Bulk segregant mapping and transcriptome analyses reveal the molecular mechanisms of spinetoram resistance in Spodoptera frugiperda.

The evolution of resistance to insecticides poses a significant threat to pest management programs. Understanding the molecular mechanisms underlying insecticide resistance is essential to design sustainable pest control and resistance management programs. The fall armyworm, Spodoptera frugiperda, is an important insect pest of many crops and has a remarkable ability to evolve resistance to insecticides. In this study, we employed bulk segregant analysis (BSA) combined with DNA and RNA sequencing to characterize the molecular basis of spinetoram resistance in S. frugiperda. Analysis of genomic data derived from spinetoram selected and unselected bulks and the spinetoram-resistant and susceptible parental strains led to the identification of a three-nucleotide deletion in the gene encoding the nicotinic acetylcholine receptor α6 subunit (nAChR α6). Transcriptome profiling identified the upregulation of few genes encoding detoxification enzymes associated with spinetoram resistance. Thus, spinetoram resistance in S. frugiperda appears to be mediated mainly by target site insensitivity with a minor role of detoxification enzymes. Our findings provide insight into the mechanisms underpinning resistance to spinetoram in S. frugiperda and will inform the development of strategies to control this highly damaging, globally distributed crop pest.

Baseline Response, Monitoring, and Cross-Resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Sodium Channel Blocker Insecticides in Brazil.

Spodoptera frugiperda (J.E. Smith) is one of the key cross-crop pests in Brazilian agroecosystems. Field-evolved resistance of S. frugiperda to some conventional insecticides and Bt proteins has already been reported. Thus, the use of insecticides with new mode of action such as sodium channel blockers (indoxacarb and metaflumizone) could be an important tool in insecticide resistance management (IRM) programs. To implement a proactive IRM, we conducted baseline response and monitoring to indoxacarb and metaflumizone in 87 field populations of S. frugiperda collected from major maize-growing regions of Brazil from 2017 to 2020, estimated the frequency of resistance alleles to indoxacarb, and evaluated cross-resistance of indoxacarb and metaflumizone to some selected insecticides and Bt proteins. Low variation in susceptibility to indoxacarb (4.6-fold) and metaflumizone (2.6-fold) was detected in populations of S. frugiperda in 2017. The frequency of the resistance allele to indoxacarb was 0.0452 (0.0382-0.0527 CI 95%), by using F2 screen method. The mean survival at diagnostic concentration, based on CL99, varied from 0.2 to 12.2% for indoxacarb and from 0.0 to 12.7% for metaflumizone, confirming high susceptibility of S. frugiperda to these insecticides in Brazil. No cross-resistance was detected between sodium channel blocker insecticides and other insecticides (organophosphate, pyrethroid, benzoylurea, spinosyn, and diamide) and Bt proteins. These findings showed that sodium channel blocker insecticides are important candidates to be exploited in IRM strategies of S. frugiperda in Brazil.

Evaluation of the Haemophilus influenzae EUCAST and CLSI disc diffusion methods to recognize aminopenicillin and amoxicillin/clavulanate resistance.

OBJECTIVES: Implementation of EUCAST susceptibility testing in an Australian hospital laboratory demonstrated higher rates of aminopenicillin and amoxicillin/clavulanate resistance in Haemophilus influenzae than previously recognized. This study aimed to better define the variability in the detection of ß-lactam resistance based on EUCAST and CLSI disc diffusion (DD) methodology, by comparison with the recommended reference method, broth microdilution (BMD), and by concordance with genomic analysis. METHODS: A total of 100 random H. influenzae isolates were assessed for ampicillin and amoxicillin/clavulanate susceptibility by EUCAST and CLSI DD and BMD. WGS was used to analyse the ftsI gene of a subset of isolates with ß-lactam resistance, other than that due to isolated ß-lactamase production. RESULTS: Of the 100 isolates, 32 were categorized as either ß-lactamase negative, ampicillin resistant (BLNAR) (n = 18) or ß-lactamase positive, amoxicillin/clavulanate resistant (BLPACR) (n = 14) by EUCAST DD. All 18 EUCAST BLNAR isolates were genotypically confirmed by WGS. Five of 18 BLNAR isolates were concordant by CLSI DD, 12 by EUCAST BMD and 4 by CLSI BMD. Nine of 14 EUCAST BLPACR isolates were confirmed by WGS; the remaining 5 were 1 mm below the EUCAST DD breakpoint. Only one isolate was detected as BLPACR by CLSI DD. Group III mutations associated with high-level ampicillin resistance were identified in 10/32 isolates. CONCLUSIONS: The EUCAST DD susceptibility method is more reliable than either CLSI or BMD for the detection of genotypically defined BLNAR resistance. However, accurate categorization of amoxicillin/clavulanate resistance remains problematic. Continuous and reproducible surveillance of resistance is needed; for this to be possible, robust susceptibility methods are required.

Geographical distribution of Vip3Aa20 resistance allele frequencies in Spodoptera frugiperda (Lepidoptera: Noctuidae) populations in Brazil.

BACKGROUND: The use of Bt plants has been the main strategy for controlling the fall armyworm Spodoptera frugiperda (J. E. Smith) in Brazil. However, many resistance cases were already registered. The resistance of S. frugiperda to the Vip3Aa20 protein was recently characterized under laboratory conditions but it is still efficient under field conditions. Here, resistance monitoring studies were conducted using phenotypic (purified protein and Bt maize leaves) and genotypic (F1 and F2 screen) methods to support insect resistance management (IRM) programs and preserve Vip3Aa20 technology on maize. RESULTS: Phenotypic monitoring with purified protein showed two populations significantly different from the susceptible strain on the second crop season in 2016. This number increased for the first and second crop seasons in 2017 in several regions. The genotypic monitoring estimated a mean frequency of the resistance allele of 0.0027 for the F1 screen and 0.0033 for the F2 screen. Three new resistant strains to Vip3Aa20 were selected from F2 screen assays. Complementation tests on these new resistant strains were positive with the previous resistant strain. CONCLUSION: Here we showed that the resistance allele of S. frugiperda to Vip3Aa20 protein is widely distributed in maize-producing regions in Brazil and its frequency increases throughout crop seasons. © 2019 Society of Chemical Industry.

Increased detection of carbapenemase-producing Enterobacteriaceae on post-clean sampling of a burns unit's wet surfaces.

Wet surface biofilms are a potential reservoir for multidrug-resistant Gram-negative organisms, including carbapenemase-producing Enterobacteriaceae (CPE). Recognition of environmental sources is important in reducing secondary patient transmission. We report the increased detection of blaIMP-4+ CPE in environmental samples from floor drains in burns unit shower rooms, when collected following cleaning as compared to pre-cleaning. We propose that disruption of biofilms during cleaning may account for the increased detection of multi-resistant organisms. The results highlight the role of the wet environment as an under-recognized potential source of CPE transmission. Environmental screening focusing on pre-cleaning samples alone will likely underestimate environmental contamination.

Incarcerated Bochdalek hernia causing bowel obstruction in an adult male patient.

Bochdalek hernias occur as a result of congenital fusion failure of the posterolateral muscular portion of the diaphragm derived from the embryonic pleuroperitoneal membrane. The vast majority of symptomatic Bochdalek hernias are diagnosed during the neonatal period. Congenital diaphragmatic hernias showing symptoms in adulthood are rare. We report the case of a 28-year-old man who presented with subacute bowel obstruction, later diagnosed to have a left-sided incarcerated Bochdalek hernia containing an ischaemic splenic flexure and the distal transverse colon. During laparotomy, he underwent primary repair of the diaphragmatic defect and an extended right hemicolectomy with ileocolic anastomosis. The patient made a good recovery and is well at three months following surgery. This case highlights the need for surgeons to be vigilant about rare types of congenital internal hernias as they may become symptomatic later in life.

Near-Isogenic Cry1F-Resistant Strain of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Investigate Fitness Cost Associated With Resistance in Brazil.

Field-evolved resistance to Cry1F maize in Spodoptera frugiperda (J.E. Smith) populations in Brazil was reported in 2014. In this study, to investigate fitness costs, we constructed a near-isogenic S. frugiperda-resistant strain (R-Cry1F) using Cry1F-resistant and Cry1F-susceptible strains sharing a close genetic background. A near-isogenic R-Cry1F strain was obtained by eight repeated backcrossings, each followed by sib-mating and selection among resistant and susceptible strains. Fitness cost parameters were evaluated by comparing the biological performance of resistant, susceptible, and heterozygous strains on artificial diet. Fitness parameters monitored included development time and survival rates of egg, larval, pupal, and egg-to-adult periods; sex ratio; adult longevity; timing of preoviposition, oviposition, and postoviposition; fecundity; and fertility. A fertility life table was also calculated. The near-isogenic R-Cry1F strain showed lower survival rate of eggs (32%), when compared with Sus and reciprocal crosses (41 and 55%, respectively). The number of R-Cry1F insects that completed the life cycle was reduced to ∼25%, compared with the Sus strain with ∼32% reaching the adult stage. The mean generation time (T) of R-Cry1F strain was ∼2 d shorter than R-Cry1F♂×Sus♀ and Sus strains. The reproductive parameters of R-Cry1F strain were similar to the Sus strain. However, fewer females were produced by R-Cry1F strain than R-Cry1F♀×Sus♂ and more females than R-Cry1F♂×Sus♀. In summary, no relevant fitness costs are observed in a near-isogenic Cry1F-resistant strain of S. frugiperda, indicating stability of resistance to Cry1F protein in Brazilian populations of this species in the absence of selection pressure.

Ultrastructural evaluation of the hybrid layer after cementation of fiber posts using adhesive systems with different curing modes.

This in vitro study evaluated the adhesive interface of intraradicular fiber glass posts and root dentin using scanning electron microscopy (SEM). Forty-eight single-rooted premolars were randomly divided into 6 groups consisting of chemical, dual, or light cured adhesive systems combined with either chemical or dual cure resin cements. Scanning electron microscopic analysis showed the best results for continuity, density and morphology of the hybrid layer and resin tags for the combination of a self-cure adhesive with self-cure cement resin, followed by a dual-cure adhesive with self-cure cement resin, and finally a light-cure adhesive with self-cure cement. For the dual-cure resin cement, the same relation may be observed. The apical third was the most critical region for evaluated the criteria for all combinations of materials (Kruskal-Wallis and Friedman tests; p<0.001). Generally, the simplification of steps in the adhesive system and the polymerization reaction of resin adhesives and cements produced a direct effect on the quality of the adhesive post/dentin substrate interface.

Ultrastructural evaluation of the hybrid layer after cementation of fiber posts using adhesive systems with different curing modes

This in vitro study evaluated the adhesive interface of intraradicular fiber glass posts and root dentin using scanning electron microscopy (SEM). Forty-eight single-rooted premolars were randomly divided into 6 groups consisting of chemical, dual, or light cured adhesive systems combined with either chemical or dual cure resin cements. Scanning electron microscopic analysis showed the best results for continuity, density and morphology of the hybrid layer and resin tags for the combination of a self-cure adhesive with self-cure cement resin, followed by a dual-cure adhesive with self-cure cement resin, and finally a light-cure adhesive with self-cure cement. For the dual-cure resin cement, the same relation may be observed. The apical third was the most critical region for evaluated the criteria for all combinations of materials (Kruskal-Wallis and Friedman tests; p<0.001). Generally, the simplification of steps in the adhesive system and the polymerization reaction of resin adhesives and cements produced a direct effect on the quality of the adhesive post/dentin substrate interface.

Este estudo in vitro avaliou as interfaces adesivas de pinos intra-radiculares de fibra de vidro e a dentina radicular utilizando microscópio eletrônico de varredura (MEV). Quarenta e oito pré-molares unirradiculares foram divididos ao acaso em seis grupos, compostos por sistemas adesivos de cura química, dual ou fotopolimerizável, associado com cimentos resinosos de polimerização química ou dual. As análises microscópicas mostraram a maior continuidade, densidade e morfologia da camada híbrida e prolongamentos resinosos para a associação entre adesivos e cimentos auto-polimerizáveis seguido pelo grupo do adesivo de dupla polimerização e cimento de resina auto-polimerizável, e finalmente pelo adesivo fotopolimerizável e cimento de resina auto-polimerizável . Para os cimentos resinosos de dupla polimerização a mesma relação pode ser observada. O terço apical mostrou ser o substrato mais crítico em relação aos critérios avaliados para todas as associações entre os materiais usados(testes de Kruskal-Wallis e Friedman p<0,001). De maneira geral, a simplificação dos passos do sistema adesivo e a reação de polimerização dos adesivos e cimentos resinosos produzem efeitos diretos na qualidade da interface adesivo pino/dentina.

THGS: a web-based database of Transmembrane Helices in Genome Sequences.

Transmembrane Helices in Genome Sequences (THGS) is an interactive web-based database, developed to search the transmembrane helices in the user-interested gene sequences available in the Genome Database (GDB). The proposed database has provision to search sequence motifs in transmembrane and globular proteins. In addition, the motif can be searched in the other sequence databases (Swiss-Prot and PIR) or in the macromolecular structure database, Protein Data Bank (PDB). Further, the 3D structure of the corresponding queried motif, if it is available in the solved protein structures deposited in the Protein Data Bank, can also be visualized using the widely used graphics package RASMOL. All the sequence databases used in the present work are updated frequently and hence the results produced are up to date. The database THGS is freely available via the world wide web and can be accessed at http:// pranag.physics.iisc.ernet.in/thgs/ or http://144.16. 71.10/thgs/.

Studies of the low dose 'hook' effect in a competitive homogeneous immunoassay.

The interactions of two monoclonal antibodies with human growth hormone (hGH) have been investigated. The individual antibodies showed normal behavior in a competitive binding assay, but mixtures of the antibodies demonstrated a 'hook' attributable to cooperative interactions. Cooperativity was observed in titrations which preceded the competitive binding assay. Size exclusion chromatographic data suggest that the cooperativity is explained by the formation of higher molecular weight complexes (up to 700 kDa). The major complex is probably linear, consisting of three antibody molecules. Circular and linear complexes with four antibody molecules (octameric complexes) are also possible. Theoretical models also support the formation of cyclic complexes in a competitive binding assay.

Multiple epitope interactions in the two-step sandwich immunoassay.

The 'hook' effect as related to the two-step sandwich immunoassay has been investigated experimentally and theoretically. The multiple epitope interactions between the analyte and the labeled antibody cause a 'hook' in the two-step sandwich immunoassay. Three different analytes and monoclonal antibodies were chosen to carefully demonstrate the effect of the analyte characteristics on this immunoassay. Two monoclonal antibodies against two different epitopes of biosynthetic human growth hormone (hGH) was the simplest model for this study. The sandwich immunoassay for hGH shows no 'hook' effect. The non-covalent dimeric form of hGH (D-hGH) possesses two repeating epitopes which is the simplest model for an analyte having a discrete number of repeating epitopes. The D-hGH assay demonstrated a 'hook' effect in the two-step sandwich immunoassay if the labeled antibody was allowed to interact with more than one epitope. In a third system multiple epitope interactions with the labeled antibody were observed using ferritin. The effect of the analyte concentration and the liquid-phase antibody have been examined to elucidate the nature of these various interactions. The cause of the 'hook' effect in the two-step sandwich immunoassay is attributed to the desorption of the bound analyte due to a conformational change after the labeled antibody interacts with several epitopes of the adsorbed analyte.

Studies of the 'hook' effect in the one-step sandwich immunoassay.

The one-step sandwich immunoassay is increasingly replacing the traditional two-step immunoassay due to obvious advantages such as assay speed. However, the one-step sandwich immunoassay suffers from the 'hook' effect irrespective of the analyte characteristics. The 'hook' effect is dependent primarily on the analyte concentration. Three different model analytes, human growth hormone (hGH), the dimeric form of hGH (D-hGH, having a discrete number of repeating epitopes) and ferritin (multiple epitopes) having different immunological properties have been employed in studies of the one-step sandwich immunoassay. The characteristics of each of the model analytes offer new insights into general guidelines for assay procedures. These guidelines permit rapid optimization of assay conditions for an immunoassay without a priori knowledge of the immunological characteristics of the antibody or antigen. Both experimental and theoretical data show several instances where high capacity solid-phase antibodies can effectively shift the 'hook' to relatively higher analyte concentrations. The effect of the concentration of labeled antibody on assay response was examined theoretically.