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RNA interference (RNAi) is a promising alternative strategy for ecologically friendly pest management. However, the identification of RNAi candidate genes is challenging owing to the absence of laboratory strains and the seasonality of most pest species. Tribolium castaneum is a well-established model, with a strong and robust RNAi response, which can be used as a high-throughput screening platform to identify potential RNAi target genes. Recently, the cactus gene was identified as a sensitive RNAi target for pest control. To explore whether the spectrum of promising RNAi targets can be expanded beyond those found by random large-scale screening, to encompass others identified using targeted knowledge-based approaches, we constructed a Cactus interaction network. We tested nine genes in this network and found that the delivery of double-stranded RNA corresponding to fusilli and cactin showed lethal effects. The silencing of cactin resulted in 100% lethality at every developmental stage from the larva to the adult. The knockdown of pelle, Dorsal-related immunity factor and short gastrulation reduced or even prevented egg hatching in the next generation. The combination of such targets with lethal and parental RNAi effects can now be tested against different pest species in field studies. [ABSTRACT FROM AUTHOR]

Transgenic maize plants expressing ds RNA targeting western corn rootworm ( WCR, Diabrotica virgifera virgifera LeConte) v- ATPase subunit C mRNA for RNAi provided significant root protection from WCR larval feeding damage in greenhouse assays compared to negative controls. Transcribed hairpin ds RNA in WCR-resistant maize plants was present as both intact hairpin-derived ds RNA and plant-processed si RNA. Therefore, the ability of ds RNA and si RNA targeting Dv v- ATPase C mRNA to cause an RNAi response was studied in both WCR larvae and adults. In 9-day diet-based feeding assays, ds RNA of at least 60 bp in length resulted in high levels of larval mortality. In contrast, 15-, 25- or 27-bp ds RNAs or pooled 21-bp si RNAs did not cause mortality of exposed larvae. When larvae were fed with diet overlaid with si RNAs, Dv v- ATPase C transcript levels did not change. Conversely, when WCR larvae were fed with diet overlaid with 184-bp ds RNA, the mRNA level was reduced by >20-fold relative to yfp ds RNA negative control. Similarly, 184-bp ds RNA caused 100% mortality of WCR adults, whereas the mortality of adults fed on diet treated with si RNAs was similar to the negative control. Feeding adults with si RNAs on diet did not affect the level of Dv v- ATPase C mRNA transcripts, whereas adults fed with the 184-bp ds RNA showed approximately 35-fold reduction in the target mRNA level. Similar results were obtained with the WCR adults injected with 184-bp ds RNA or 21-bp si RNA. These results suggest that only long ds RNA or hairpin-derived ds RNA is effective in causing lethal knock-down of Dv v- ATPase C mRNA. These results have implications for efficacious plant-delivered ds RNA for the protection of transgenic maize from WCR feeding damage and for the risk assessment of transgenic maize expressing insecticidal ds RNA. [ABSTRACT FROM AUTHOR]

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) is a destructive pest of the cultivated potato, Solanum tuberosum. Members of this species are well-suited to agricultural habitats because of a suite of physiological adaptations and their ability to evolve resistance to multiple insecticides. Recently, a novel double-stranded RNA (dsRNA) insecticide (Calantha, active ingredient ledprona) has been demonstrated as an effective tool to manage Colorado potato beetle populations through RNA interference (RNAi). Previous studies have demonstrated the lethality of the high doses of ledprona but had not assessed possible effects of low doses that may happen due to product degradation in the environment, incomplete spray coverage, and foliage growth. Exposure of fourth instar larvae to low concentrations of ledprona interfered with their pupation. Exposure of adults significantly reduced their mobility after seven days, as well as their fertility. Reproductive effects were stronger in females, especially when exposed before reaching sexual maturity. The observed effects of low doses of ledprona may aid in the overall management of Colorado potato beetles by reducing the size of resident populations, inhibiting beetle movement within and between fields, and reducing the population growth rate., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) is one of the most notorious pests of the potato, Solanum tuberosum. Potato beetles are capable of developing resistance to various insecticides in relatively few generations. Novel and effective means of controlling Colorado potato beetle populations are constantly required to protect potato crops and prevent loss of yield. The knockdown of gene function through ribonucleic acid interference has been demonstrated in Colorado potato beetles, suggesting the use of this technology as a means of beetle management. A novel double-stranded RNA-based insecticide with the active ingredient, ledprona, has been tested in variable dose laboratory bioassays, followed by field studies., Results: Exposure to ledprona resulted in both increased beetle mortality and decreased foliage consumption in all four instars and adult beetles. Effects decreased from earlier to later life stages. No ovicidal activity was detected. Onset of mortality was slower compared with existing chemical insecticides. Nevertheless, field applications of formulated ledprona to potato plots resulted in their protection comparable with that provided by spinosad and chlorantraniliprole., Conclusion: Based on the results of this study, formulated ledprona has attributes to become a useful tool in controlling Colorado potato beetle populations that is likely to be a good fit in integrated pest management protocols. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper) are two important defoliation pests of soybeans. In the present study, we have investigated the susceptibility and brush border membrane-binding properties of both species to Bacillus thuringiensis Cry1Ea toxin. Bioassays performed in first-instar larvae demonstrated potent activity against both soybean pests in terms of mortality or practical mortality. Competition-binding studies carried out with 125 Iodine-labelled Cry1Ea, demonstrated the presence of specific binding sites on the midgut brush border membrane vesicles (BBMV) of both insect species. Heterologous competition-binding experiments indicated that Cry1Ea does not share binding sites with Cry1Ac or Cry1Fa in either soybean pest. This study contributes to the knowledge of Cry1Ea toxicity and midgut binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ea with other Bt proteins aimed at controlling lepidopteran pests in soybeans.

Evolution of resistance threatens sustainability of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The fall armyworm (Spodoptera frugiperda) is a devastating pest of corn in the Western Hemisphere initially controlled by transgenic Bt corn producing the Cry1Fa insecticidal protein (event TC1507). However field-evolved resistance to TC1507 was observed in Puerto Rico in 2007 and has subsequently been reported in a number of locations in North and South America. Early studies on Puerto Rico fall armyworm populations found that the resistance phenotype was associated with reduced expression of alkaline phosphatase. However, in this work we show that field-evolved resistance to Cry1Fa Bt corn in Puerto Rico is closely linked to a mutation in an ATP Binding Cassette subfamily C2 (ABCC2) gene that functions as a Cry1Fa receptor in susceptible insects. Furthermore, we report a DNA-based genotyping test used to demonstrate the presence of the resistant (SfABCC2mut) allele in Puerto Rico populations in 2007, coincident with the first reports of damage to TC1507 corn. These DNA-based field screening data provide strong evidence that resistance to TC1507 in fall armyworm maps to the SfABCC2 gene and provides a useful molecular marker for detecting the SfABCC2mut allele in resistant fall armyworm.

Cry6Aa1 is a Bacillus thuringiensis ( Bt ) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 μg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5-9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

With Next Generation Sequencing technologies, high-throughput RNA sequencing (RNAseq) was conducted to examine gene expression in neonates of Diabrotica virgifera virgifera (LeConte) (Western Corn Rootworm, WCR) challenged with individual proteins of the binary Bacillus thuringiensis insecticidal proteins, Cry34Ab1 and Cry35Ab1, and the combination of Cry34/Cry35Ab1, which together are active against rootworm larvae. Integrated results of three different statistical comparisons identified 114 and 1300 differentially expressed transcripts (DETs) in the Cry34Ab1 and Cry34/35Ab1 treatment, respectively, as compared to the control. No DETs were identified in the Cry35Ab1 treatment. Putative Bt binding receptors previously identified in other insect species were not identified in DETs in this study. The majority of DETs (75% with Cry34Ab1 and 68.3% with Cry34/35Ab1 treatments) had no significant hits in the NCBI nr database. In addition, 92 DETs were shared between Cry34Ab1 and Cry34/35Ab1 treatments. Further analysis revealed that the most abundant DETs in both Cry34Ab1 and Cry34/35Ab1 treatments were associated with binding and catalytic activity. Results from this study confirmed the nature of these binary toxins against WCR larvae and provide a fundamental profile of expression pattern of genes in response to challenge of the Cry34/35Ab1 toxin, which may provide insight into potential resistance mechanisms.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important maize pest throughout most of the U.S. Corn Belt. Bacillus thuringiensis (Bt) insecticidal proteins including modified Cry3Aa and Cry34/35Ab1 have been expressed in transgenic maize to protect against WCR feeding damage. To date, there is limited information regarding the WCR midgut target sites for these proteins. In this study, we examined whether a cadherin-like gene from Diabrotica virgifera virgifera (DvvCad; GenBank accession # EF531715) associated with WCR larval midgut tissue is necessary for Cry3Aa or Cry34/35Ab1 toxicity. Experiments were designed to examine the sensitivity of WCR to trypsin activated Cry3Aa and Cry34/35Ab1 after oral feeding of the DvvCad dsRNA to knockdown gene expression. Quantitative real-time PCR confirmed that DvvCad mRNA transcript levels were reduced in larvae treated with cadherin dsRNA. Relative cadherin expression by immunoblot analysis and nano-liquid chromatography - mass spectrometry (nanoLC-MS) of WCR neonate brush border membrane vesicle (BBMV) preparations exposed to DvvCad dsRNA confirmed reduced cadherin expression when compared to BBMV from untreated larvae. However, the larval mortality and growth inhibition of WCR neonates exposed to cadherin dsRNA for two days followed by feeding exposure to either Cry3Aa or Cry34/35Ab1 for four days was not significantly different to that observed in insects exposed to either Cry3Aa or Cry34/35Ab1 alone. In combination, these results suggest that cadherin is unlikely to be involved in the toxicity of Cry3Aa or Cry34/35Ab1 to WCR., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: Fiber-reinforced composite (FRC) root canal posts have been introduced to be used instead of metal alloys and ceramics. The aim of this study was to investigate the flexural properties of different types of FRC posts and compare those values with a novel FRC material for dental applications., Methods: Seventeen different FRC posts of various brands (Snowpost, Carbopost, Parapost, C-post, Glassix, Carbonite) and diameters, (1.0-2.1 mm) and a continuous unidirectional E-glass FRC polymerized by light activation to a cylindrical form (everStick, diameter 1.5 mm) as a control material were tested. The posts (n=5) were stored at room's humidity or thermocycled (12.000 x, 5 degrees C/55 degrees C) and stored in water for 2 weeks before testing. A three-point bending test (span=10 mm) was used to measure the flexural strength and modulus of FRC post specimens., Results: Analysis of ANOVA revealed that thermocycling, brand of material and diameter of specimen had a significant effect (p<0.001) on the fracture load and flexural strength. The highest flexural strength was obtained with the control material (everStick, 1144.9+/-99.9 MPa). There was a linear relationship between fracture load and diameter of posts for both glass fiber and carbon fiber posts. Thermocycling decreased the flexural modulus of the tested specimens by approximately 10%. Strength and fracture load decreased approximately 18% as a result of thermocycling., Significance: Considerable variation can be found in the calculated strength values of the studied post brands. Commercial prefabricated FRC posts showed lower flexural properties than an individually polymerised FRC material.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

Purpose: The aim of this study was to evaluate clinical usefulness and durability of continuous glass-fiber reinforcement in repair of acrylic resin removable dentures., Materials and Methods: Fractured removable dentures without reinforcement, with conventional metal-wire reinforcement, or with mesh reinforcement were collected from two dental schools in Finland. The total number of dentures was 51 and the number of patients was 48. During the repair, the dentures were reinforced with a polymer-preimpregnated E-glass fiber at the region of the fracture. The fibers were used as partial fiber reinforcement, i.e., only the weakest part of the denture was reinforced. Follow-up time varied from 4 months to 4.1 years. After the follow-up period, possible fractures and discoloring were visually inspected. Possible irritation of oral mucosa by glass fibers and the general shape of the denture were also evaluated., Results: In 88% of the cases, there was no need for adjustment at the region of partial fiber reinforcement, and the clinical condition of the dentures was good. Glass fibers did not irritate the oral mucosa. In the case of refracture or hairline fracture, positioning of the partial fiber reinforcement was incorrect or the reinforcement had been used incorrectly (the wetting of the reinforcement with denture base resin was inadequate)., Conclusion: Polymer-preimpregnated partial fiber reinforcement seems to be useful in eliminating fractures of acrylic resin removable dentures. However, this study emphasizes the importance of correct positioning and accurate laboratory technique when partial fiber reinforcement is used.

Different polymer matrices are used for dental glass fibre composites. The aims of this study were to determine water sorption and solubility of glass fibre composites with different polymer matrices. In addition, the effect of post-curing of matrix polymers with heat on the water sorption and solubility values was investigated. Commercial one-phase and two-phase (powder-liquid) monomer systems were used in polymer matrix of E-glass fibre composite. Rhombic unreinforced and fibre reinforced test specimens were polymerized by autopolymerization or by light only, or additionally post-cured with heat. Water sorption and solubility determination method was based on ISO/DIS 1567-1997 draft for international standard with 7 d immersion time. In addition, water sorption was measured at second time for 30 d immersion time to determine saturation time of test specimens by water. Five test specimens of unreinforced polymer and reinforced polymer were tested and the quantity of fibres was determined by combustion analysis. Water sorption values of different brands of polymer matrices ranged from 0.9 to 8.3 wt% (P < 0.001, ANOVA). High sorption values were explained by microscopic voids in the polymer matrix and by composition of polymer matrix. Solubility values ranged from 0.02 to 2.5 wt% (P < 0.001, ANOVA). Generally, fibre inclusion and post-curing of polymer matrix reduced water sorption and solubility. The results of this study suggest that the water sorption and solubility of fibre composites varies according to the brand of polymer matrix and homogenity of polymer matrix. Water sorption of polymer matrix might influence hydrolytic stability of polymer-glass fibre composite.

The effect of fiber reinforcement of autopolymerizing poly(methyl methacrylate) was investigated. The impact strength of continuous E-glass fiber-poly(methyl methacrylate) composite was determined. Rectangular test specimens (n = 10 per group) were modified by incorporating an additional fiber reinforcement of untreated E-glass fibers, silanized E-glass fibers, or aramid fibers in the test specimens. Controls were either unreinforced or reinforced from the middle of the test specimen only. The impact strength of the specimens was measured by using a charpy-type pendulum impact tester after the specimens had been stored in water at 37 degrees C for 4 weeks. After the impact strength test, the length of the delamination of poly(methyl methacrylate) from the fibers was measured and plotted to the impact strength of the test specimens by using a linear regression model. The impact strength of unreinforced autopolymerizing poly(methyl methacrylate) was 7.8 kl/m2, while incorporation of glass fiber reinforcement with a fiber concentration of 12.4 wt% increased the impact strength to 74.7 kl/m2 (P = .000). The additional fiber reinforcement of the test specimen did not affect the impact strength (P = .363). Delamination negatively correlated with the impact strength of the test specimens (r = -.72, P = .000). The results of this study suggest that glass fiber reinforcement enhanced the impact strength of autopolymerizing poly(methyl methacrylate), while the use of additional fiber reinforcement made of aramid or glass fibers in the test specimens did not have an effect on the impact strength.

The gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A was cloned and expressed in E. coli plasmid pUC8. Plasmid (pNC-1A.14.8) encoded M.CviBIII methylates adenine in TCGA sequences both in vivo in E. coli and in vitro. Transposon Tn5 mutagenesis localized the M.CviBIII functional domain to a 1.5 kbp region of pNC-1A.14.8 and also indicated that a virus promoter directs transcription of the gene in E. coli. The 2.1 kbp insert containing the M.CviBIII gene was sequenced and a single open reading frame of 1131 bp was identified within the domain determined by Tn5 mutagenesis. When the M.CviBIII gene was fused in-frame with the 19 amino-terminal codons of lacZ a 45 kD polypeptide was identified in maxicells as predicted by the DNA sequence. The M.CviBIII gene was not essential for virus replication since a virus M.CviBIII deletion mutant also replicated in Chlorella.

The sequences of the genes coding for M.CviBIII (from virus NC-1A which infects a eukaryotic alga) [Narva et al., Nucleic Acids Res. 15 (1987) 9807-9823] and M.TaqI (from the bacterium Thermus aquaticus) [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] have been determined recently. Both enzymes methylate adenine in the sequence TCGA. We have compared the predicted amino acid sequences of these two methyltransferases (MTases), with each other and with ten other N6 A-MTases and find regions of similarity. M.CviBIII and M.TaqI were most closely related followed by M.PaeR7, whose recognition sequence (CTCGAG) contains the M.TaqI/M.CviBIII recognition sequence TCGA, and M.PstI, whose recognition sequence is CTGCAG. All of the N6-MTases contain the sequence Asp/Asn-Pro-Pro-Tyr (B-P-P-Y) referred to by Hattman et al. [J. Bacteriol. 164 (1985) 932-937] as region IV. The predicted secondary structure of this region forms a finger-like structure ('beta finger') containing a beta-pleated sheet (...XXXB), two beta-turns (P-P) followed by another beta-pleated sheet [Y/FXXX...].