Diverse patterns of constitutive and inducible overexpression of detoxifying enzyme genes among resistant Aphis glycines populations.

Understanding the mechanisms of pyrethroid resistance is essential to the effective management of pesticide resistance in Aphis glycines Matsumura (Hemiptera: Aphididae). We mined putative detoxifying enzyme genes in the draft genome sequence of A. glycines for cytochrome oxidase P450 (CYP), glutathione-S-transferase (GST) and esterases (E4 and carboxylesterases-CES). Aphids from clonal populations resistant to pyrethroids from three sites in Minnesota, USA, were screened against a diagnostic LC99 concentration of either λ-cyhalothrin or bifenthrin and detoxifying enzyme genes expression in survivors was analyzed by qPCR. Their expression profiles were compared relative to a susceptible clonal population. We found 61 CYP (40 full-length), seven GST (all full-length), seven E4 (five full-length) and three CES (two full-length) genes, including 24 possible pseudogenes. The detoxifying enzymes had different expression profiles across resistant aphid populations, possibly reflecting differences in the genetic background and pyrethroid selection pressures as the number of constitutively overexpressed detoxifying enzyme genes was correlated with the level of resistance. Our findings will strengthen the understanding of the pyrethroid resistance mechanisms in A. glycines.

Competitive release and outbreaks of non-target pests associated with transgenic Bt cotton.

The adoption of transgenic Bt cotton has, in some cases, led to environmental and economic benefits through reduced insecticide use. However, the distribution of these benefits and associated risks among cotton growers and cotton-growing regions has been uneven due in part to outbreaks of non-target or secondary pests, thereby requiring the continued use of synthetic insecticides. In the southeastern USA, Bt cotton adoption has resulted in increased abundance of and damage from stink bug pests, Euschistus servus and Nezara viridula (Heteroptera: Pentatomidae). While the impact of increased stink bug abundance has been well-documented, the causes have remained unclear. We hypothesize that release from competition with Bt-susceptible target pests may drive stink bug outbreaks in Bt cotton. We first examined the evidence for competitive release of stink bugs through meta-analysis of previous studies. We then experimentally tested if herbivory by Bt-susceptible Helicoverpa zea increases stink bug leaving rates and deters oviposition on non-Bt cotton. Consistent with previous studies, we found differences in leaving rates only for E servus, but we found that both species strongly avoided ovipositing on H. zea-damaged plants. Considering all available evidence, competitive release of stink bug populations in Bt cotton likely contributes to outbreaks, though the relative importance of competitive release remains an open question. Ecological risk assessments of Bt crops and other transgenic insecticidal crops would benefit from greater understanding of the ecological mechanisms underlying non-target pest outbreaks and greater attention to indirect ecological effects more broadly.

Limitations in dose-response and surrogate species methodologies for risk assessment of Cry toxins on arthropod natural enemies.

Dose-response assays and surrogate species are standard methods for risk analysis for environmental chemicals. These assume that individuals within a species have unimodal responses and that a surrogate species can predict responses of other related taxa. We exposed immature individuals of closely related aphidophagous coccinellid predators, Cycloneda sanguinea and Harmonia axyridis, to Cry1Ac and Cry1F toxins through uniform and constant artificial tritrophic exposure through Myzus persicae aphids. Both toxins were detected in coccinellid pupae, with individual and interspecific variation. Uptake was significantly higher in H. axyridis than in C. sanguinea, both in the proportion of individuals and the concentrations per individual. We also observed bimodal uptake of the Cry toxins by H. axyridis, which indicated that some individuals had low bioaccumulation and some had high bioaccumulation. This suggests that standard dose-response assays need to be interpreted with caution and future assays should examine the modality of the responses. In addition, the similarity in the biological effects of the Cry toxins in the two predators was due to different biological exposure mechanisms. The majority of H. axyridis were exposed both internally and in the gut, while C. sanguinea was exposed primarily in the gut. Thus, despite their close phylogenetic relatedness, these species would not be good surrogates for each other and the surrogate species methodology should be tested more rigorously.

Artificial tritrophic exposure system for environmental risk analysis on aphidophagous predators.

We evaluated an artificial tritrophic exposure system for use in ecotoxicological evaluations of environmental stressors on aphidophagous predators. It consists of an acrylic tube with a Parafilm M sachet containing liquid aphid diet, into which can be added environmental stressors. Immature Cycloneda sanguinea, Harmonia axyridis and Chrysoperla externa, and adult H. axyridis were reared on Myzus persicae. Larval and pupal development and survival of all species and reproductive parameters of H. axyridis were similar to published results. The system provides a suitable tritrophic exposure route, enables ex-ante evaluation of stressors, and improves the accuracy of the assessment.

Early Detection and Mitigation of Resistance to Bt Maize by Western Corn Rootworm (Coleoptera: Chrysomelidae).

Transgenic Bt maize that produces less than a high-dose has been widely adopted and presents considerable insect resistance management (IRM) challenges. Western corn rootworm, Diabrotica virgifera virgifera LeConte, has rapidly evolved resistance to Bt maize in the field, leading to local loss of efficacy for some corn rootworm Bt maize events. Documenting and responding to this resistance has been complicated by a lack of rapid diagnostic bioassays and by regulatory triggers that hinder timely and effective management responses. These failures are of great concern to the scientific and agricultural community. Specific challenges posed by western corn rootworm resistance to Bt maize, and more general concerns around Bt crops that produce less than a high-dose of Bt toxin, have caused uncertainty around current IRM protocols. More than 15 years of experience with IRM has shown that high-dose and refuge-based IRM is not applicable to Bt crops that produce less than a high-dose. Adaptive IRM approaches and pro-active, integrated IRM-pest management strategies are needed and should be in place before release of new technologies that produce less than a high-dose. We suggest changes in IRM strategies to preserve the utility of corn rootworm Bt maize by 1) targeting local resistance management earlier in the sequence of responses to resistance and 2) developing area-wide criteria to address widespread economic losses. We also favor consideration of policies and programs to counteract economic forces that are contributing to rapid resistance evolution.

Recruitment and Retention of Volunteers in a Citizen Science Network to Detect Invasive Species on Private Lands.

Volunteer citizen monitoring is an increasingly important source of scientific data. We developed a volunteer program for early detection of new invasive species by private landowners on their own land. Early detection of an invasive species, however, subjects the landowner to the potentially costly risk of government intervention to control the invasive species. We hypothesized that an adult experiential learning module could increase recruitment and retention because private landowners could learn more about and understand the social benefits of early detection and more accurately gauge the level of personal risk. The experiential learning module emphasized group discussion and individual reflection of risks and benefits of volunteering and included interactions with experts and regulatory personnel. A population of woodland owners with >2 ha of managed oak woodland in central Minnesota were randomly assigned to recruitment treatments: (a) the experiential learning module or (b) a letter inviting their participation. The recruitment and retention rates and data quality were similar for the two methods. However, volunteers who experienced the learning module were more likely to recruit new volunteers than those who merely received an invitation letter. Thus the module may indirectly affect recruitment of new volunteers. The data collection was complex and required the volunteers to complete timely activities, yet the volunteers provided sufficiently high quality data that was useful to the organizers. Volunteers can collect complex data and are willing to assume personal risk to contribute to early detection of invasive species.

A simple definition of food resources for the highly polyphagous Lygus lineolaris (Hemiptera: Miridae) enables prediction of adult abundance.

Lygus lineolaris (Palisot de Beauvois) is a highly polyphagous herbivore with more than 300 known host plants. The high polyphagy has created logistical challenges for understanding its population dynamics. I hypothesized that the primary food resource of this species can be characterized simply, cutting across the multiple host plant species, and enabling a simpler understanding of its population dynamics. The food resource was defined as the apical buds and meristematic tissue and terminal flowers and young seeds. Adult abundance in a habitat was related to the relative abundance of food in the habitat, abundance on a host plant stem was related to the amount of food resource on the stem, and the rate of emigration was lower from host plant patches with higher amounts of food resources. These results suggest that the population dynamics of L. lineolaris depends less on the identity of the host plants and more on the amount of food resource the various host plant species provide.

Estimating predation rates from molecular gut content analysis.

Several methods have been published to estimate per capita predation rates from molecular gut content analysis relying on intuitive understanding of predation, but none have been formally derived. We provide a theoretical framework for estimating predation rates to identify an accurate method and lay bare its assumptions. Per capita predation can be estimated by multiplying the prey decay rate and the prey quantity in the predators. This assumes that variation in per capita predation rate is approximately normally distributed, prey decay occurs exponentially, and predation is in steady state. We described several ways to estimate steady state predation, including using only qualitative presence-absence data to estimate the decay rate and in addition, we provided a method for estimating per capita predation rate when predation is not in steady state. We used previously published data on aphid consumption by a ladybird beetle in a feeding trial to calculate the predation rate and compare published methods with this theoretically derived method. The estimated predation rate (3.29 ± 0.27 aphids/h) using our derived method was not significantly different from the actual predation rate, 3.11 aphids/h. In contrast, previously published methods were less accurate, underestimating the predation rate (0.33 ± 0.02 to 1.66 ± 0.8 aphids/h) or overestimating it (3.64 ± 0.30 aphids/h). In summary, we provide methods to estimate predation rates even when variation in predation rates is not exactly normally distributed and not in steady state and demonstrate that the prey decay rate, and not the prey detection period, is required.

Quantitative prey species detection in predator guts across multiple trophic levels by mapping unassembled shotgun reads.

Quantifying species trophic interaction strengths is crucial for understanding community dynamics and has significant implications for pest management and species conservation. DNA-based methods to identify species interactions have revolutionized these efforts, but a significant limitation is the poor ability to quantify the strength of trophic interactions, that is the biomass or number of prey consumed. We present an improved pipeline, called Lazaro, to map unassembled shotgun reads to a comprehensive arthropod mitogenome database and show that the number of prey reads detected is quantitatively predicted from the prey biomass consumed, even for indirect predation. Two feeding bioassays were performed: starved coccinellid larvae consuming different numbers of aphids (Prey Quantity bioassay), and starved coccinellid larvae consuming a chrysopid larvae that had consumed aphids (Direct and Indirect Predation bioassay). Prey taxonomic assignment against a mitochondrial genome database had high accuracy (99.8% positive predictive value) and the number of prey reads was directly related to the number of prey consumed and inversely related to the elapsed time since consumption with high significance (r2  = .932, p = 4.92E-6). Aphids were detected up to 6 h after direct predation plus 3 h after indirect predation (9 h in total) and detection was related to the predator-specific decay rates. Lazaro enabled quantitative predictions of prey consumption across multiple trophic levels with high taxonomic resolution while eliminating all false positives, except for a few confirmed contaminants, and may be valuable for characterizing prey consumed by field-sampled predators. Moreover, Lazaro is readily applicable for species diversity determination from any degraded environmental DNA.

Organization of the macroinvertebrate community in a tropical annual agroecosystem into modules.

The structure of macroinvertebrate communities in agroecosystems has been assumed to be modular and organized around key herbivore pests. We characterized the macroinvertebrate community in the annual organic brassica agroecosystem in tropical central Brazil to determine if the community was a random assemblage of independent populations or was organized into repeatable multi-species components. We sampled 36 macroinvertebrate taxa associated with six organic brassica farms at biweekly intervals during the dry season during two years in the Distrito Federal, Brazil. We used an unconstrained ordination based on latent variable modeling (boral) with negative binomial population counts to analyze community composition independent of variation in sample abundance. We evaluated observed community structure by comparing it with randomized alternatives. We found that the community was not a random assemblage and consistently organized itself into two modules based around the major herbivores; one with lepidoptera and whiteflies and their associated natural enemies which was gradually replaced during the season by one with brassica aphids, aphid parasitoids and coccinellids. This analysis suggests that the historical and present-day focus on pest herbivores and their associated species in agroecosystems may be justified based on community structure.

Metabarcoding versus mapping unassembled shotgun reads for identification of prey consumed by arthropod epigeal predators.

BACKGROUND: A central challenge of DNA gut content analysis is to identify prey in a highly degraded DNA community. In this study, we evaluated prey detection using metabarcoding and a method of mapping unassembled shotgun reads (Lazaro). RESULTS: In a mock prey community, metabarcoding did not detect any prey, probably owing to primer choice and/or preferential predator DNA amplification, while Lazaro detected prey with accuracy 43-71%. Gut content analysis of field-collected arthropod epigeal predators (3 ants, 1 dermapteran, and 1 carabid) from agricultural habitats in Brazil (27 samples, 46-273 individuals per sample) revealed that 64% of the prey species detections by either method were not confirmed by melting curve analysis and 87% of the true prey were detected in common. We hypothesized that Lazaro would detect fewer true- and false-positive and more false-negative prey with greater taxonomic resolution than metabarcoding but found that the methods were similar in sensitivity, specificity, false discovery rate, false omission rate, and accuracy. There was a positive correlation between the relative prey DNA concentration in the samples and the number of prey reads detected by Lazaro, while this was inconsistent for metabarcoding. CONCLUSIONS: Metabarcoding and Lazaro had similar, but partially complementary, detection of prey in arthropod predator guts. However, while Lazaro was almost 2× more expensive, the number of reads was related to the amount of prey DNA, suggesting that Lazaro may provide quantitative prey information while metabarcoding did not.

Predation on sentinel prey increases with increasing latitude in Brassica-dominated agroecosystems.

In natural ecosystems, arthropod predation on herbivore prey is higher at lower latitudes, mirroring the latitudinal diversity gradient observed across many taxa. This pattern has not been systematically examined in human-dominated ecosystems, where frequent disturbances can shift the identity and abundance of local predators, altering predation rates from those observed in natural ecosystems. We investigated how latitude, biogeographical, and local ecological factors influenced arthropod predation in Brassica oleracea-dominated agroecosystems in 55 plots spread among 5 sites in the United States and 4 sites in Brazil, spanning at least 15° latitude in each country. In both the United States and Brazil, arthropod predator attacks on sentinel model caterpillar prey were highest at the highest latitude studied and declined at lower latitudes. The rate of increased arthropod attacks per degree latitude was higher in the United States and the overall gradient was shifted poleward as compared to Brazil. PiecewiseSEM analysis revealed that aridity mediates the effect of latitude on arthropod predation and largely explains the differences in the intensity of the latitudinal gradient between study countries. Neither predator richness, predator density, nor predator resource availability predicted variation in predator attack rates. Only greater non-crop plant density drove greater predation rates, though this effect was weaker than the effect of aridity. We conclude that climatic factors rather than ecological community structure shape latitudinal arthropod predation patterns and that high levels of aridity in agroecosystems may dampen the ability of arthropod predators to provide herbivore control services as compared to natural ecosystems.

The evolution of resistance to two-toxin pyramid transgenic crops.

Pyramid transgenic crops that express two Bacillus thuringiensis (Bt) toxins hold great potential for reducing insect damage and slowing the evolution of resistance to the toxins. Here, we analyzed a suite of models for pyramid Bt crops to illustrate factors that should be considered when implementing the high dose-refuge strategy for resistance management; this strategy involves the high expression of toxins in Bt plants and use of non-Bt plants as refuges. Although resistance evolution to pyramid Bt varieties should in general be slower, resistance to pyramid Bt varieties is nonetheless driven by the same evolutionary processes as single Bt-toxin varieties. The main advantage of pyramid varieties is the low survival of insects heterozygous for resistance alleles. We show that there are two modes of resistance evolution. When populations of purely susceptible insects persist, leading to density dependence, the speed of resistance evolution changes slowly with the proportion of refuges. However, once the proportion of non-Bt plants crosses the threshold below which a susceptible population cannot persist, the speed of resistance evolution increases rapidly. This suggests that adaptive management be used to guarantee persistence of susceptible populations. We compared the use of seed mixtures in which Bt and non-Bt plants are sown in the same fields to the use of spatial refuges. As found for single Bt varieties, seed mixtures can speed resistance evolution if larvae move among plants. Devising optimal management plans for deploying spatial refuges is difficult because they depend on crop rotation patterns, whether males or females have limited dispersal, and other characteristics. Nonetheless, the effects of spatial refuges on resistance evolution can be understood by considering the three mechanisms determining the rate of resistance evolution: the force of selection (the proportion of insects killed by Bt), assortative mating (deviations of the proportion of heterozygotes from Hardy-Weinberg equilibrium at the total population level), and male mating success (when males carrying resistance alleles find fewer mates). Of these three, assortative mating is often the least important, even though this mechanism is the most frequently cited explanation for the efficacy of the high dose-refuge strategy.

Investigating the Movement Components of Host Preference in a Highly Mobile Insect Herbivore, Nephotettix cincticeps (Hemiptera: Cicadellidae).

Effective insect management strategies require a firm understanding of the factors determining host preference, particularly in highly mobile insect herbivores. Host preference studies commonly employ average or first position as a proxy for preference. Yet few studies have explored host preference in relation to transitory attraction and leaving rates, yet these are both components of host plant selection. We investigated the transitory dynamics of preference by the green rice leafhopper, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae) by conducting experiments on groups of females, males, or mixed-sex leafhoppers, and recording their position over time between low-N and normal-N rice plants. Utilizing a log-linear model and variants of a biostatistical model we used these positional data to extract attraction, leaving and tenure rates to better understand the process of host-plant selection. We found a general preference for normal-N over low-N plants at equilibrium. However, between sexes there was variation in the relative significance of attraction or leaving rates on that preference. Female leafhoppers were more attracted to host plants with higher nitrogen content. Male leafhoppers were less discriminate in their initial attraction to hosts but left low-N hosts at a faster rate. Whereas estimated tenure times on both normal- and low-N plants exceeded transmission times for the leafhopper-transmitted rice dwarf virus, longer tenure on normal-N plants likely increases the likelihood of virus acquisition from these plants. Our findings support previous recommendations that growers can mitigate the risks of leafhopper damage and pathogen transmission by optimizing their application of nitrogenous fertilizers.

Transgenic insecticidal crops and natural enemies: a detailed review of laboratory studies.

This review uses a data-driven, quantitative method to summarize the published, peer-reviewed literature about the impact of genetically modified (GM) plants on arthropod natural enemies in laboratory experiments. The method is similar to meta-analysis, and, in contrast to a simple author-vote counting method used by several earlier reviews, gives an objective, data-driven summary of existing knowledge about these effects. Significantly more non-neutral responses were observed than expected at random in 75% of the comparisons of natural enemy groups and response classes. These observations indicate that Cry toxins and proteinase inhibitors often have non-neutral effects on natural enemies. This synthesis identifies a continued bias toward studies on a few predator species, especially the green lacewing, Chrysoperla carnea Stephens, which may be more sensitive to GM insecticidal plants (16.8% of the quantified parameter responses were significantly negative) than predators in general (10.9% significantly negative effects without C. carnea). Parasitoids were more susceptible than predators to the effects of both Cry toxins and proteinase inhibitors, with fewer positive effects (18.0%, significant and nonsignificant positive effects combined) than negative ones (66.1%, significant and nonsignificant negative effects combined). GM plants can have a positive effect on natural enemies (4.8% of responses were significantly positive), although significant negative (21.2%) effects were more common. Although there are data on 48 natural enemy species, the database is still far from adequate to predict the effect of a Bt toxin or proteinase inhibitor on natural enemies.

Using an F(2) screen to monitor frequency of resistance alleles to Bt cotton in field populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).

BACKGROUND: In an effort to control the most devastating cotton pest, Helicoverpa armigera (Hübner), in Qiuxian County (Hebei, China), Bt cotton has been planted almost exclusively since 1998 in this area. Owing to the high insecticidal selection pressures in this region, monitoring of Bt resistance in H. armigera is necessary so that proactive actions can be implemented before field control measures fail. From 2003 to 2005, an F(2) screen was conducted in order to monitor Bt resistance in H. armigera populations collected from this area. RESULTS: The F(2) screen showed that 15 out of 278 isofemale lines carried resistance alleles to Bt cotton. The resistance allele frequency in field populations of H. armigera ranged from 0.0119 to 0.0297, with an overall frequency of 0.0146 and a 95% confidence interval of 0.0084-0.0225 for the 3 year period. This value is greater than the value reported from 1999 (P < 0.05). CONCLUSION: A fluctuating but overall increase in resistance allele frequency was detected in the field populations of H. armigera in Qiuxian County from 1999 to 2005. To prevent further increases in Bt resistance frequency in this pest, it is necessary to introduce Bt cotton expressing multiple Bt toxins and integrate this technology with other tactics for management of this key pest.

Bacillus thuringiensis Cry1Ac resistance frequency in tobacco budworm (Lepidoptera: Noctuidae).

The tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is one of the most important pests of cotton, Gossypium hirsutum L, that has become resistant to a wide range of synthetic insecticides. CrylAc-expressing cotton has proven its effectiveness against this insect since its introduction in North America in 1996. However, the constant exposure of tobacco budworm to this protein toxin may result in the development of resistance to it. To estimate the frequency of alleles that confer resistance to a 1.0 microg of Bacillus thuringiensis Cry1Ac diagnostic concentration in field-collected insects, the second generation (F2) of 1,001 single-pair families from seven geographical regions representing 2,202 alleles from natural populations was screened in 2006 and 2007 without finding major resistant alleles. Neonates of 56 single-pair families were able to develop to second instar on the diagnostic concentration in the initial screen, but only seven of these lines did so again in a second confirmatory screen. Minor resistance alleles to Cry1Ac may be quite common in natural populations of H. virescens. Our estimated resistance allele frequencies (0.0036-0.0263) were not significantly different from a previously published estimate from 1993. There is no evidence that H. virescens populations have become more resistant to Cry1Ac.

An empirical test of the F2 screen for detection of Bacillus thuringiensis-resistance alleles in tobacco budworm (Lepidoptera: Noctuidae).

Insects exposed to genetically modified crops expressing Bacillus thuringiensis (Bt) toxins are under intense selection pressure that could result on widespread Bt resistance. Screening for early indications of Bt resistance developing in targeted Lepidoptera is conducted in many of the regions where genetically modified cotton and corn have been commercialized. Heliothis virescens (F.) (Lepidoptera: Noctuidae) has been selected in the laboratory to have a gene for resistance to Cry1Ac. We used this laboratory line to test the assumptions and theoretical predictions related to detection of recessive Bt-resistant alleles in field populations based on a second generation (F2) screen. By creating single-pair families from mating a heterozygous Cry1Ac-resistant moth with a Cry1Ac-susceptible moth, we simulated the most common genotype when Bt-resistance alleles are at low frequency in the field. The second generation (F2) neonates of single-pair families were screened daily with diagnostic concentration bioassays. Cry1Ac-resistant homozygous larvae were detected, but the proportion of resistant larvae was generally below the theoretical expectation of 6.25% and was influenced by the moth F1 sib-mating density and by the day of oviposition of F2 eggs. Logistical considerations such as F1 sib-mating density and F2 neonate screening are important for the successful implementation of a reliable method.

Co-occurrence Among Intraguild Predators: Avoidance or Aggregation?

Intraguild predation (IGP) may interact with prey availability to structure predator communities. However, if predators are able to avoid each other, its effect on predator community structure may be minimized or absent. To determine whether co-occurrence among IG predators is limited, we estimated co-occurrence among predators in experimental plots of maize and soybean. These crops provide high densities of shared resources (aphids) as well as known IG predators (primarily coccinellids). Despite documented intraspecific and interspecific avoidance behaviors, aggregation to patchy resources could bring IG predators into contact. We, therefore, hypothesized that despite documented intraspecific avoidance behaviors, aphidophagous IG coccinellid predators would not significantly avoid co-occurrence, making IGP likely. Co-occurrence was estimated from visual counts of aphid predators and their prey on randomly selected plants over the course of the growing season. For each habitat, we used maximum likelihood analysis to determine whether observed co-occurrence deviated significantly from that expected for each possible pairwise combination of IG predators. We repeated this analysis using published data on co-occurrence among aphid predators on tansy. We found that most co-occurrence among IG predators was random, suggesting that avoidance does not limit co-occurrence. Failure to limit co-occurrence could be the result of ineffective avoidance mechanisms or because predators balance aggregating on shared resources with avoiding IG predators.

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.