Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).

The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.

Sugar transporters enable a leaf beetle to accumulate plant defense compounds.

Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates glucosinolate defense compounds from Brassicaceae in the hemolymph. Using phylogenetic analyses of coleopteran major facilitator superfamily transporters, we identify a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTRs are predominantly expressed in the excretory system, the Malpighian tubules. Silencing of PaGTRs leads to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays corroborated the selective retention of glucosinolates by Malpighian tubules of P. armoraciae in situ. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion.

De Novo Whole-Genome Assembly of the Swede Midge (Contarinia nasturtii), a Specialist of Brassicaceae, Using Linked-Read Sequencing.

The swede midge, Contarinia nasturtii, is a cecidomyiid fly that feeds specifically on plants within the Brassicaceae. Plants in this family employ a glucosinolate-myrosinase defense system, which can be highly toxic to nonspecialist feeders. Feeding by C. nasturtii larvae induces gall formation, which can cause substantial yield losses thus making it a significant agricultural pest. A lack of genomic resources, in particular a reference genome, has limited deciphering the mechanisms underlying glucosinolate tolerance in C. nasturtii, which is of particular importance for managing this species. Here, we present an annotated, scaffolded reference genome of C. nasturtii using linked-read sequencing from a single individual and explore systems involved in glucosinolate detoxification. The C. nasturtii genome is similar in size and annotation completeness to that of the Hessian fly, Mayetiola destructor, but has greater contiguity. Several genes encoding enzymes involved in glucosinolate detoxification in other insect pests, including myrosinases, sulfatases, and glutathione S-transferases, were found, suggesting that C. nasturtii has developed similar strategies for feeding on Brassicaceae. The C. nasturtii genome will, therefore, be integral to continued research on plant-insect interactions in this system and contribute to effective pest management strategies.

Acuities into tolerance mechanisms via different bioassay during Brassicaceae-Alternaria brassicicola interaction and its impact on yield.

Heavy losses by dark leaf spot disease in oilseed Brassica have incited research towards identifying sources of genetic tolerance against causal pathogen, Alternaria brassicicola. Several morpho-molecular parameters were evaluated to test the performance of field mustard and rapeseed genotypes under artificial inoculation with this pathogen. During Brassica-Alternaria interaction, physio-biochemical defense response was witnessed in tolerant genotypes. Two tolerant genotypes (one for field mustard and one for rapeseed), i.e., EC250407 and EC1494 were identified. However, necrotic lesions were more prominent in susceptible genotypes with minimum chlorophyll (chlorophyll a, chlorophyll b and total chlorophyll) and carotenoids contents. Contrary to photosynthetic pigments, increase in total soluble protein (TSP) contents was observed with disease progression in susceptible genotypes. Tolerant genotypes of field mustard and rapeseed displayed remarkable increase in the activities of redox enzyme in infected leaves with least yield loss (6.47% and 5.74%) and disease severity index (DSI) of 2.9 and 2.1, respectively. However, yield/plant showed close association with other morpho-yield parameters, photosynthetic pigments and redox enzymes (superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)) activities except silique length and TSP. Based on the results of morpho-biochemical analyses, redox enzymes and morphological parameters; their interplay is proposed to determine the tolerance outcome of the Brassica-A. brassicicola interaction.

Eradicating the large white butterfly from New Zealand eliminates a threat to endemic Brassicaceae.

In May 2010 the large white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae), was discovered to have established in New Zealand. It is a Palearctic species that-due to its wide host plant range within the Brassicaceae-was regarded as a risk to New Zealand's native brassicas. New Zealand has 83 native species of Brassicaceae including 81 that are endemic, and many are threatened by both habitat loss and herbivory by other organisms. Initially a program was implemented to slow its spread, then an eradication attempt commenced in November 2012. The P. brassicae population was distributed over an area of approximately 100 km2 primarily in urban residential gardens. The eradication attempt involved promoting public engagement and reports of sightings, including offering a bounty for a two week period, systematically searching gardens for P. brassicae and its host plants, removing host plants, ground-based spraying of insecticide to kill eggs and larvae, searching for pupae, capturing adults with nets, and augmenting natural enemy populations. The attempt was supported by research that helped to progressively refine the eradication strategy and evaluate its performance. The last New Zealand detection of P. brassicae occurred on 16 December 2014, the eradication program ceased on 4 June 2016 and P. brassicae was officially declared eradicated from New Zealand on 22 November 2016, 6.5 years after it was first detected and 4 years after the eradication attempt commenced. This is the first species of butterfly ever to have been eradicated worldwide.

Evolution of phenotypic plasticity: Genetic differentiation and additive genetic variation for induced plant defence in wild arugula Eruca sativa.

Phenotypic plasticity is the primary mechanism of organismal resilience to abiotic and biotic stress, and genetic differentiation in plasticity can evolve if stresses differ among populations. Inducible defence is a common form of adaptive phenotypic plasticity, and long-standing theory predicts that its evolution is shaped by costs of the defensive traits, costs of plasticity and a trade-off in allocation to constitutive versus induced traits. We used a common garden to study the evolution of defence in two native populations of wild arugula Eruca sativa (Brassicaceae) from contrasting desert and Mediterranean habitats that differ in attack by caterpillars and aphids. We report genetic differentiation and additive genetic variance for phenology, growth and three defensive traits (toxic glucosinolates, anti-nutritive protease inhibitors and physical trichome barriers) as well their inducibility in response to the plant hormone jasmonic acid. The two populations were strongly differentiated for plasticity in nearly all traits. There was little evidence for costs of defence or plasticity, but constitutive and induced traits showed a consistent additive genetic trade-off within each population for the three defensive traits. We conclude that these populations have evolutionarily diverged in inducible defence and retain ample potential for the future evolution of phenotypic plasticity in defence.

Molecular signatures of selection associated with host plant differences in Pieris butterflies.

Adaptive traits that enable organisms to conquer novel niches and experience subsequent diversification are ecologically and evolutionarily important. The larvae of Pieris butterflies express nitrile-specifier proteins (NSPs), a key innovation for overcoming the glucosinolate (GLS)-myrosinase-based defence system of their Brassicales host plants. Nitrile-specifier proteins are a member of the NSP-like gene family, which includes the major allergen (MA) protein, a paralog of NSP with a GLS-disarming function, and a single domain major allergen (SDMA) protein, whose function is unknown. The arms-race between GLS-based defences and the NSP-like gene family is suggested to mediate diversification in both Pierid butterflies and Brassicales plants. Here, we tested whether the expected strong selection on NSP-like gene family correlates with shifts in host plant spectra among Pierid butterflies. We combined feeding experiments using 25 Brassicaceae plants and five Pieris species with larval transcriptome data to investigate the patterns of selection acting on NSP-like gene family members. Although we observed significantly elevated nonsynonymous to synonymous substitution rate ratios in NSPs on branches associated with changes in patterns of host plant usage, no such pattern was observed in MAs or SDMAs. Furthermore, we found evidence for positive selection of NSP at a phylogenetic branch which reflects different host plant spectra. Our data indicate that the NSP-related gene members have evolved differently: NSPs have accumulated more amino acid changes in response to shifting preferences for host plants, whereas MAs and SDMAs appear to be more conserved. Further detailed functional assays of these genes would provide important insights to understand their role in the chemical arms-race between Pieris butterflies and their Brassicales host plants.

Specific Genes Identified in Pathotype 4 of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot is an important disease of cruciferous crops caused by Plasmodiophora brassicae, and pathotypes are classified based on the response of differential hosts. This study was conducted to identify genetic markers able to differentiate pathotypes. Differential expression of genes between pathotype 4 (P4) and pathotype 7 (P7) was assessed according to transcriptome data of molecular marker screening. Among the pathotypes (P2, P4, P5, P7, P9, P10, and P11) tested, six genes were exclusive to P4, dividing the isolates into three types: PBRA_003263 and PBRA_003268 were present in all P4 isolates, PBRA_000003/Novel512 were found in a type of P4 (P4-1), and Novel137/PBRA_005772 were found in another P4 type, P4-2. Amplicons for all six genes were produced for only one isolate, which we named P4-3. This study is the first to establish a molecular identification system for P4 the, predominant pathotype in China. The genes identified might serve as molecular markers for differentiation of P4 from other pathotypes and may also distinguish different types of P4.

Insecticide-resistance mechanism of Plutella xylostella (L.) associated with amino acid substitutions in acetylcholinesterase-1: A molecular docking and molecular dynamics investigation.

Acetylcholinesterase-1 (AChE1) is a vital enzyme involved in neurotransmission and represents an attractive insecticide-target for organophosphates and carbamates in Plutella xylostella (Linneaus), an important pest of cruciferous crops worldwide. However, insecticide-resistance often occurs due to mutations, making many organophosphates and carbamates ineffective. In particular, A298S and G324A mutations in AChE1 significantly lower the binding affinity of insecticides. In the present study, the wild-type and mutant AChE1 structures were constructed and their structural stabilities, residual flexibilities were investigated through molecular dynamics simulations. Subsequently, the structural and energetic changes responsible for the insecticide-resistance in AChE1 were analyzed using molecular docking. The results of molecular dynamics simulation showed that the mutant AChE1 shows little structural deviation than the wild-type, indicate the structural instability. Furthermore, the docking results demonstrated that these mutations break the intermolecular hydrogen bonding interactions and thereby affect the prothiofos as well as all insecticide binding. Hence, the results could provide some insights into the resistance mechanism of AChE1 in insecticides binding and helpful in the development of novel insecticides that are less susceptible to insecticide-resistance.

Characterization of Five Molecular Markers for Pathotype Identification of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot disease is an important disease on cruciferous crops caused by Plasmodiophora brassicae infections. The pathotypes have been classified based on the reactions of differential hosts. However, molecular markers of particular pathotypes for P. brassicae are limited. In this study, we found five genetic markers in association with different pathotypes. Different gene expression patterns among different pathotypes (P4, P7, P9, and P11) were assayed according to the transcriptome data. The assay indicated that molecular markers PBRA_007750 and PBRA_009348 could be used to distinguish P11 from P4, P7, and P9; PBRA_009348 and Novel342 could distinguish P9 from P4, P7, and P11; and PBRA_008439 and Novel342 could represent a kind of P4. Polymerase chain reaction cycles ranging from 25 to 30 were able to identify the predominant pathotype in general. Therefore, these molecular markers would be a valuable tool to identify and discriminate pathotypes in P. brassicae population.

Mining of Brassica-Specific Genes (BSGs) and Their Induction in Different Developmental Stages and under Plasmodiophora brassicae Stress in Brassica rapa.

Orphan genes, also called lineage-specific genes (LSGs), are important for responses to biotic and abiotic stresses, and are associated with lineage-specific structures and biological functions. To date, there have been no studies investigating gene number, gene features, or gene expression patterns of orphan genes in Brassica rapa. In this study, 1540 Brassica-specific genes (BSGs) and 1824 Cruciferae-specific genes (CSGs) were identified based on the genome of Brassica rapa. The genic features analysis indicated that BSGs and CSGs possessed a lower percentage of multi-exon genes, higher GC content, and shorter gene length than evolutionary-conserved genes (ECGs). In addition, five types of BSGs were obtained and 145 out of 529 real A subgenome-specific BSGs were verified by PCR in 51 species. In silico and semi-qPCR, gene expression analysis of BSGs suggested that BSGs are expressed in various tissue and can be induced by Plasmodiophora brassicae. Moreover, an A/C subgenome-specific BSG, BSGs1, was specifically expressed during the heading stage, indicating that the gene might be associated with leafy head formation. Our results provide valuable biological information for studying the molecular function of BSGs for Brassica-specific phenotypes and biotic stress in B. rapa.

Crab spiders impact floral-signal evolution indirectly through removal of florivores.

The puzzling diversity of flowers is primarily shaped by selection and evolutionary change caused by the plant's interaction with animals. The contribution of individual animal species to net selection, however, may vary depending on the network of interacting organisms. Here we document that in the buckler mustard, Biscutella laevigata, the crab spider Thomisus onustus reduces bee visits to flowers but also benefits plants by feeding on florivores. Uninfested plants experience a trade-off between pollinator and spider attraction as both bees and crab spiders are attracted by the floral volatile ß-ocimene. This trade-off is reduced by the induced emission of ß-ocimene after florivore infestation, which is stronger in plant populations where crab spiders are present than where they are absent, suggesting that plants are locally adapted to the presence of crab spiders. Our study demonstrates the context-dependence of selection and shows how crab spiders impact on floral evolution.

Parasitoid wasps' exposure to host-infested plant volatiles affects their olfactory cognition of host-infested plants.

Using Cotesia vestalis, a parasitoid wasp of diamondback moth larvae and three crucifer plant species (cabbage, komatsuna, and Japanese radish), we examined the effects of exposure to host-infested plant volatiles from one plant species on a newly emerged wasp's subsequent olfactory cognition of host-infested plant volatiles from the same or different plant species. The preference of C. vestalis between infested and uninfested plant volatiles was tested in a choice chamber. Volatile-inexperienced wasps significantly preferred infested cabbage and infested radish volatiles, but not infested komatsuna volatiles. After exposure to infested cabbage volatiles, wasps showed a significant preference for infested cabbage volatiles, while the significant preference for infested radish volatiles that had been observed in inexperienced wasps was no longer observed. After exposure to infested komatsuna volatiles, wasps significantly preferred infested komatsuna volatiles, and the pre-exposure significant preferences for infested cabbage volatiles and infested radish volatiles remained. After exposure to infested radish volatiles, the significant preferences for infested cabbage and infested radish volatiles did not change. Furthermore, wasps showed a significant preference for infested komatsuna volatiles. The compound compositions of the volatile blends from the three infested plant species were grouped separately in a principal coordinates analysis. The experience-based cognition of C. vestalis for host-infested plant volatiles of three plant species is discussed.

Phenetic and phylogenetic relationships among Aceria spp. (Acari: Eriophyoidea) inhabiting species within the family Brassicaceae in Serbia.

We examined morphological and genetic differences among Aceria spp. inhabiting six Brassicaceae species in Serbia. Five of them have been already mentioned in the literature as original or alternate hosts of Aceria spp. (Berteroa incana (L.) DC., Capsella bursa-pastoris (L.) Medik., Cardamine hirsuta L., Lepidium draba L. and Sisymbrium orientale L.), whereas Aurinia petraea (Ard.) Schur was registered here for the first time as a host for Aceria sp. Results of morphometric analyses indicated clear differentiation of Aceria spp. from B. incana, L. draba, A. petraea and S. orientale, whereas the other two entities were less diverse and clustered together. Molecular analyses indicated that the average mean divergence over all sequence pairs was 18.3% (11.8-25.9%) and disclosed that the observed mtCOI distances between the six host-associated Aceria entities are large enough to represent differences between species. The Aceria sp. hosted by B. incana was morphologically and genetically most distinctive, whereas discrepancy between phenetic and phylogenetic relationships was found in the position of Aceria sp. hosted by L. draba when compared with those inhabiting C. hirsuta, S. orientale and C. bursa-pastoris.

De Novo Assembly and Developmental Transcriptome Analysis of the Small White Butterfly Pieris rapae.

The small white butterfly Pieris rapae is one of the most destructive pests of Brassicaceae. Yet little is understood about its genes involved in development. To facilitate research on P. rapae, we sequenced the transcriptome of P. rapae during six developmental stages, including the egg, three larval stages, the pupa, and the adult. In total, 240 million high-quality reads were obtained. De novo assembly generated 96,069 unigenes with an average length of 1353 nt. Of these, 31,629 unigenes had homologs as determined by a blastx search against the NR database with a cut-off e-value of 10-5. Clusters of Orthologous Groups of proteins (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to functionally annotate those genes. Then, 849 genes involved in seven canonical development signaling pathway were identified, including dozens of key genes such as Hippo, Notch, and JAK2. A total of 21,883 differentially expressed (cut-off of 2-fold) unigenes were detected across the developmental stages, most of which were found between the egg and first larval stages. Interestingly, only 34 differentially expressed unigenes, most of which are cuticle protein related genes, were detected with a cut-off of 210-fold. Furthermore, we identified 32 heat shock protein (Hsp) genes that were expressed with complete open reading frames. Based on phylogenetic trees of the Hsp genes, we found that Hsp genes with close evolutionary relationships had similar expression pattern. Additionally, partial pattern recognition receptors genes were found to be developmental regulated. This study provides comprehensive sequence resources for P. rapae and numerous differential expressed genes, and these findings will lay the foundation for future functional genomics studies on this species.

Long-Chain Omega-3 Polyunsaturated Fatty Acids Have Developmental Effects on the Crop Pest, the Cabbage White Butterfly Pieris rapae.

Nutritional enhancement of crops using genetic engineering can potentially affect herbivorous pests. Recently, oilseed crops have been genetically engineered to produce the long-chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at levels similar to that found in fish oil; to provide a more sustainable source of these compounds than is currently available from wild fish capture. We examined some of the growth and development impacts of adding EPA and DHA to an artificial diet of Pieris rapae, a common pest of Brassicaceae plants. We replaced 1% canola oil with EPA: DHA (11:7 ratio) in larval diets, and examined morphological traits and growth of larvae and ensuing adults across 5 dietary treatments. Diets containing increasing amounts of EPA and DHA did not affect developmental phenology, larval or pupal weight, food consumption, nor larval mortality. However, the addition of EPA and DHA in larval diets resulted in progressively heavier adults (F 4, 108 = 6.78; p = 0.011), with smaller wings (p < 0.05) and a higher frequency of wing deformities (R = 0.988; p = 0.001). We conclude that the presence of EPA and DHA in diets of larval P. rapae may alter adult mass and wing morphology; therefore, further research on the environmental impacts of EPA and DHA production on terrestrial biota is advisable.

High Diversity Revealed in Leaf-Associated Protists (Rhizaria: Cercozoa) of Brassicaceae.

The largest biological surface on earth is formed by plant leaves. These leaf surfaces are colonized by a specialized suite of leaf-inhabiting microorganisms, recently termed "phyllosphere microbiome". Microbial prey, however, attract microbial predators. Protists in particular have been shown to structure bacterial communities on plant surfaces, but virtually nothing is known about the community composition of protists on leaves. Using newly designed specific primers targeting the 18S rDNA gene of Cercozoa, we investigated the species richness of this common protist group on leaves of four Brassicaceae species from two different locations in a cloning-based approach. The generated sequences revealed a broad diversity of leaf-associated Cercozoa, mostly bacterial feeders, but also including known plant pathogens and a taxon of potential endophytes that were recently described as algal predators in freshwater systems. This initial study shows that protists must be regarded as an integral part of the microbial diversity in the phyllosphere of plants.

The butterfly plant arms-race escalated by gene and genome duplications.

Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits.

Root parasitic plant Orobanche aegyptiaca and shoot parasitic plant Cuscuta australis obtained Brassicaceae-specific strictosidine synthase-like genes by horizontal gene transfer.

BACKGROUND: Besides gene duplication and de novo gene generation, horizontal gene transfer (HGT) is another important way of acquiring new genes. HGT may endow the recipients with novel phenotypic traits that are important for species evolution and adaption to new ecological niches. Parasitic systems expectedly allow the occurrence of HGT at relatively high frequencies due to their long-term physical contact. In plants, a number of HGT events have been reported between the organelles of parasites and the hosts, but HGT between host and parasite nuclear genomes has rarely been found. RESULTS: A thorough transcriptome screening revealed that a strictosidine synthase-like (SSL) gene in the root parasitic plant Orobanche aegyptiaca and the shoot parasitic plant Cuscuta australis showed much higher sequence similarities with those in Brassicaceae than with those in their close relatives, suggesting independent gene horizontal transfer events from Brassicaceae to these parasites. These findings were strongly supported by phylogenetic analysis and their identical unique amino acid residues and deletions. Intriguingly, the nucleus-located SSL genes in Brassicaceae belonged to a new member of SSL gene family, which were originated from gene duplication. The presence of introns indicated that the transfer occurred directly by DNA integration in both parasites. Furthermore, positive selection was detected in the foreign SSL gene in O. aegyptiaca but not in C. australis. The expression of the foreign SSL genes in these two parasitic plants was detected in multiple development stages and tissues, and the foreign SSL gene was induced after wounding treatment in C. australis stems. These data imply that the foreign genes may still retain certain functions in the recipient species. CONCLUSIONS: Our study strongly supports that parasitic plants can gain novel nuclear genes from distantly related host species by HGT and the foreign genes may execute certain functions in the new hosts.

Fitness costs associated with field-evolved resistance to chlorantraniliprole in Plutella xylostella (Lepidoptera: Plutellidae).

Plutella xylostella (L.) is the most important pest of Brassicaceae worldwide, with a recent estimate of US$ 4-5 billion expenditure for the control of this insect. A case of very high resistance of this pest to chlorantraniliprole was recently associated with reduced efficacy in a Brazilian field of Brassica spp. Although diamide resistance has been characterized, the fitness of insects due to such resistance has yet to be examined. Therefore, in this study, biological parameters were assessed in both susceptible and resistant strains of P. xylostella subjected to sublethal chlorantraniliprole concentrations. The field strain showed high resistance to chlorantraniliprole (RR50=27,793-fold), although resistance rapidly decreased in the first generations, showing instability. The exposure of susceptible and resistant larvae to their respective LC1, LC10, and LC25 values led to an increased duration of the larval and pupae phases and reduced weight in both strains; however, no significant differences in pupal viability across the treatments were observed. The resistant insects presented significantly lower larval weight and fecundity and higher larval and pupal periods, hatchability, and male longevity when not exposed to chlorantraniliprole, suggesting a fitness cost associated with resistance. In addition, resistant females showed a significantly higher egg-laying period and longevity at LC25, whereas the males lived longer at LC1. Chlorantraniliprole negatively impacted the biological parameters of both strains tested, although these effects were more relevant to the resistant insects. Resistant P. xylostella showed negative and positive biological trade-offs when compared with the susceptible individuals in both the absence and presence of chlorantraniliprole. Despite the important role that these trade-offs may play in the evolution of resistance to chlorantraniliprole, practical applications still depend on such information as the dominance of fitness costs and resistance.