Isolation, screening and identification of key components having intense insect repellent activity against Plodia interpunctella from four different medicinal plant materials.

BACKGROUND: Global warming and the indiscriminate use of pesticides have increased the propagation of the stored-product insect pests, leading to enormous losses in the agriculture and food industries. The most used insect repellents are synthetic derivatives; however, these have an adverse effect on human health as well as on the environment. Therefore, we attempted to find materials with insect repellent activity in natural products. The present study aimed to identify the single chemical component with intense insect repellent activity in extracts from four different Oriental medicinal plant materials: (i) Anethum graveolens L. (dill) seeds; (ii) Artemisia capillaris Thunb. (capillary wormwood) leaves; (iii) smoked Prunus mume Siebold & Zucc. (mume) fruits; and (iv) Rhus javanica L. (galls). RESULTS: As a result of the bioassay-guided fractionation of each extract against the Plodia interpunctella, stored-product insect, the n-hexane fraction of dill seeds extract was confirmed as the optimal fraction between all of the fractions. In total, 32 chemical components were identified from the n-hexane fraction of dill seeds by gas chromatography-mass spectrometry analysis, and the two main components were dillapiole (47.51%) and carvone (26.76%). Of the two components, dillapiole was confirmed as the key component playing an essential role in insect repellent activity. CONCLUSION: Our study suggests that dillapiole has the potential to be used as a natural insect repellent for the control of P. interpunctella infestation in agricultural and food products during distribution and storage. © 2021 Society of Chemical Industry.

Genetic Knockouts Indicate That the ABCC2 Protein in the Bollworm Helicoverpa zea Is Not a Major Receptor for the Cry1Ac Insecticidal Protein.

Members of the insect ATP binding cassette transporter subfamily C2 (ABCC2) in several moth species are known as receptors for the Cry1Ac insecticidal protein from Bacillus thuringiensis (Bt). Mutations that abolish the functional domains of ABCC2 are known to cause resistance to Cry1Ac, although the reported levels of resistance vary widely depending on insect species. In this study, the function of the ABCC2 gene as a putative Cry1Ac receptor in Helicoverpa zea, a major pest of over 300 crops, was evaluated using CRISPR/Cas9 to progressively eliminate different functional ABCC2 domains. Results from bioassays with edited insect lines support that mutations in ABCC2 were associated with Cry1Ac resistance ratios (RR) ranging from 7.3- to 39.8-fold. No significant differences in susceptibility to Cry1Ac were detected between H. zea with partial or complete ABCC2 knockout, although the highest levels of tolerance were observed when knocking out half of ABCC2. Based on >500-1000-fold RRs reported in similar studies for closely related moth species, the low RRs observed in H. zea knockouts support that ABCC2 is not a major Cry1Ac receptor in this insect.

Molecular genotypic diversity of populations of brinjal shoot and fruit borer, Leucinodes orbonalis and development of SCAR marker for pesticide resistance.

BACKGROUND: The brinjal shoot and fruit borer, Leucinodes orbonalis is a destructive pest of Solanum melongena. The control of L. orbonalis with extensive application of synthetic chemical insecticides resulted in the development of resistance with known genetic heterogeneity among populations. Understanding the genetic diversity of their populations is important in developing strategies for their management. The present investigation was performed to characterize populations of L. orbonalis for their genetic diversity in the entire region of Tamil Nadu, South India using random amplified polymorphic DNA (RAPD) primers as a tool of the molecular marker. METHODS AND RESULTS: Among 60 random 10-mer primers, only ten primers generated reproducible and scorable banding profile. Among the ten different random primers, the primers namely OPG 7, OPG 8, OPS 2 and OPS 7 generated the highest genetic variation with over 80% genetic polymorphism. Phylogram analysis produced 18 clusters with eight major and ten minor clusters. Cluster analysis, statistical fitness, population structure and analysis of molecular variance confirmed the significant genetic variation among different populations. A trait specific marker obtained through RAPD was cloned, sequenced and used to develop a stable diagnostic SCAR marker for DNA fingerprinting to distinguish the populations. Amplification of this locus in the samples of 20 different populations indicated recognition of the trait for pesticide resistance in 12 populations. CONCLUSIONS: The results suggest that the biochemical nature of host plant varieties of this insect pest and variation in the application of different insecticides are essential contributing factors for the genotypic variations observed among populations of L. orbonalis.

A cis-Acting Mutation in the PxABCG1 Promoter Is Associated with Cry1Ac Resistance in Plutella xylostella (L.).

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.

The Impact of Graphite Oxide Nanocomposites on the Antibacterial Activity of Serum.

Nanoparticles can interact with the complement system and modulate the inflammatory response. The effect of these interactions on the complement activity strongly depends on physicochemical properties of nanoparticles. The interactions of silver nanoparticles with serum proteins (particularly with the complement system components) have the potential to significantly affect the antibacterial activity of serum, with serious implications for human health. The aim of the study was to assess the influence of graphite oxide (GO) nanocomposites (GO, GO-PcZr(Lys)2-Ag, GO-Ag, GO-PcZr(Lys)2) on the antibacterial activity of normal human serum (NHS), serum activity against bacteria isolated from alveoli treated with nanocomposites, and nanocomposite sensitivity of bacteria exposed to serum in vitro (using normal human serum). Additionally, the in vivo cytotoxic effect of the GO compounds was determined with application of a Galleria mellonella larvae model. GO-PcZr(Lys)2, without IR irradiation enhance the antimicrobial efficacy of the human serum. IR irradiation enhances bactericidal activity of serum in the case of the GO-PcZr(Lys)2-Ag sample. Bacteria exposed to nanocomposites become more sensitive to the action of serum. Bacteria exposed to serum become more sensitive to the GO-Ag sample. None of the tested GO nanocomposites displayed a cytotoxicity towards larvae.

Glyphosate inhibits melanization and increases susceptibility to infection in insects.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab.

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.

Exogenous nitrogen enhances poplar resistance to leaf herbivory and pathogen infection after exposure to soil cadmium stress.

Elemental defense hypothesis suggests that toxic metals accumulated in plant tissues could enhance plant defense against herbivores and pathogens. Since over-accumulation of metals in plant organs will pose negative effects on plant health, it is necessary to find a way to alleviate metal-induced toxicity in plants while keeping or even improving plant resistance. Exogenous nitrogen (N) application was reported to have such alleviation effect while stimulating metal accumulation in plant tissues. In this study, we examined whether soil N addition in three different doses to a poplar species under cadmium (Cd) stress can simultaneously improve plant growth and resistance to four herbivorous insects and a leaf pathogen. The results showed that N application to Cd-amended soil prominently enhanced plant growth and leaf Cd accumulation. While N addition in three doses all remarkably reduced herbivore growth than control plants, only the highest N dose exerted stronger inhibition than the sole Cd-treated plants. In the paired-choice experiment, plants supplied with the highest N dose showed an enhanced deterrent effect on herbivore preference than plants exposed to sole Cd. Furthermore, plant resistance to the leaf pathogen infection was strongly enhanced as the levels of N addition increased. Leaf sugar and three main defensive chemicals were not affected by N application implied that such enhanced effect of N on plant resistance was due to increased leaf Cd accumulation. Our results suggested that the application of exogenous N over a certain amount could enhance the resistance of Cd-treated plants to leaf herbivory and pathogen infection.

Exploration of insecticidal potential of Cry protein purified from Bacillus thuringiensis VIID1.

Insect pests are a threat to agriculture as they cause a loss of 15-22% to economically important crops every year. Bacillus thuringiensis produces parasporal crystal inclusions that have insecticidal 'Cry' proteins which are toxic to insect larvae of the order Lepidoptera, Coleoptera and Diptera, etc. In the present study, 40 different soil samples from Amritsar and its surrounding areas were selected for isolation of B. thuringiensis. The rod shaped, gram-positive bacterial isolates were further analyzed for characteristic crystal formation using phase contrast and scanning electron microscopy. 6 Bacillus samples containing cry genes were identified using the universal primers for cry genes, of which one isolate exhibited a protein band of ~95 kDa. This protein was purified using a Sephadex G-75 column. The insecticidal assays conducted with purified Cry protein on insect larvae of lepidopteran and dipteran orders viz. Spodoptera litura, Galleria malonella, Bactrocera cucurbitae and Culex pipens revealed considerable detrimental effects. A significant increase in larval mortality was observed for the larvae of all insects in a concentration dependent manner when treated with Cry protein purified from B. thuringenisis VIID1. The purified Cry protein did not have any significant effect on honey bee larvae.

Effect of diflubenzuron on the chitin biosynthesis pathway in Conopomorpha sinensis eggs.

Conopomorpha sinensis is the dominant borer pest of Litchi chinensis (litchi) and Euphoria longan (longan) in China. Control of C. sinensis is difficult because of its cryptic life habit; thus, an effective ovicide could be beneficial. The larvicidal effects of diflubenzuron (DFB) have been documented in many insect pest species. Therefore, DFB might be a useful ovicide to control C. sinensis. However, the detailed mode of action of DFB interference with insect molting and egg hatching is unclear. Thus, we studied alterations in expression of all genes potentially affected by DFB treatment using a transcriptome approach in 2-d-old C. sinensis eggs. Clean reads were assembled to generate 203 455 unigenes and 440 558 transcripts. A total of 4625 differently expressed genes, which included 2670 up-regulated and 1955 down-regulated unigenes, were identified. Chitin binding and chitin metabolic processes were among the most significant enriched pathways according to Gene Ontology analyses. Most of the genes that encode enzymes involved in the chitin biosynthesis pathway were unaffected, whereas genes that presumably encode cuticle proteins were up-regulated. Furthermore, altered expression patterns of 10 genes involved in the chitin biosynthesis pathway of C. sinensis embryos were observed in response to DFB treatment at different time points by quantitative reverse transcription polymerase chain reaction. We also observed abnormal development; there was reduced chitin content and modulated chitin distribution of newly hatched larvae, and altered egg hatching. Our findings illustrate an ovicidal effect of DFB on C. sinensis, and reveal more molecular consequences of DFB treatment on insects.

Host-Induced Plant Volatiles Mediate Ability of the Parasitoid Microplitis croceipes to Discriminate Between Unparasitized and Parasitized Heliothis virescens Larvae and Avoid Superparasitism.

In solitary endoparasitoids, oviposition in a host previously parasitized by a conspecific (superparasitism) leads to intraspecific competition, resulting in the elimination of all but one parasitoid offspring. Therefore, avoidance of parasitized hosts presents a strong selective advantage for such parasitoid species. Parasitoids use herbivore-induced plant volatiles (HIPVs) to find their hosts. In this study, we evaluated the ability of Microplitis croceipes (Hymenoptera: Braconidae) to discriminate between unparasitized and parasitized Heliothis virescens (Lepidoptera: Noctuidae) larvae using cotton plant odors as cues. A combination of behavioral and analytical techniques were used to test two hypotheses: (i) parasitoids will show preference for plant odors induced by unparasitized hosts over odors induced by parasitized hosts, and (ii) the parasitism status of herbivores affects HIPV emission in plants. Heliothis virescens larvae were parasitized for varying durations (0, 2 and 6-days after parasitism (DAP)). In four-choice olfactometer bioassays, female M. croceipes showed greater attraction to plant odors induced by unparasitized hosts compared to plant odors induced by parasitized hosts (2 and 6-DAP). Comparative gas chromatography-mass spectrometry analyses of cotton volatiles indicated reduced emission of 10 out of 21 identified compounds from plants infested by parasitized hosts compared with plants infested by unparasitized hosts. The results suggest that changes in plant volatile emission due to the parasitism status of infesting herbivores affect recruitment of parasitoids. Avoidance of superparasitism using plant odors optimizes host foraging in M. croceipes, and this strategy may be widespread in solitary parasitoid species.

Differences in midgut transcriptomes between resistant and susceptible strains of Chilo suppressalis to Cry1C toxin.

BACKGROUND: Chilo suppressalis is a widespread rice pest that poses a major threat to food security in China. This pest can develop resistance to Cry toxins from Bacillus thuringiensis (Bt), threatening the sustainable use of insect-resistant transgenic Bt rice. However, the molecular basis for the resistance mechanisms of C. suppressalis to Cry1C toxin remains unknown. This study aimed to identify genes associated with the mechanism of Cry1C resistance in C. suppressalis by comparing the midgut transcriptomic responses of resistant and susceptible C. suppressalis strains to Cry1C toxin and to provide information for insect resistance management. RESULTS: A C. suppressalis midgut transcriptome of 139,206 unigenes was de novo assembled from 373 million Illumina HiSeq and Roche 454 clean reads. Comparative analysis identified 5328 significantly differentially expressed unigenes (DEGs) between C. suppressalis Cry1C-resistant and -susceptible strains. DEGs encoding Bt Cry toxin receptors, aminopeptidase-P like protein, the ABC subfamily and alkaline phosphatase were downregulated, suggesting an association with C. suppressalis Cry1C resistance. Additionally, Cry1C resistance in C. suppressalis may be related to changes in the transcription levels of enzymes involved in hydrolysis, digestive, catalytic and detoxification processes. CONCLUSION: Our study identified genes potentially involved in Cry1C resistance in C. suppressalis by comparative transcriptome analysis. The assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of C. suppressalis resistance to Cry toxins.

Analysis of Cry1Ah Toxin-Binding Reliability to Midgut Membrane Proteins of the Asian Corn Borer.

Evolution of insect resistance to Bt toxins challenges the use of Cry toxins to control agricultural pests. In lepidopterans, Cry toxin affinity towards multiple midgut epithelial receptors has become a matter of dispute. Cry1Ah toxin-binding proteins were identified in the larval midgut of susceptible (ACB-BtS) and resistant (ACB-AhR) strains of the Asian corn borer (ACB). A pull-down assay was performed using biotinylated Cry1Ah toxin, and the binding proteins were identified by employing liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study aimed to find the binding consistency of the midgut epithelial protein to the Cry1Ah toxin. The binding proteins from different fractions of SDS-PAGE showed a different pattern. We observed an isoform of prophenoloxidase PPO1b (UniProt Acc No. A0A1Q1MKI0), which was found only in the ACB-AhR fractions. Prophenoloxidase (proPO) is an extraordinary defense molecule activated in insect species during pathogen invasion and the wound healing process. Importantly, this prophenoloxidase might have direct/indirect interaction with the Cry1Ah toxin. Our data also suggest that factors like techniques, enrichment of binding proteins in the sample and the reversible and irreversible nature of the brush border membrane vesicles (BBMVs) to Cry toxins could cause the inconsistency in the protein-protein interactions. Moreover, inside the larva midgut, the influence of the Cry toxins under physiological conditions might be different from the laboratory procedures.

Evaluation of the Toxicity of Supernatant Cultures and Spore-Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria.

Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect's resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore-crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of "functional mortality" was used to discriminate between "nontoxic" and "toxic" isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore-crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130 kDa whereas the Bt isolate Bl5NA expresses a protein of 65-70 kDa. The LC-MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce ß-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

Field-evolved resistance to chitin synthesis inhibitor insecticides by soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae), in Brazil.

Soybean looper (SBL), Chrysodeixis includens (Walker), is an economically important soybean and cotton pest in Brazil. Here, we selected an SBL strain resistant to teflubenzuron using F2 screening, estimated the resistance allele frequency, characterized the inheritance of resistance, investigated fitness costs, evaluated patterns of cross-resistance, and determined the magnitude of resistance. The teflubenzuron-resistant strain (Teflu-R) was selected from field-collected populations with an estimated allele frequency of 0.1700. Estimated LC50 values were 0.010 and 363.61 µg a.i. cm-2 for the susceptible (Sus) and Teflu-R strains, respectively, representing a 36,361-fold resistance ratio (RR). The LC50 values of reciprocal crosses were 1.02 and 0.59 µg a.i. cm-2, suggesting that resistance is autosomally inherited. The low survival of reciprocal crosses (16 and 20%) on teflubenzuron-sprayed leaves indicates incomplete recessive resistance. The number of segregations influencing resistance was 2.72, suggesting a polygenic effect. The Teflu-R strain showed longer development periods as well as lower survival and population growth than the Sus strain, revealing fitness costs. The Teflu-R strain also showed high cross-resistancesto other chitin inhibitor insecticides, such as novaluron (RR = 6147-fold) and lufenuron (RR = 953-fold), but low cross-resistance to methoxyfenozide, flubendiamide, and indoxacarb (RR < 3.45-fold). On discriminatory concentrations of teflubenzuron and novaluron, populations of SBL showed survival rates from 15 to 52%, indicating field resistance to these insecticides. Our findings indicated that resistance to teflubenzuron in SBL is autosomal, recessive, polygenic, and associated with fitness cost. We also found a high cross-resistance to other benzoylphenylureas and a high frequency of resistance to this mode-of-action in SBL in Brazil.

New Silver(I) Coordination Compound Loaded into Polymeric Nanoparticles as a Strategy to Improve In Vitro Anti-Helicobacter pylori Activity.

Helicobacter pylori inhabits the gastric epithelium and can promote the development of gastric disorders, such as peptic ulcers, acute and chronic gastritis, mucosal lymphoid tissue (MALT), and gastric adenocarcinomas. To use nanotechnology as a tool to increase the antibacterial activity of silver I [Ag(I)] compounds, this study suggests a new strategy for H. pylori infections, which have hitherto been difficult to control. [Ag (PhTSC·HCl)2] (NO3)·H2O (compound 1) was synthesized, characterized, and loaded into polymeric nanoparticles (PN1). PN1 had been developed by nanoprecipitation with poly(ε-caprolactone) polymer and poloxamer 407 surfactant. System characterization assays showed that the PNs had adequate particle sizes and ζ-potentials. Transmission electron microscopy confirmed the formation of polymeric nanoparticles (PNs). Compound 1 had a minimum inhibitory concentration for H. pylori of 3.90 µg/mL, which was potentiated to 0.781 µg/mL after loading. The minimum bactericidal concentration of 7.81 µg/mL was potentiated 5-fold to 1.56 µg/mL in PN. Compound 1 loaded in PN1 displayed better activity for H. pylori biofilm formation and mature biofilm. PN1 reduced the toxicity of compound 1 to MRC-5 cells. Loading compound 1 into PN1 inhibited the mutagenicity of the free compound. In vivo, the system allowed survival of Galleria mellonella larvae at a concentration of 200 µg/mL. This is the first demonstration of the antibacterial activity of a silver complex enclosed in polymeric nanoparticles against H. pylori.

Anti-inflammatory and antioxidant potential, in vivo toxicity, and polyphenolic composition of Eugenia selloi B.D.Jacks. (pitangatuba), a Brazilian native fruit.

Brazilian native fruits are a rich source of polyphenolic compounds that can act as anti-inflammatory and antioxidant agents. Here, we determined the polyphenolic composition, anti-inflammatory mechanism of action, antioxidant activity and systemic toxicity in Galleria mellonella larvae of Eugenia selloi B.D.Jacks. (synonym Eugenia neonitida Sobral) extract (Ese) and its polyphenol-rich fraction (F3) obtained through bioassay-guided fractionation. Phenolic compounds present in Ese and F3 were identified by LC-ESI-QTOF-MS. The anti-inflammatory activity of Ese and F3 was tested in vitro and in vivo through NF-κB activation, cytokine release and neutrophil migration assays. The samples were tested for their effects against reactive species (ROO•, O2•-, HOCl and NO•) and for their toxicity in Galleria mellonella larvae model. The presence of hydroxybenzoic acid, ellagitannins and flavonoids was identified. Ese and F3 reduced NF-κB activation, cytokine release and neutrophil migration, with F3 being three-fold more potent. Overall, F3 exhibited strong antioxidant effects against biologically relevant radicals, and neither Ese nor F3 were toxic to G. mellonella larvae. In conclusion, Ese and F3 revealed the presence of phenolic compounds that decreased the inflammatory parameters evaluated and inhibited reactive oxygen/nitrogen species. E. selloi is a novel source of bioactive compounds that may provide benefits for human health.

Application of natural products as insecticide candidates: Semisynthesis and biological evaluation of some novel osthole-based esters.

Natural products are very important sources for the development of new pesticides. Osthole, derived from many medical plants such as Cnidium, Angelica and Citrus plants, is a naturally occurring coumarin compound. To discover the new natural products-based insecticides, thirty-one osthole-based esters containing O-acyl-hydroxylamine groups were prepared, and their structures were identified by different spectral analysis methods. Derivatives A7, A17, A20 and A25 displayed more potent growth inhibitory (GI) activity than the botanical insecticide, toosendanin. Over half of target osthole derivatives had more effective larvicidal effect on P. xylostella than toosendanin. Among all title derivatives, compound A18 displayed more pronounced larvicidal activity (LC50 = 0.64 µmol mL-1) when compared with toosendanin (LC50 = 0.94 µmol mL-1). Some interesting results of structure-activity relationships (SARs) of these osthole derivatives were also discussed. In addition, the hemolysis and cytotoxicity assays indicated that these osthole derivatives showed very low toxicity toward normal mammalian cells.

Toxicology study of fraxinellone as ovicidal agents against Mythimna separata Walker and Bombyx mori Linaeus.

Fraxinellone is a naturally occurring degraded limonoid isolated from many species of plants in Meliaceae and Rutaceae. Besides structural modification of the lead compounds, the toxicology study of the lead compounds is also a very important procedure to develop insecticidal agents. Herein the toxicology study of fraxinellone was carried out as the ovicidal agent against the eggs of two lepidopteran insects Mythimna separata Walker and Bombyx mori Linaeus. Fraxinellone selectively exhibited an ovicidal activity against the eggs of M. separata. After treatment with fraxinellone, the eggshells of M. separata were shrinked, whereas those of B. mori had no obvious change. The dynamic process of M. separata embryo development demonstrated that the distinct difference between the treated eggs and the control ones was obvious at the second day after treatment, especially, the control embryo finished blastokinesis, whereas the treated ones were still laid at pre-reversion status and a lot of yolk can be seen around the embryo. It ultimately resulted in the eggshell withered and the egg hatching inhibited.

Proteomic analysis reveals the damaging role of low redox laccase from Yersinia enterocolitica strain 8081 in the midgut of Helicoverpa armigera.

OBJECTIVE: Earlier, we have found that the enteropathogenic Yersinia enterocolitica have evolved the survival mechanisms that regulate the expression of laccase-encoding genes in the gut. The present study aims to characterize the purified recombinant laccase from Y. enterocolitica strain 8081 biovar 1B and understand its effect on the midgut of cotton bollworm, Helicoverpa armigera (Hübner) larvae. RESULTS: The recombinant laccase protein showed high purity fold and low molecular mass (~ 43 kDa). H. armigera larvae fed with laccase protein showed a significant decrease in body weight and damage in the midgut. Further, transmission electron microscopy (TEM) studies revealed the negative effect of laccase protein on trachea, malpighian tubules, and villi of the insect. The proteome comparison between control and laccase-fed larvae of cotton bollworm showed significant expression of proteolytic enzymes, oxidoreductases, cytoskeletal proteins, ribosomal proteins; and proteins for citrate (TCA cycle) cycle, glycolysis, stress response, cell redox homeostasis, xenobiotic degradation, and insect defence. Moreover, it also resulted in the reduction of antioxidants, increased melanization (insect innate immune response), and enhanced free radical generation. CONCLUSIONS: All these data collectively suggest that H. armigera (Hübner) larvae can be used to study the effect of microbes and their metabolites on the host physiology, anatomy, and survival.