442, and L445). A phylogenetic analysis indicated that CAT from lepidopteran species could be assigned to one well-supported cluster. Regarding its stage- and tissue-specific expression profiles, HvCAT was expressed at high levels in fifth-instar larvae, fat body of larvae, and abdomen of adults. Furthermore, when fifth-instar larvae were exposed to thermal stress at 35, 37, and 39 °C, hydrogen peroxide and malondialdehyde content significantly increased. HvCAT mRNA was upregulated when the larvae were exposed to temperatures of 31, 33, 35, 37, and 39 °C. The enzymatic activity of HvCAT was significantly elevated following thermal stress (35 and 37 °C). After the knockdown of HvCAT by double-stranded RNA interference, the expression of thioredoxin peroxidase (Tpx) increased, whereas that of copper zinc superoxide dismutase (Cu/ZnSOD) decreased. Additionally, knocking down HvCAT transcripts in fifth-instar larvae resulted in accelerated death following thermal stress at 35 °C. In summary, the results suggest that HvCAT plays a major role in the thermotolerance of H. vitessoides.

CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon.

Tyrosine hydroxylase (TH) is involved in insect melanin and the catecholamine biosynthesis pathway. TH as an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine is the first step reaction in the pathway. Although TH has been proven to affect the pigmentation of the epidermis and development in many insects, there is no report about physiological function of the TH gene in Agrotis ipsilon. Here we cloned the TH gene from A. ipsilon. Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages. Moreover, its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development. Then, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system to target the AiTH gene: deletion events were detected at the target sites. Compared with the control group, a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch; the other hatched embryos were seriously dehydrated after hatching and died within the first day. Quantitative real-time PCR analysis revealed that TH was down-regulated in AiTH mutants. Here, our work demonstrated that AiTH plays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy for A. ipsilon.