Performance of Sesamia nonagrioides on cultivated and wild host plants: Implications for Bt maize resistance management.

BACKGROUND: Sesamia nonagrioides is an important maize pest in the Mediterranean basin that is effectively controlled by Cry1Ab-expressing maize (Bt maize). The continued cultivation of Bt maize in Spain exerts high selection pressure on the target pests, which could lead to the development of resistance. Provision of refuges of non-Bt plants is an essential component in the high-dose/refuge (HDR) strategy to delay resistance evolution. Here we analyze the suitability of cultivated (rice and sorghum) and wild (Johnsongrass, cattail, common reed and giant reed) plants, reported as hosts of S. nonagrioides, for larval development and oviposition of this pest compared to maize, and we evaluate their potential role in delaying resistance development to Bt maize. RESULTS: Bioassays conducted with plant pieces or whole plants showed that the larval cycle could only be completed in the three cultivated plants and in Johnsongrass. Females showed a strong preference for ovipositing on maize in comparison with sorghum or rice. Although young larvae consumed more sorghum than maize in two-choice bioassays, both larvae and adults had a better performance (shorter larval period and higher pupal weight, fecundity and fertility) when larvae fed on maize throughout their larval stage than when they fed on sorghum or rice. CONCLUSION: None of the alternative hosts of S. nonagrioides tested here should be considered as natural unstructured refuges within the HDR strategy for Bt maize and this pest in Spain, as some of the necessary requirements to fulfill this strategy would not be met. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Multiple mutations in the nicotinic acetylcholine receptor Ccα6 gene associated with resistance to spinosad in medfly.

Spinosad is an insecticide widely used for the control of insect pest species, including Mediterranean fruit fly, Ceratitis capitata. Its target site is the α6 subunit of the nicotinic acetylcholine receptors, and different mutations in this subunit confer resistance to spinosad in diverse insect species. The insect α6 gene contains 12 exons, with mutually exclusive versions of exons 3 (3a, 3b) and 8 (8a, 8b, 8c). We report here the selection of a medfly strain highly resistant to spinosad, JW-100 s, and we identify three recessive Ccα6 mutant alleles in the JW-100 s population: (i) Ccα63aQ68* containing a point mutation that generates a premature stop codon on exon 3a (3aQ68*); (ii) Ccα63aAG>AT containing a point mutation in the 5' splicing site of exon 3a (3aAG > AT); and (iii) Ccα63aQ68*-K352* that contains the mutation 3aQ68* and another point mutation on exon 10 (K352*). Though our analysis of the susceptibility to spinosad in field populations indicates that resistance has not yet evolved, a better understanding of the mechanism of action of spinosad is essential to implement sustainable management practices to avoid the development of resistance in field populations.

Plant-Mediated Effects of Water Deficit on the Performance of Tetranychus evansi on Tomato Drought-Adapted Accessions.

Climate change is expected to increase drought periods and the performance and dispersal of some invasive species such as Tetranychus evansi, which has been reported to take advantage of the nutritional changes induced by water-shortage on the tomato cultivar Moneymaker (MM). We have examined the implications for mite's biology of four accessions of the drought-adapted tomatoes, "Tomàtiga de Ramellet" (TR), under moderate drought stress. Mite performance was enhanced by drought in two accessions (TR61 and TR154), but not in the other two accessions (TR58 and TR126). We selected one accession of each outcome (i.e., TR154 and TR126) to further analyze plant nutritional parameters. We found that free sugars and most essential amino acids for mites were induced by drought and/or mite infestation on MM and TR154 plants, whereas sugars were not altered and a reduced number of essential amino acids were induced by drought in TR126. Remarkably, mite performance was enhanced by leaf infiltration of free sugars, essential amino acids mixture, and L-proline on well-watered MM and by free sugars on drought-stressed TR126 plants. These results indicate a positive link between the induction of soluble carbohydrates and amino acids used by the plant for osmotic adjustment and mite performance. The effects of drought and/or mite infestation on the defense response of plants was analyzed at three levels: phytohormone accumulation, the transcript levels of marker genes linked to jasmonates (JAs), salicylic acid (SA), and abscisic acid (ABA) pathways, and the activity of defense proteins. The ability of T. evansi to downregulate the accumulation of defense-related phytohormones was noted on MM and the two TR accessions analyzed (TR126 and TR154), though differences in the induction of protein defense genes and activities by drought and/or mite infestation were observed among them. These results emphasize the importance of studying plant biotic and abiotic stress factors in combination and provides an experimental framework for screening drought-tolerant tomato accessions that will be also resistant to herbivore mites.

First detection of a Sesamia nonagrioides resistance allele to Bt maize in Europe.

The Ebro Valley (Spain) is the only hotspot area in Europe where resistance evolution of target pests to Cry1Ab protein is most likely, owing to the high and regular adoption of Bt maize (>60%). The high-dose/refuge (HDR) strategy was implemented to delay resistance evolution, and to be effective it requires the frequency of resistance alleles to be very low (<0.001). An F2 screen was performed in 2016 to estimate the frequency of resistance alleles in Sesamia nonagrioides from this area and to evaluate if the HDR strategy is still working effectively. Out of the 137 isofemale lines screened on Cry1Ab maize leaf tissue, molted larvae and extensive feeding were observed for two consecutive generations in one line, indicating this line carried a resistance allele. The frequency of resistance alleles in 2016 was 0.0036 (CI 95% 0.0004-0.0100), higher but not statistically different from the value obtained in 2004-2005. Resistance does not seem to be evolving faster than predicted by a S. nonagrioides resistance evolution model, but the frequency of resistance is now triple the value recommended for an effective implementation of the HDR strategy. Owing to this, complementary measures should be considered to further delay resistance evolution in the Ebro Valley.

Weeds and ground-dwelling predators' response to two different weed management systems in glyphosate-tolerant cotton: A farm-scale study.

The use of glyphosate, as a post-emergence broad-spectrum herbicide in genetically modified glyphosate-tolerant (GT) cotton, supposes a big change in weed management programs with respect to a conventional regime. Thus, alterations in arable flora and arthropod fauna must be considered when evaluating their potential impacts. A 3-year farm-scale study was conducted in a 2-ha GT cotton crop, in southern Spain, to compare the effects of conventional and glyphosate herbicide regimes on weed abundance and diversity and their consequences for ground-dwelling predators. Surveys reveal that weed density was relatively low within all treatments with a few dominant species, with significantly higher weed densities and modifications of the floristic composition in glyphosate-treated plots that led to an increase in the abundance of Portulaca oleracea and to a reduction in plant diversity. The activity-density of the main predatory arthropod taxa (spiders, ground beetles, rove beetles and earwigs) varied among years, but no significant differences were obtained between conventional and glyphosate herbicide regimes. However, significant differences between treatments were obtained for ground beetles species richness and diversity, being higher under the glyphosate herbicide regime, and a positive correlation with weed density could be established for both parameters. The implications of these findings to weed control in GT cotton are discussed.

Monitoring of Sesamia nonagrioides resistance to MON 810 maize in the European Union: lessons from a long-term harmonized plan.

BACKGROUND: Use of MON 810 maize (Zea mays), which expresses the insecticidal protein Cry1Ab from Bacillus thuringiensis (Bt maize), is a highly effective method to control Sesamia nonagrioides (Lefèbvre), a key maize pest in Mediterranean countries. Monitoring programs to assess the potential development of resistance of target pests to Bt maize are mandatory in the European Union (EU). Here we report the results of the S. nonagrioides resistance monitoring plan implemented for MON 810 maize in the EU between 2004 and 2015 and reassess the different components of this long-term harmonized plan. RESULTS: No major shifts in the susceptibility of S. nonagrioides to the Cry1Ab protein have occurred over time. The reassessment of this long-term program has identified some practical and technical constraints, allowing us to provide specific recommendations for improvement: use reference strains instead of susceptibility baselines as comparators for field-collected populations; shift from dose-response bioassays to diagnostic concentrations; and focus monitoring on areas with high adoption rates, such as the Ebro basin in Spain. CONCLUSION: There are no signs of field resistance of S. nonagrioides to the Cry1Ab protein of MON 810 maize. Specific recommendations for improvement are provided, based on the knowledge and experience accumulated through the implementation of this unique EU-wide harmonized plan. © 2017 Society of Chemical Industry.

The impact of restrictions on neonicotinoid and fipronil insecticides on pest management in maize, oilseed rape and sunflower in eight European Union regions.

BACKGROUND: In 2013, the European Commission restricted the use of three neonicotinoids (clothianidin, imidacloprid and thiamethoxam) and the pyrazole fipronil, which are widely used to control early-season pests. Here, we used original farm survey data to examine the impact of the restrictions on pest management practices in eight regional case studies including maize, oilseed rape and sunflower in seven European Union (EU) countries. RESULTS: In four case studies, farmers switched to using untreated seeds as no alternative seed treatments were available. In three case studies, farmers switched to using unrestricted neonicotinoid- or pyrethroid-treated seeds. In five case studies, farmers increased the use of soil or foliar treatments, with pyrethroids as the principal insecticide class. Other changes in pest management practices ranged from increased sowing density to more frequent scouting for pests. Many farmers perceived that the time, cost and amount of insecticides required to protect crops increased, along with pest pressure. Alternative seed treatments were mostly perceived as being less effective than the restricted seed treatments. CONCLUSION: Farmers generally relied on alternative seed treatments or more soil/foliar treatments in the first growing season after the restrictions took effect. Further study is required to assess the effectiveness and sustainability of these alternatives compared with the restricted insecticides. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Farm-scale evaluation of the impact of Cry1Ab Bt maize on canopy nontarget arthropods: a 3-year study.

The cultivation of Cry1Ab-expressing genetically modified MON810 (Bt maize) has led to public concern in Europe, regarding its impact on nontarget arthropods (NTAs). We have assessed the potential effects of DKC 6451 YG (MON810) maize on canopy NTAs in a farm-scale study performed in Central Spain during 3 years. The study focused on hemipteran herbivores (leafhoppers and planthoppers) and hymenopteran parasitic wasps (mymarids) collected by yellow sticky traps, which accounted for 72% of the total number of insects studied. The dynamics and abundance of these groups varied among years, but no significant differences were found between Bt and non-Bt maize, indicating that Bt maize had no negative effect on these taxa. Nonetheless, the Cry1Ab toxin was detected in 2 different arthropods collected from Bt maize foliage, the cicadellids Zyginidia scutellaris and Empoasca spp. A retrospective power analysis on the arthropod abundance data for our field trials has determined that Z. scutellaris and the family Mymaridae have high capacity to detect differences between the Bt maize and its isogenic counterpart. The use of these canopy NTAs as surrogates for assessing environmental impacts of Bt maize is discussed.

Drought stress promotes the colonization success of a herbivorous mite that manipulates plant defenses.

Climate change is expected to bring longer periods of drought and this may affect the plant's ability to resist pests. We assessed if water deficit affects the tomato russet mite (TRM; Aculops lycopersici), a key tomato-pest. TRM thrives on tomato by suppressing the plant's jamonate defenses while these defenses typically are modulated by drought stress. We observed that the TRM population grows faster and causes more damage on drought-stressed plants. To explain this observation we measured several nutrients, phytohormones, defense-gene expression and the activity of defensive proteins in plants with or without drought stress or TRM. TRM increased the levels of total protein and several free amino acids. It also promoted the SA-response and upregulated the accumulation of jasmonates but down-regulated the downstream marker genes while promoting the activity of cysteine-but not serine-protease inhibitors, polyphenol oxidase and of peroxidase (POD). Drought stress, in turn, retained the down regulation of JA-marker genes and reduced the activity of serine protease inhibitors and POD, and altered the levels of some free-amino acids. When combined, drought stress antagonized the accumulation of POD and JA by TRM and synergized accumulation of free sugars and SA. Our data show that drought stress interacts with pest-induced primary and secondary metabolic changes and promotes pest performance.

Comparative analysis of the genetic basis of Cry1F resistance in two strains of Spodoptera frugiperda originated from Puerto Rico and Florida.

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target pest of Bacillus thuringiensis (Bt) maize and cotton in America. Since the commercialization of Cry1F maize (event TC1507) in 2003, resistance to Cry1F maize in field populations of S. frugiperda has occurred in Puerto Rico, Brazil and the southeast region of the United States. In this paper, we conducted a comparative analysis of the inheritance of two Cry1F-resistant colonies of S. frugiperda originated from Puerto Rico (PR) and Florida (FL), respectively. The objective of the analysis was to determine if the genetic basis of the resistance was similar in the two different originated colonies. To accomplish the objective, besides PR, FL, and a known Cry1F-susceptible colony, 14 additional colonies were developed by reciprocal crosses among the three parents, F1 by F1 crosses, backcrosses, and intercolony-crosses between PR and FL. Larval mortalities of the 17 colonies were assayed on both Cry1F maize leaf tissue and Cry1F-treated diet at the concentrations of 3.16, 10.00, and 31.60µg/g. Resistance to Cry1F in both PR and FL was autosomal and recessive or incompletely recessive. Segregations in F2 and backcrossed generations associated with FL fitted the Mendelian monogenic model well, while with PR the segregations did not follow the single gene model in some bioassays. Further analyses with the intercolony complementation tests showed a similar level of resistance in the F1 progeny as their parents FL and PR. Together with the data, it was likely that a single (or a few tightly-linked) gene was involved in FL; PR shared the same locus of the major resistance gene as FL, but the resistance in PR might also be associated with additional minor factors. Information generated from this study should be useful in understanding the origin of Cry1F resistance in the U.S. mainland and developing effective strategies for Bt resistance management in S. frugiperda.

Drought stress in tomato increases the performance of adapted and non-adapted strains of Tetranychus urticae.

The performance of the two-spotted spider mite, Tetranychus urticae Koch, on plants depends on the rate of adaptation of mite populations to each particular host and can be influenced by environmental conditions. We have tested the effects of drought stress, caused by water deficiency, in the interaction of tomato plants with tomato adapted (TA) and tomato non-adapted (TNA) strains of T. urticae. Our data revealed that mite performance was enhanced for the TA strain when reared on drought-stressed tomato plants, rising population growth and leaf damage. Population growth in the case of the TNA strain was negative, but they laid more eggs and the number of mobile forms was higher on drought-stressed tomato plants than on control plants. Water stress resulted in tomato plants with increased concentrations of essential amino acids and free sugars, improving the nutritional value of drought-stressed tomato plants for T. urticae. Mite infestation alone had almost no effect on the nutritional composition of tomato leaves, with the exception of an increase of free sugars. Tomato plant defense proteins were induced by both drought stress and mite infestation. However, the induction of protease inhibitors was higher in tomatoes exposed to mites from the TNA strain than in tomatoes that were fed upon by mites from the TA strain. The better performance of the TA strain could be associated to both changes in the digestive (cysteine and aspartyl protease and α-amylase activities) and detoxification (esterase activity) physiology of the mites and the attenuation of some of the plant defenses (protease inhibitors). Taken together, our results suggest that drought stress might favor outbreaks of T. urticae on tomato, by enhancing population growth of adapted populations and increasing the suitability of tomato as a host for non-adapted ones.

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control. RESULTS: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT. CONCLUSIONS: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.

Correction: Sixteen Years of Bt Maize in the EU Hotspot: Why Has Resistance Not Evolved?

[This corrects the article DOI: 10.1371/journal.pone.0154200.].

Sixteen Years of Bt Maize in the EU Hotspot: Why Has Resistance Not Evolved?

The majority of Bt maize production in the European Union (EU) is concentrated in northeast Spain, which is Europe's only hotspot where resistance might evolve, and the main target pest, Sesamia nonagrioides, has been exposed to Cry1Ab maize continuously since 1998. The cropping system in northeast Spain has some similar characteristics to those that probably led to rapid resistance failures in two other target noctuid maize pests. These include repeated cultivation of Bt maize in the same fields, low use of refuges, recurring exposure of larvae to non-high dose concentrations of Cry1Ab toxin during the first years of cultivation, low migratory potential, and production concentrated in an irrigated region with few alternative hosts. Available data reveal no evidence of resistance in S. nonagrioides after 16 years of use. We explore the possible reasons for this resistance management success using evolutionary models to consider factors expected to accelerate resistance, and those expected to delay resistance. Low initial adoption rates and the EU policy decision to replace Event 176 with MON 810 Bt maize were key to delaying resistance evolution. Model results suggest that if refuge compliance continues at the present 90%, Bt maize might be used sustainably in northeast Spain for at least 20 more years before resistance might occur. However, obtaining good estimates of the present R allele frequency and level of local assortative mating are crucial to reduce uncertainty about the future success of resistance management.

Drought-Stressed Tomato Plants Trigger Bottom-Up Effects on the Invasive Tetranychus evansi.

Climate change will bring more drought periods that will have an impact on the irrigation practices of some crops like tomato, from standard water regime to deficit irrigation. This will promote changes in plant metabolism and alter their interactions with biotic stressors. We have tested if mild or moderate drought-stressed tomato plants (simulating deficit irrigation) have an effect on the biological traits of the invasive tomato red spider mite, Tetranychus evansi. Our data reveal that T evansi caused more leaf damage to drought-stressed tomato plants (≥1.5 fold for both drought scenarios). Mite performance was also enhanced, as revealed by significant increases of eggs laid (≥2 fold) at 4 days post infestation (dpi), and of mobile forms (≥2 fold and 1.5 fold for moderate and mild drought, respectively) at 10 dpi. The levels of several essential amino acids (histidine, isoleucine, leucine, tyrosine, valine) and free sugars in tomato leaves were significantly induced by drought in combination with mites. The non-essential amino acid proline was also strongly induced, stimulating mite feeding and egg laying when added to tomato leaf disks at levels equivalent to that estimated on drought-infested tomato plants at 10 dpi. Tomato plant defense proteins were also affected by drought and/or mite infestation, but T. evansi was capable of circumventing their potential adverse effects. Altogether, our data indicate that significant increases of available free sugars and essential amino acids, jointly with their phagostimulant effect, created a favorable environment for a better T. evansi performance on drought-stressed tomato leaves. Thus, drought-stressed tomato plants, even at mild levels, may be more prone to T evansi outbreaks in a climate change scenario, which might negatively affect tomato production on area-wide scales.

Digestive proteases in bodies and faeces of the two-spotted spider mite, Tetranychus urticae.

Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC-nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.

Inheritance, fitness costs, incomplete resistance and feeding preferences in a laboratory-selected MON810-resistant strain of the true armyworm Mythimna unipuncta.

BACKGROUND: The low efficacy of MON810 maize against Mythimna unipuncta represents a scenario of non-compliance with the 'high-dose' strategy, raising concerns about potential resistance development and outbreaks of this secondary pest. The present study offers insight into the different components related to resistance in a laboratory-selected MON810-resistant (MR) strain of M. unipuncta. RESULTS: The resistance in the MR strain is autosomal and inherited as a partially dominant trait. We have found a lack of fitness costs in this strain for essential life history traits, reproductive potential and most of the population growth parameters analysed, the only exception being an increment in the mean generation time. Larvae of the MR strain reared on Bacillus thuringiensis (Bt) maize took longer to develop, presented a high adult cumulative emergence time and had lower growth rate than those reared on non-Bt maize, suggesting the existence of incomplete resistance. Feeding preference assays reveal a low discrimination between Bt and conventional maize. CONCLUSION: Both resistant and heterozygous larvae of M. unipuncta survive the Cry1Ab toxin expressed on Bt maize, with a weak fitness cost for the homozygous larvae, indicating the potential risk of field-evolved resistance and its relevance to resistance monitoring. © 2015 Society of Chemical Industry.

Resistance to lambda-cyhalothrin in Spanish field populations of Ceratitis capitata and metabolic resistance mediated by P450 in a resistant strain.

BACKGROUND: The withdrawal of malathion in the European Union in 2009 resulted in a large increase in lambda-cyhalothrin applications for the control of the Mediterranean fruit fly, Ceratitis capitata, in Spanish citrus crops. RESULTS: Spanish field populations of C. capitata have developed resistance to lambda-cyhalothrin (6-14-fold), achieving LC50 values (129-287 ppm) higher than the recommended concentration for field treatments (125 ppm). These results contrast with the high susceptibility to lambda-cyhalothrin found in three Tunisian field populations. We have studied the mechanism of resistance in the laboratory-selected resistant strain W-1Kλ (205-fold resistance). Bioassays with synergists showed that resistance was almost completely suppressed by the P450 inhibitor PBO. The study of the expression of 53 P450 genes belonging to the CYP4, CYP6, CYP9 and CYP12 families in C. capitata revealed that CYP6A51 was overexpressed (13-18-fold) in the resistant strain. The W-1Kλ strain also showed high levels of cross-resistance to etofenprox (240-fold) and deltamethrin (150-fold). CONCLUSION: Field-evolved resistance to lambda-cyhalothrin has been found in C. capitata. Metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant strain W-1Kλ. The levels of cross-resistance found may compromise the effectiveness of other pyrethroids for the control of this species. © 2014 Society of Chemical Industry.