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.

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.

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.

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.

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.

The steroid hormone 20-hydroxyecdysone induces phosphorylation and aggregation of stromal interacting molecule 1 for store-operated calcium entry.

Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm (Helicoverpa armigera) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis.

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.

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.

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.

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.

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.

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.

Contaminants of emerging concern affect Trichoplusia ni growth and development on artificial diets and a key host plant.

Many countries are utilizing reclaimed wastewater for agriculture because drought, rising temperatures, and expanding human populations are increasing water demands. Unfortunately, wastewater often contains biologically active, pseudopersistent pharmaceuticals, even after treatment. Runoff from farms and output from wastewater treatment plants also contribute high concentrations of pharmaceuticals to the environment. This study assessed the effects of common pharmaceuticals on an agricultural pest, Trichoplusia ni (Lepidoptera: Noctuidae). Larvae were reared on artificial diets spiked with contaminants of emerging concern (CECs) at environmentally relevant concentrations. Trichoplusia ni showed increased developmental time and mortality when reared on artificial diets containing antibiotics, hormones, or a mixture of contaminants. Mortality was also increased when T. ni were reared on tomatoes grown hydroponically with the same concentrations of antibiotics. The antibiotic-treated plants translocated ciprofloxacin through their tissues to roots, shoots, and leaves. Microbial communities of T. ni changed substantially between developmental stages and when exposed to CECs in their diets. Our results suggest that use of reclaimed wastewater for irrigation of crops can affect the developmental biology and microbial communities of an insect of agricultural importance.

Production of the Inaccessible Sesquiterpene (-)-5-Epieremophilene by Metabolically Engineered Escherichia coli.

(-)-5-Epieremophilene, an epimer of the versatile sesquiterpene (+)-valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1-3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (-)-5-epieremophilene production with yields of 42.4-76.0 mg/L in shake-flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1-FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (-)-5-epieremophilene were achieved. Biological assay indicated that (-)-5-epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC50 =1.25 µg/cm2 ), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (-)-5-epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology.

Formulation and evaluation of norcanthridin nanoemulsions against the Plutella xylostella (Lepidotera: Plutellidae).

BACKGROUND: Norcantharidin (NCTD), a demethylated derivative of cantharidin (defensive toxin of blister beetles), has been reported to exhibit insecticidal activity against various types of agricultural pests. However, NCTD applications are limited by its poor water solubility and high dosage requirement. Nanoemulsions have attracted much attentions due to the transparent or translucence appearance, physical stability, high bioavailability and non-irritant in nature. In general, nanoemulsions with small droplet size can enhance the bioavailability of drugs, whereas this phenomenon is likely system dependent. In present study, NCTD nanoemulsions were developed and optimized to evaluate and improve the insecticidal activity of NCTD against Plutella xylostella (Lepidotera: Plutellidae) by a spontaneous emulsification method. RESULTS: Triacetin, Cremophor EL and butanol were selected as the constituents of NCTD nanoemulsions via solubility determination, emulsification efficiency and ternary phase diagram construction. Insecticidal activity of NCTD nanoemulsion was associated with the content of surfactant and cosurfactant: (1) Higher effective toxicity exhibited at Smix (surfactant to cosurfactant mass ratio) = 3:1 that may be associated with the changes in interfacial tension; (2) NCTD nanoemulsion at 3:7 < SOR (surfactant to oil mass ratio) < 6:4 was more effective at lower surfactant level, which was attributed to the relatively slow diffusion rate of NCTD hindering by excess surfactant. Interestingly, nanoemulsions with smaller droplets were not found to be more effective in our study. CONCLUSIONS: The optimized NCTD nanoemulsion (triacetin/Cremophor EL/butanol (60/20/20, w/w)) exhibited effective insecticidal activity (LC50 60.414 mg/l, LC90 185.530 mg/l, 48 h) than the NCTD acetone solution (LC50 175.602 mg/L, LC90 303.050 mg/L, 48 h). Spontaneous emulsifying nanoemulsion employed to formulate this poor water-soluble pesticide is a potential system for agriculture application.

Genome sequencing of Bacillus thuringiensis isolate T414 toxic to pink bollworm (Pectinophora gossypiella Saunders) and its insecticidal genes.

Bacillus thuringiensis is a spore-forming bacterium that is pathogenic towards a range of insect and nematode species and had been widely used as a biopesticide. In this study, we present the morphological, molecular and genetic characteristics of an indigenous Bt isolate T414 which displayed an effective toxicity against Pectinophora gossypiella. Scanning electron microscopy revealed the presence of bipyramidal, spherical and cubic shaped protein crystals in its spore-crystal suspension. SDS-PAGE analysis of its spore-crystal mixture showed the presence of two major protein bands viz.130 and 65 kDa. Whole genome sequencing with MiSeq divulged that it contains a chromosome and many plasmids. The assembled genome finally contained 6493494bp. Automated annotation of this genome draft predicted 6877 coding sequences and 152 RNAs (rRNAs, tRNAs and ncRNAs). NCBI blast analysis showed that assembled genome was distributed in a chromosome and 15 different types of plasmids. Further analysis of draft sequence revealed it harbors parasporal crystal protein genes (cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab and cyt1), vegetative insecticidal protein gene (vip3Aa), all plasmid borne and various additional virulence factors such as chitinases, proteases, bacteriocins and hemolysins. From the analysis it is evident that all the Cry, Cyt or Vip toxins are plasmid borne and are present on two types of plasmids named as p414A and p414E in the present study. A cry2A type gene was cloned and sequenced. It was named as cry2Aa21 by Bt nomenclature committee.

Functional textiles impregnated with biogenic silver nanoparticles from Bionectria ochroleuca and its antimicrobial activity.

Biogenic silver nanoparticles (AgNPs) were obtained throughout the fungal biosynthesis using extracellular filtrate of the epiphytic fungus B. ochroleuca and were incorporated in cotton and polyester fabrics by common impregnation procedure that was repeated once, twice or four times. Both fabrics were analyzed by scanning electron microscopy (SEM), and the effectiveness of impregnation was determined using inductively coupled plasma optical emission spectrometry (ICP OES). The AgNPs loaded fabrics showed potent antimicrobial activity on Staphylococcus aureus and Escherichia coli as well as on clinically relevant Candida albicans, Candida glabrata, and Candida parapsilosis, indicating that the AgNPs impregnation of cotton and polyester fabrics was efficient. AgNPs effectively inhibited the biofilm formation by Pseudomonas aeruginosa and was not toxic to Galleria mellonella larvae indicating a promising probability of biotechnological application.

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure.

Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.

The insect, Galleria mellonella, is a compatible model for evaluating the toxicology of okadaic acid.

The polyether toxin, okadaic acid, causes diarrhetic shellfish poisoning in humans. Despite extensive research into its cellular targets using rodent models, we know little about its putative effect(s) on innate immunity. We inoculated larvae of the greater wax moth, Galleria mellonella, with physiologically relevant doses of okadaic acid by direct injection into the haemocoel (body cavity) and/or gavage (force-feeding). We monitored larval survival and employed a range of cellular and biochemical assays to assess the potential harmful effects of okadaic acid. Okadaic acid at concentrations ≥ 75 ng/larva (≥ 242 µg/kg) led to significant reductions in larval survival (> 65%) and circulating haemocyte (blood cell) numbers (> 50%) within 24 h post-inoculation. In the haemolymph, okadaic acid reduced haemocyte viability and increased phenoloxidase activities. In the midgut, okadaic acid induced oxidative damage as determined by increases in superoxide dismutase activity and levels of malondialdehyde (i.e. lipid peroxidation). Our observations of insect larvae correspond broadly to data published using rodent models of shellfish-poisoning toxidrome, including complementary LD50 values: 206-242 µg/kg in mice, ~ 239 µg/kg in G. mellonella. These data support the use of this insect as a surrogate model for the investigation of marine toxins, which offers distinct ethical and financial incentives.