The Molecular Resistance Mechanisms of European Earwigs from Apple Orchards Subjected to Different Management Strategies.

To date, apple orchards are among the most treated crops in Europe with up to 35 chemical treatments per year. Combining control methods that reduce the number of pesticide treatments is essential for agriculture and more respectful of the environment, and the use of predatory insects such as earwigs may be valuable to achieve this goal. European earwigs, Forficula auricularia (Dermaptera: Forficulidae) are considered beneficial insects in apple orchards where they can feed on many pests like aphids. The aim of this study was to investigate the potential impact of orchards' insecticide treatments on resistance-associated molecular processes in natural populations of earwigs. Because very few molecular data are presently available on earwigs, our first goal was to identify earwig resistance-associated genes and potential mutations. Using earwigs from organic, integrated pest management or conventional orchards, we identified mutations in acetylcholinesterase 2, α1 and ß2 nicotinic acetylcholine receptors. In addition, the expression level of these targets and of some essential detoxification genes were monitored using RT-qPCR. Unexpectedly, earwigs collected in organic orchards showed the highest expression for acetylcholinesterase 2. Four cytochromes P450, one esterase and one glutathione S-transferases were over-expressed in earwigs exposed to various management strategies in orchards. This first study on resistance-associated genes in Forficula auricularia paves the way for future experimental studies aimed at better understanding the potential competition between natural enemies in apple orchards in order to optimize the efficiency of biocontrol.

Investigating the role of the ROS/CncC signaling pathway in the response to xenobiotics in Spodoptera frugiperda using Sf9 cells.

Spodoptera frugiperda (fall armyworm, FAW) is an invasive polyphagous lepidopteran pest that has developed sophisticated resistance mechanisms involving detoxification enzymes to eliminate toxic compounds it encounters in its diet including insecticides. Although its inventory of detoxification enzymes is known, the mechanisms that enable an adapted response depending on the toxic compound remain largely unexplored. Sf9 cells were used to investigate the role of the transcription factors, Cap n' collar isoform C (CncC) and musculoaponeurotic fibrosarcoma (Maf) in the regulation of the detoxification response. We overexpressed CncC, Maf or both genes, and knocked out (KO) CncC or its repressor Kelch-like ECH associated protein 1 (Keap1). Joint overexpression of CncC and Maf is required to confer increased tolerance to indole 3-carbinol (I3C), a plant secondary metabolite, and to methoprene, an insecticide. Both molecules induce reactive oxygen species (ROS) pulses in the different cell lines. The use of an antioxidant reversed ROS pulses and restored the tolerance to I3C and methoprene. The activity of detoxification enzymes varied according to the cell line. Suppression of Keap1 significantly increased the activity of cytochrome P450s, carboxylesterases and glutathione S-transferases. RNAseq experiments showed that CncC mainly regulates the expression of detoxification genes but is also at the crossroads of several signaling pathways (reproduction and immunity) maintaining homeostasis. We present new data in Sf9 cell lines suggesting that the CncC:Maf pathway plays a central role in FAW response to natural and synthetic xenobiotics. This knowledge helps to better understand detoxification gene expression and may help to design next-generation pest insect control measures.

Insecticidal efficacy and possibility of Combretum trifoliatum Vent. (Myrtales: Combretaceae) extracts in controlling Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

BACKGROUND: The fall armyworm Spodoptera frugiperda (J.E. Smith), is an important pest of agronomical crops. It is interesting to discover secondary metabolites in plants that are environmentally safer than synthetic pesticides. For this purpose, Combretum trifoliatum crude extract and its isolated compounds were investigated for their insecticidal activities against S. frugiperda. RESULTS: The median lethal dose (LD50 ) was evaluated in the second-instar larvae using the topical application method. The isolated compounds, apigenin and camphor, demonstrated a highly toxic effect on larvae at a lower LD50 dose than crude extract. Moreover, when the larvae were exposed to crude extract concentrations, the development to pupa and adult stages was reduced by more than 50%. The ovicidal toxicity was examined using a hand sprayer. The extract concentration 5, 10, and 20 µg/egg significantly decreased the egg hatchability. In addition, crude extract showed a significant difference in inhibiting acetylcholinesterase (AChE) activity while crude extract and camphor showed significant inhibitory effects on carboxylesterase (CE) and glutathione-S-transferase (GST) activities. CONCLUSION: The crude ethanol extract of Combretum trifoliatum was toxic to S. frugiperda in terms of larval mortality, negatively affecting biological parameters, and decreasing egg hatchability. Additionally, the activities of cholinergic and detoxifying enzymes were affected by crude extract and its isolated compounds. These results highlight that Combretum trifoliatum might be efficient as a bioinsecticide to control S. frugiperda. © 2023 Society of Chemical Industry.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

Spodoptera frugiperda Sf9 cells as a model system to investigate the role of detoxification gene expression in response to xenobiotics.

Spodoptera frugiperda (fall armyworm) is a highly destructive invasive pest that feeds on numerous crops including maize and rice. It has developed sophisticated mechanisms to detoxify xenobiotics such as secondary plant metabolites as well as manmade insecticides. The aim of the study was to explore the detoxification response to plant secondary metabolites and insecticides employing a S. frugiperda Sf9 cell model exposed to indole 3-carbinol (I3C) and methoprene. The cell Inhibitory Concentration 50 (IC50) for these molecules was determined and IC10, IC20 and IC30 doses were used to monitor the induction profiles of detoxification genes. Cytochrome P450 monooxygenases (P450s) of the CYP9A subfamily were the most inducible genes of the seven examined. Our results also showed the induction of the transcription factor Cap'n'collar isoform C (CncC). Transient transformation of Sf9 cells overexpressing CncC and its partner muscle aponeurosis fibromatosis (Maf) induces overexpression of CYP4M14, CYP4M15, CYP321A9 and GSTE1 while CYP9As were not induced. Next, we determined the capacity of recombinantly expressed CYP9A30, CYP9A31 and CYP9A32 to interact with methoprene and I3C. Fluorescence-based biochemical assays revealed an interaction of methoprene with functionally expressed CYP9A30, CYP9A31 and CYP9A32 whereas almost no interaction was detected for I3C, suggesting the ability of CYP9As to metabolize methoprene. Our results showed that Sf9 cells could be a useful model to decipher detoxification pathways of S. frugiperda.

Comparative analysis of the detoxification gene inventory of four major Spodoptera pest species in response to xenobiotics.

The genus Spodoptera (Lepidoptera: Noctuidae) comprises some of the most polyphagous and destructive agricultural pests worldwide. The success of many species of this genus is due to their striking abilities to adapt to a broad range of host plants. Superfamilies of detoxification genes play a crucial role in the adaption to overcome plant defense mechanisms mediated by numerous secondary metabolites and toxins. Over the past decade, a substantial amount of expression data in Spodoptera larvae was produced for those genes in response to xenobiotics such as plant secondary metabolites, but also insecticide exposure. However, this information is scattered throughout the literature and in most cases does not allow to clearly identify candidate genes involved in host-plant adaptation and insecticide resistance. In the present review, we analyzed and compiled information on close to 600 pairs of inducers (xenobiotics) and induced genes from four main Spodoptera species: S. exigua, S. frugiperda, S. littoralis and S. litura. The cytochrome P450 monooxygenases (P450s; encoded by CYP genes) were the most upregulated detoxification genes across the literature for all four species. Most of the data was provided from studies on S. litura, followed by S. exigua, S. frugiperda and S. littoralis. We examined whether these detoxification genes were reported for larval survival under xenobiotic challenge in forward and reverse genetic studies. We further analyzed whether biochemical assays were carried out showing the ability of corresponding enzymes and transporters to breakdown and excrete xenobiotics, respectively. This revealed a clear disparity between species and the lack of genetic and biochemical information in S. frugiperda. Finally, we discussed the biological importance of detoxification genes for this genus and propose a workflow to study the involvement of these enzymes in an ecological and agricultural context.

Recent Advances in the Understanding of Molecular Mechanisms of Resistance in Noctuid Pests.

Noctuid moths are among the most devastating crop pests on the planet [...].

Resistance in the Genus Spodoptera: Key Insect Detoxification Genes.

The genus Spodoptera (Lepidoptera: Noctuidae) includes species that are among the most important crop pests in the world. These polyphagous species are able to feed on many plants, including corn, rice and cotton. In addition to their ability to adapt to toxic compounds produced by plants, they have developed resistance to the chemical insecticides used for their control. One of the main mechanisms developed by insects to become resistant involves detoxification enzymes. In this review, we illustrate some examples of the role of major families of detoxification enzymes such as cytochromes P450, carboxyl/cholinesterases, glutathione S-transferases (GST) and transporters such as ATP-binding cassette (ABC) transporters in insecticide resistance. We compare available data for four species, Spodoptera exigua, S. frugiperda, S. littoralis and S. litura. Molecular mechanisms underlying the involvement of these genes in resistance will be described, including the duplication of the CYP9A cluster, over-expression of GST epsilon or point mutations in acetylcholinesterase and ABCC2. This review is not intended to be exhaustive but to highlight the key roles of certain genes.

Detoxification gene families in Phylloxera: Endogenous functions and roles in response to the environment.

Phylloxera, Daktulosphaira vitifoliae, is an agronomic pest that feeds monophagously on grapevine, Vitis spp. host plants. Phylloxera manipulates primary and secondary plant metabolism to establish either leaf or root galls. We manually annotated 198 detoxification genes potentially involved in plant host manipulation, including cytochrome P450 (66 CYPs), carboxylesterase (20 CCEs), glutathione-S-transferase (10 GSTs), uridine diphosphate-glycosyltransferase (35 UGTs) and ABC transporter (67 ABCs) families. Transcriptomic expression patterns of these detoxification genes were analyzed for root and leaf galls. In addition to these transcriptomic analyses, we reanalyzed recent data from L1 and L2-3 stages feeding on tolerant and resistant rootstock. Data from two agricultural pest aphids, the generalist Myzus persicae and the Fabaceae specialist Acyrthosiphon pisum, and from the true bug vector of Chagas disease, Rhodnius prolixus, were used to perform phylogenetic analyses for each detoxification gene family. We found expansions of several gene sub-families in the genome of D. vitifoliae. Phylogenetically close genes were found to be organized in clusters in the same genomic position and orientation suggesting recent successive duplications. These results highlight the roles of the phylloxera detoxification gene repertoire in insect physiology and in adaptation to plant secondary metabolites, and provide gene candidates for further functional analyses.

Transcriptional regulation of xenobiotic detoxification genes in insects - An overview.

Arthropods have well adapted to the vast array of chemicals they encounter in their environment. Whether these xenobiotics are plant allelochemicals or anthropogenic insecticides one of the strategies they have developed to defend themselves is the induction of detoxification enzymes. Although upregulation of detoxification enzymes and efflux transporters in response to specific inducers has been well described, in insects, yet, little is known on the transcriptional regulation of these genes. Over the past twenty years, an increasing number of studies with insects have used advanced genetic tools such as RNAi, CRISPR/Cas9 and reporter gene assays to dissect the genomic grounds of their xenobiotic response and hence contributed substantially in improving our knowledge on the players involved. Xenobiotics are partly recognized by various "xenobiotic sensors" such as membrane-bound or nuclear receptors. This initiates a molecular reaction cascade ultimately leading to the translocation of a transcription factor to the nucleus that recognizes and binds to short sequences located upstream their target genes to activate transcription. To date, a number of signaling pathways were shown to mediate the upregulation of detoxification enzymes in arthropods and to play a role in either metabolic resistance to insecticides or host-plant adaptation. These include nuclear receptors AhR/ARNT and HR96, GPCRs, CncC and MAPK/CREB. Recent work reveals that upregulation and activation of some components of these pathways as well as polymorphism in the binding motifs of transcription factors are linked to insects' adaptive processes. The aim of this mini-review is to summarize and describe recent work that shed some light on the main regulatory routes of detoxification gene expression in insects.

Chromosomal scale assembly of parasitic wasp genome reveals symbiotic virus colonization.

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

Perceptions of Tailored Dietary Advice to Improve the Nutrient Adequacy of the Diet in French Pregnant Women.

Tailored dietary counseling could be specifically efficient during pregnancy, a period accompanied by a rise in nutrition awareness, but little is known about the expectations of pregnant women in this regard. We studied these expectations regarding tailored dietary advice in French women during their pregnancy, as well as their motivations and the perceived barriers and enablers. In French pregnant women, we evaluated the perceptions of tailored dietary advice provided by stepwise dietary counseling based on three types of dietary changes, consisting of: (1) a modification of the amounts consumed, (2) substitutions within the food subgroups, and (3) substitutions between food subgroups. A sequential explanatory mixed-method approach was designed. Using qualitative data from a focus group study (n = 40), we intended to explore in depth the women's expectations regarding dietary advice and adherence to a tailored approach. These were combined with quantitative and qualitative data from a 6-week online longitudinal study (n = 115), using questionnaires designed to assess the modifications of dietary habits during pregnancy and to evaluate each type of dietary change. Both studies confirmed that most women in our samples did indeed intend to institute changes regarding healthier dietary practices during pregnancy. The principal motivation behind changes to their habits was to ensure the health and well-being of both their babies and themselves. The proposal of dietary advice that is tailored to both the current diet and the specific needs of pregnant women, but that is also positive and credible, was perceived as enabling implementing healthier dietary practices during pregnancy. Regarding the implementation of the dietary changes proposed, the enablers and barriers identified differed between modifications of the amounts consumed and substitutions. The women displayed interest in all types of dietary changes. This gave relevance to combining different types of changes in order to propose dietary counseling during pregnancy. Tailored dietary counseling was identified by French pregnant women in our samples as enabling them to adopt a healthier diet. However, perceived barriers might limit the implementation of dietary changes, especially when they involved marked modifications to their usual diet.

A Novel Insecticidal Molecule Extracted from Alpinia galanga with Potential to Control the Pest Insect Spodoptera frugiperda.

Spodoptera frugiperda, a highly polyphagous insect pest from America, has recently invaded and widely spread throughout Africa and Asia. Effective and environmentally safe tools are needed for successful pest management of this invasive species. Natural molecules extracted from plants offer this possibility. Our study aimed to determine the insecticidal efficacy of a new molecule extracted from Alpinia galanga rhizome, the 1'S-1'-acetoxychavicol acetate (ACA). The toxicity of ACA was assessed by topical application on early third-instar larvae of S. frugiperda. Results showed that ACA caused significant larval growth inhibition and larval developmental abnormalities. In order to further explore the effects of this molecule, experiments have been performed at the cellular level using Sf9 model cells. ACA exhibited higher toxicity on Sf9 cells as compared to azadirachtin and was 38-fold less toxic on HepG2 cells. Inhibition of cell proliferation was observed at sublethal concentrations of ACA and was associated with cellular morphological changes and nuclear condensation. In addition, ACA induced caspase-3 activity. RT-qPCR experiments reveal that ACA induces the expression of several caspase genes. This first study on the effects of ACA on S. frugiperda larvae and cells provides evidence that ACA may have potential as a botanical insecticide for the control of S. frugiperda.

Correction to: The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

An amendment to this paper has been published and can be accessed via the original article.

The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

BACKGROUND: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture. RESULTS: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world. CONCLUSIONS: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges.

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Resistance evolution in Drosophila: the case of CYP6G1.

The massive use of DDT as an insecticide between 1940 and 1970 has resulted in the emergence of a resistant population of insects. One of the main metabolic mechanisms developed by resistant insects involves detoxification enzymes such as cytochrome P450s. These enzymes can metabolise the insecticide to render it less toxic and facilitate its elimination from the organism. The P450 Cyp6g1 was identified as the major factor responsible for DDT resistance in Drosophila melanogaster field populations. In this article, we review the data available for this gene since it was associated with resistance in 2002. The knowledge gained on Cyp6g1 allows a better understanding of the evolution of insecticide resistance mechanisms and highlights the major role of transposable elements in evolutionary processes. © 2016 Society of Chemical Industry.

Concerns, attitudes, beliefs and information seeking practices with respect to nutrition-related issues: a qualitative study in French pregnant women.

BACKGROUND: From a life course perspective, pregnancy leads to a rise in nutrition awareness and an increase in information flow in favour of adopting healthier eating behaviours. This qualitative study was designed to better understand the determinants of eating behaviours in French pregnant women by focusing on their concerns, attitudes and beliefs and their nutrition-related information seeking practices. METHODS: Seven focus groups were conducted, involving a total of 40 French pregnant women. An inductive thematic approach, adapted from the grounded theory, was adopted to analyse the data. Two major themes were identified: eating behaviour and nutrition-related information behaviour. RESULTS: The eating behaviour theme was divided into four sub-themes using the attribution theory. Three external causes affected the eating behaviour of pregnant women (food restrictions, physiological changes and weight gain), and led to frustration and a perceived loss of control. By contrast the adoption of a healthier diet was perceived as internal by pregnant women, and resulted in self-fulfilment and empowerment regarding the health and the well-being of their baby and themselves, and their weight gain management. Greater attention was paid to nutrition-related information obtained from healthcare providers, the social environment and the mass media. Information was passively absorbed or actively sought by pregnant women, but most was perceived as contradictory, which led to confusion. CONCLUSION: Pregnancy is accompanied by a rise in nutrition awareness, substantiated by eating behaviour modifications due to external and internal causes. However, conflicts between and within information sources result in confusion that can limit the adoption of healthier eating behaviour.

Development and Validation of a Fully Automated Platform for Extended Blood Group Genotyping.

Thirty-five blood group systems, containing >300 antigens, are listed by the International Society of Blood Transfusion. Most of these antigens result from a single nucleotide polymorphism. Blood group typing is conventionally performed by serology. However, this technique has some limitations and cannot respond to the growing demand of blood products typed for a large number of antigens. The knowledge of the molecular basis of these red blood cell systems allowed the implementation of molecular biology methods in immunohematology laboratories. Here, we describe a blood group genotyping assay based on the use of TKL immobilization support and microarray-based HIFI technology that takes approximately 4 hours and 30 minutes from whole-blood samples to results analysis. Targets amplified by multiplex PCR were hybridized on the chip, and a revelation step allowed the simultaneous identification of up to 24 blood group antigens, leading to the determination of extended genotypes. Two panels of multiplex PCR were developed: Panel 1 (KEL1/2, KEL3/4; JK1/2; FY1/2; MNS1/2, MNS3/4, FY*Fy et FY*X) and Panel 2 (YT1/2; CO1/2; DO1/2, HY+, Jo(a+); LU1/2; DI1/2). We present the results of the evaluation of our platform on a panel of 583 and 190 blood donor samples for Panel 1 and 2, respectively. Good correlations (99% to 100%) with reference were obtained.