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.

Cross effects of heat stress and three insecticides on the survival of the codling moth Cydia pomonella (L.): Investigating the molecular and biochemical mechanisms.

As temperature is expected to strongly increase in the future, understanding temperature-mediated toxicity of insecticides is determinant to assess pest management efficiency in a warming world. Investigating molecular and biochemical mechanisms associated with cross mechanisms of temperature and insecticides on pests' tolerance would also be useful in this context. This study aimed to investigate cross effects between temperature and insecticides on the survival of a major pest, the codling moth Cydia pomonella, and their underlying mechanisms. The effect of three insecticidal active ingredients, i.e. chlorantraniliprole, emamectin and spinosad, was assessed at different temperatures on: (i) C. pomonella larval survival; (ii) detoxification enzymes activities (cytochrome P450 multi-function oxygenases, carboxylesterases and glutathione S-transferases) and (iii) genes expression of some detoxification enzymes, heat shock proteins and receptors targeted by the insecticides. We observed a decreased efficiency of emamectin and spinosad at high temperature to control the codling moth while no influence of temperature on chlorantraniliprole efficacy was observed. Detoxification enzymes activities were improved by heat stress alone but not by double stress (temperature + insecticides). Moreover, two detoxification genes (Cyp9A61 and Gst1) were over-expressed by a single stress but not by two stresses while Hsp70 and Cyp6B2 genes may be involved in tolerance to two stresses in C. pomonella. These results confirmed the cross effects of temperature and insecticides on C. pomonella for emamectin and spinosad and provided clues to understand how temperature affects the susceptibility of C. pomonella to insecticides. They illustrate however the complexity of molecular and biochemical responses of individuals facing multiple stresses.

Exposure to a sublethal dose of imidacloprid induces cellular and physiological changes in Periplaneta americana: Involvement of α2 nicotinic acetylcholine subunit in imidacloprid sensitivity.

Neonicotinoids are the most important class of insecticides used as pest management tools during several decades. Exposition of insect to sublethal dose of insecticide induces physiological and cellular changes that could contribute to the adaptation of the insects in order to loss their sensitivity to insecticides. The aim of our study is to demonstrate that a subchronic exposure to a sublethal dose of a neonicotinoid imidacloprid is sufficient to induce molecular changes leading to a loss of imidacloprid sensitivity. We report that in the cockroach, Periplaneta americana, subchronic exposure to a sublethal dose of imidacloprid induced weak changes in detoxification enzyme activity and a significant decrease of the nicotinic acetylcholine α2 mRNA. This molecular effect is correlated to a decrease of imidacloprid sensitivity of cockroaches. Using RNA interference, we shown the key role of nicotinic acetylcholine α2 subunit in imidacloprid sensitivity. Thus, quantitative changes in insecticide targets lead to decreased sensitivity to insecticides. This parameter needs to be considered in order to develop sustainable insect resistance management strategies.

How varying parameters impact insecticide resistance bioassay: An example on the worldwide invasive pest Drosophila suzukii.

Monitoring pesticide resistance is essential for effective and sustainable agricultural practices. Bioassays are the basis for pesticide-resistance testing, but devising a reliable and reproducible method can be challenging because these tests are carried out on living organisms. Here, we investigated five critical parameters and how they affected the evaluation of resistance to the organophosphate phosmet or the pyrethroid lambda-cyhalothrin using a tarsal-contact protocol on Drosophila suzukii, a worldwide invasive pest. Three of the parameters were related to insect biology: (i) sex, (ii) age of the imago (adult stage) and (iii) genetic diversity of the tested population. The two remaining parameters were linked to the experimental setup: (iv) the number of individuals tested per dose and (v) the duration of exposure to the active ingredient. Results showed that response to insecticide differed depending on sex, males being twice as susceptible to phosmet as females. Age principally affected young females' susceptibility to phosmet, because 0-24 hour-old flies were twice as susceptible as 24-48 hour-old and 72-96 hour-old females. Genetic diversity had no observable effect on resistance levels. The precision and accuracy of the median lethal dose (LD50) were greatly affected by the number of individuals tested per dose with a threshold effect. Finally, optimal duration of exposure to the active ingredient was 24 h, as we found an underestimation of mortality when assessed between 1 and 5 h after exposure to lambda-cyhalothrin. None of the main known point mutations on the para sodium channel gene associated with a knockdown effect were observed. Our study demonstrates the importance of calibrating the various parameters of a bioassay to develop a reliable method. It also provides a valuable and transferable protocol for monitoring D. suzukii resistance worldwide.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

Importance of the Host Phenotype on the Preservation of the Genetic Diversity in Codling Moth Granulovirus.

To test the importance of the host genotype in maintaining virus genetic diversity, five experimental populations were constructed by mixing two Cydiapomonella granulovirus isolates, the Mexican isolate CpGV-M and the CpGV-R5, in ratios of 99% M + 1% R, 95% M + 5% R, 90% M + 10% R, 50% M + 50% R, and 10% M + 90% R. CpGV-M and CpGV-R5 differ in their ability to replicate in codling moth larvae carrying the type I resistance. This ability is associated with a genetic marker located in the virus pe38 gene. Six successive cycles of replication were carried out with each virus population on a fully-permissive codling moth colony (CpNPP), as well as on a host colony (RGV) that carries the type I resistance, and thus blocks CpGV-M replication. The infectivity of offspring viruses was tested on both hosts. Replication on the CpNPP leads to virus lineages preserving the pe38 markers characteristic of both isolates, while replication on the RGV colony drastically reduces the frequency of the CpGV-M pe38 marker. Virus progeny obtained after replication on CpNPP show consistently higher pathogenicity than that of progeny viruses obtained by replication on RGV, independently of the host used for testing.

Metabolic mechanisms and acetylcholinesterase sensitivity involved in tolerance to chlorpyrifos-ethyl in the earwig Forficula auricularia.

Apple orchards are highly treated crops, in which organophosphorus (OP) are among the most heavily sprayed insecticides. These pesticides are toxic to non-target arthropods and their repeated use increases the risk of resistance. We studied mechanisms involved in tolerance and resistance to OP insecticides in the earwig Forficula auricularia, an effective generalist predator in pomefruit orchards. Adult earwigs were sampled in three apple orchards managed under contrasting strategies: conventional, Integrated Pest Management, and organic. The threshold activities of enzyme families involved in pesticides tolerance: Glutathione-S-transferases (GSTs) and Carboxylesterases (CbEs) were measured in earwig extracts. Acetylcholinesterase (AChE) was monitored as a toxicological endpoint. Variations in these activities were assessed prior to and after exposure to chlorpyrifos-ethyl at the normal application rate. We observed that the mortality of earwigs exposed to chlorpyrifos-ethyl depended on the management strategy of orchards. Significantly lower mortality was seen in individuals sampled from conventional orchard. The basal activities of CbEs and GSTs of collected organisms were higher in conventional orchard. After in vivo exposure, AChE activity appeared to be inhibited in surviving males with no difference between orchards. However an in vitro inhibition trial with chlorpyrifos-oxon showed that AChE from earwigs collected in organic and IPM orchards were more sensitive than from conventional ones. These observations support the hypothesis of a molecular target modification in AChE and highlight the possible role of CbEs in effective protection of AChE. Our findings suggest that the earwigs with a high historic level of insecticide exposure could acquire resistance to chlorpyrifos-ethyl.

Microbial Pest Control Agents: Are they a Specific And Safe Tool for Insect Pest Management?

Microorganisms (viruses, bacteria and fungi) or their bioactive agents can be used as active substances and therefore are referred as Microbial Pest Control Agents (MPCA). They are used as alternative strategies to chemical insecticides to counteract the development of resistances and to reduce adverse effects on both environment and human health. These natural entomopathogenic agents, which have specific modes of action, are generally considered safer as compared to conventional chemical insecticides. Baculoviruses are the only viruses being used as the safest biological control agents. They infect insects and have narrow host ranges. Bacillus thuringiensis (Bt) is the most widely and successfully used bioinsecticide in the integrated pest management programs in the world. Bt mainly produces crystal delta-endotoxins and secreted toxins. However, the Bt toxins are not stable for a very long time and are highly sensitive to solar UV. So genetically modified plants that express toxins have been developed and represent a large part of the phytosanitary biological products. Finally, entomopathogenic fungi and particularly, Beauveria bassiana and Metarhizium anisopliae, are also used for their insecticidal properties. Most studies on various aspects of the safety of MPCA to human, non-target organisms and environment have only reported acute but not chronic toxicity. This paper reviews the modes of action of MPCA, their toxicological risks to human health and ecotoxicological profiles together with their environmental persistence. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity".

Biochemical and Molecular Mechanisms Associated With the Resistance of the European Corn Borer (Lepidoptera: Crambidae) to Lambda-Cyhalothrin and First Monitoring Tool.

The European corn borer (Ostrinia nubilalis (Hübner)) is one of the most serious corn pest in Europe where it is controlled with pesticides, in particular, pyrethroids. First control failures with this chemical family occurred on the field in 2008 in the center of France, and the first resistance case was described in 2012. In the present study, we investigate resistance mechanisms involved in seven French populations of O. nubilalis collected in the field. Resistances to deltamethrin and lambda-cyhalothrin were confirmed, with a higher resistance ratio for lambda-cyhalothrin (63.79 compared to 7.67). Resistance to the two active compounds was correlated except for one population, indicating a high probability of cross-resistance. Analyses of the activity of three major families of detoxification enzymes in resistant individuals showed a significant increase of the average MFO activity in males of four populations (activity ratios of 2.76-5.73) and higher GST activity in females of two other populations (activity ratios 4.48 and 5.21). Molecular investigation of the sodium channel gene sequence showed the presence of the kdr mutation in a highly resistant individual. We designed a PCR-RFLP screening tool to search for this mutation in the field, and we found it in five populations but not in the susceptible one. The resistance of O. nubilalis to pyrethroids in France seems to result from a combination of resistance mechanisms, possibly as a consequence of a selection pressure with an exceptional duration (almost 40 yr old).

Biological Characteristics of Experimental Genotype Mixtures of Cydia Pomonella Granulovirus (CpGV): Ability to Control Susceptible and Resistant Pest Populations.

The detection of resistance in codling moth (Cydia pomonella) populations against the Mexican isolate of its granulovirus (CpGV-M), raised questions on the sustainability of the use of this biological insecticide. In resistant host cells, CpGV-M is not able to complete its replication cycle because replication is blocked at an early step. Virus isolates able to overcome this resistance have been characterized-among them, the CpGV-R5 isolate. In mixed infections on resistant insects, both CpGV-M and CpGV-R5 viruses replicate, while CpGV-M alone does not induce mortality. Genetically heterogeneous virus populations, containing 50% of each CpGV-M and CpGV-R5 appear to control resistant host populations as well as CpGV-R5 alone at the same final concentration, even if the concentration of CpGV-R5 is only half in the former. The use of mixed genotype virus preparations instead of genotypically homogeneous populations may constitute a better approach than traditional methods for the development of baculovirus-based biological insecticides.

Differentiating Oriental Fruit Moth and Codling Moth (Lepidoptera: Tortricidae) Larvae Using Near-Infrared Spectroscopy.

Cydia pomonella (L.) and Cydia molesta (Busck) (Lepidoptera: Tortricidae) are two important lepidopteran pests that may co-occur in apple orchards and are difficult to differentiate in the larval stage. We investigate the possibility of using near-infrared spectroscopy (NIRS) coupled with partial least squares analysis to distinguish the larvae of the two species. We further assess whether wild individuals can be differentiated using laboratory strains of the two species for model calibration. The NIRS spectra of C. molesta and C. pomonella differed most in the wavelengths between 1,142 and 1,338 nm. Using these wavelengths, partial least squares analysis allowed the differentiation of C. molesta and C. pomonella at the larval stage with very low error, but only as long as both the calibration and prediction sets for individuals had the same origin (either both from the laboratory or both from the field). Errors that appeared when using laboratory individuals for calibration were owing to the divergence of the C. pomonella laboratory strain, most likely following evolution during rearing. Thus, NIRS appears to be a promising tool for the easy and rapid identification of individuals in the field, provided that it is calibrated based on a subset of field individuals.

Is the efficacy of biological control against plant diseases likely to be more durable than that of chemical pesticides?

The durability of a control method for plant protection is defined as the persistence of its efficacy in space and time. It depends on (i) the selection pressure exerted by it on populations of plant pathogens and (ii) on the capacity of these pathogens to adapt to the control method. Erosion of effectiveness of conventional plant protection methods has been widely studied in the past. For example, apparition of resistance to chemical pesticides in plant pathogens or pests has been extensively documented. The durability of biological control has often been assumed to be higher than that of chemical control. Results concerning pest management in agricultural systems have shown that this assumption may not always be justified. Resistance of various pests to one or several toxins of Bacillus thuringiensis and apparition of resistance of the codling moth Cydia pomonella to the C. pomonella granulovirus have, for example, been described. In contrast with the situation for pests, the durability of biological control of plant diseases has hardly been studied and no scientific reports proving the loss of efficiency of biological control agents against plant pathogens in practice has been published so far. Knowledge concerning the possible erosion of effectiveness of biological control is essential to ensure a durable efficacy of biological control agents on target plant pathogens. This knowledge will result in identifying risk factors that can foster the selection of strains of plant pathogens resistant to biological control agents. It will also result in identifying types of biological control agents with lower risk of efficacy loss, i.e., modes of action of biological control agents that does not favor the selection of resistant isolates in natural populations of plant pathogens. An analysis of the scientific literature was then conducted to assess the potential for plant pathogens to become resistant to biological control agents.

Resistance to bio-insecticides or how to enhance their sustainability: a review.

After more than 70 years of chemical pesticide use, modern agriculture is increasingly using biological control products. Resistances to conventional insecticides are wide spread, while those to bio-insecticides have raised less attention, and resistance management is frequently neglected. However, a good knowledge of the limitations of a new technique often provides greater sustainability. In this review, we compile cases of resistance to widely used bio-insecticides and describe the associated resistance mechanisms. This overview shows that all widely used bio-insecticides ultimately select resistant individuals. For example, at least 27 species of insects have been described as resistant to Bacillus thuringiensis toxins. The resistance mechanisms are at least as diverse as those that are involved in resistance to chemical insecticides, some of them being common to bio-insecticides and chemical insecticides. This analysis highlights the specific properties of bio-insecticides that the scientific community should use to provide a better sustainability of these products.

Progressive adaptation of a CpGV isolate to codling moth populations resistant to CpGV-M.

The NPP-R1 isolate of CpGV is able to replicate on CpGV-M-resistant codling moths. However, its efficacy is not sufficient to provide acceptable levels of control in natural (orchard) conditions. A laboratory colony derived from resistant codling moths was established, which exhibited a homogeneous genetic background and a resistance level more than 7000 fold. By successive cycles of replication of NPP-R1 in this colony, we observed a progressive increase in efficacy. After 16 cycles (isolate 2016-r16), the efficacy of the virus isolate was equivalent to that of CpGV-M on susceptible insects. This isolate was able to control both CpGV-M-susceptible and CpGV-M-resistant insects with similar efficacy. No reduction in the levels of occlusion body production in susceptible larvae was observed for 2016-r16 compared to CpGV-M.

A single sex-linked dominant gene does not fully explain the codling moth's resistance to granulovirus.

BACKGROUND: In 2004, resistance to a commercial formulation of the Cydia pomonella granulovirus (CpGV) was identified in a field population of Cydia pomonella from an organic orchard in southern France. The genetic inheritance of this resistance was analysed in the resistant laboratory strain RGV. This strain was obtained using successive crosses between the resistant field population and a susceptible laboratory strain, SV, with selection for CpGV resistance at each generation. RESULTS: After eight generations of introgression of the resistant trait into SV, the RGV-8 strain exhibited 7000-fold higher resistance than SV. Mass-crossing experiments showed that resistance to CpGV is strongly dominant, sex dependent and under the control of a single major gene. However, the contribution of other genes is required to explain all of the data obtained in this study. These additional genes do not follow the laws of classical Mendelian transmission. CONCLUSION: Transmission of granulovirus resistance in the RGV-8 strain of C. pomonella cannot be fully explained by the effect of a locus located on the Z chromosome. The action of other factors needs to be considered.

Multiple origins of the sodium channel kdr mutations in codling moth populations.

Resistance to insecticides is one interesting example of a rapid current evolutionary change. DNA variability in the voltage-gated sodium channel gene (trans-membrane segments 5 and 6 in domain II) was investigated in order to estimate resistance evolution to pyrethroid in codling moth populations at the World level. DNA variation among 38 sequences revealed a unique kdr mutation (L1014F) involved in pyrethroid resistance in this gene region, which likely resulted from several convergent substitutions. The analysis of codling moth samples from 52 apple orchards in 19 countries using a simple PCR-RFLP confirmed that this kdr mutation is almost worldwide distributed. The proportions of kdr mutation were negatively correlated with the annual temperatures in the sampled regions. Homozygous kdr genotypes in the French apple orchards showed lower P450 cytochrome oxidase activities than other genotypes. The most plausible interpretation of the geographic distribution of kdr in codling moth populations is that it has both multiple independent origins and a spreading limited by low temperature and negative interaction with the presence of alternative resistance mechanisms to pyrethroid in the populations.