The genome-wide response of Dermatophagoides pteronyssinus to cystatin A, a peptidase inhibitor from human skin, sheds light on its digestive physiology and allergenicity.

The digestive physiology of house dust mites (HDMs) is particularly relevant for their allergenicity since many of their allergens participate in digestion and are excreted into faecal pellets, a main source of exposure for allergic subjects. To gain insight into the mite dietary digestion, the genome of the HDM Dermatophagoides pteronyssinus was screened for genes encoding peptidases (n = 320), glycosylases (n = 77), lipases and esterases (n = 320), peptidase inhibitors (n = 65) and allergen-related proteins (n = 52). Basal gene expression and transcriptional responses of mites to dietary cystatin A, a cysteine endopeptidase inhibitor with previously shown antinutritional effect on mites, were analysed by RNAseq. The ingestion of cystatin A resulted in significant regulation of different cysteine endopeptidase and glycosylase genes. One Der p 1-like and two cathepsin B-like cysteine endopeptidase genes of high basal expression were induced, which suggests their prominent role in proteolytic digestion together with major allergen Der p 1. A number of genes putatively participating in the interaction of mites with their microbiota and acquired by horizontal gene transfer were repressed, including genes encoding the peptidase Der p 38, two 1,3-beta-glucanases, a lysozyme and a GH19 chitinase. Finally, the disruption of mite digestion resulted in the regulation of up to 17 allergen and isoallergen genes. Altogether, our results shed light on the putative role of specific genes in digestion and illustrate the connection between the digestive physiology of HDM and allergy.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

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.

Phytocystatins: Defense Proteins against Phytophagous Insects and Acari.

This review deals with phytocystatins, focussing on their potential role as defence proteins against phytophagous arthropods. Information about the evolutionary, molecular and biochemical features and inhibitory properties of phytocystatins are presented. Cystatin ability to inhibit heterologous cysteine protease activities is commented on as well as some approaches of tailoring cystatin specificity to enhance their defence function towards pests. A general landscape on the digestive proteases of phytophagous insects and acari and the remarkable plasticity of their digestive physiology after feeding on cystatins are highlighted. Biotechnological approaches to produce recombinant cystatins to be added to artificial diets or to be sprayed as insecticide-acaricide compounds and the of use cystatins as transgenes are discussed. Multiple examples and applications are included to end with some conclusions and future perspectives.

Effects of pesticides used on citrus grown in Spain on the mortality of Ceratitis capitata (Diptera: Tephritidae) Vienna-8 strain sterile males.

Vienna-8 sterile males are currently released in Spain to reduce wild populations of the medfly. Because pesticides are required to maintain populations of some citrus key pests below economic thresholds, there is a need to evaluate the effects of pesticides commonly used in citrus on Vienna-8 males. Males were exposed to differently aged residues of eight pesticides. Abamectin, etofenprox, etoxazole, petroleum spray oil, pymetrozine, and pyriproxyfen resulted harmless to Vienna-8 males. However, fresh residues of chlorpyrifos and spinosad caused high mortalities and had residual effects until 21 and 28 d after treatment, respectively. Following the same method, the lethal effects of chlorpyrifos and spinosad on Ceratitis capitata (Wiedemann) wild-type (wt) males were determined. Surprisingly, these pesticides resulted more toxic for wt than for Vienna-8 males. To determine whether these results could be attributed to intrinsic characteristics of the pesticides or to behavioral differences among Vienna-8 and wt males, a topical application trial was conducted. Vienna-8 males were twice as susceptible to chlorpyrifos as wt males, whereas their susceptibility to spinosad was slightly lower. These results in intrinsic toxicity did not directly explain the differences observed in the extended-laboratory tests with these pesticides. We hypothesize that the lower flight activity ofVienna-8 males relative to wt conspecifics can explain the lower risk observed for Vienna-8 males in the residual tests. Our results should be taken into account when planning area-wide Sterile Insect Technique programs against C. capitata especially in those areas where treatments with chlorpyrifos are approved.

Monitoring Insect Resistance to Bt Maize in the European Union: Update, Challenges, and Future Prospects.

Transgenic maize producing the Cry1Ab toxin of Bacillus thuringiensis (Bt maize) was approved for cultivation in the European Union (EU) in 1998 to control the corn borers Sesamia nonagrioides (Lefèbvre) and Ostrinia nubilalis (Hübner). In the EU since then, Cry1Ab is the only Bt toxin produced by Bt maize and Spain is the only country where Bt maize has been planted every year. In 2021, about 100,000 hectares of Bt maize producing Cry1Ab were cultivated in the EU, with Spain accounting for 96% and Portugal 4% of this area. In both countries, Bt maize represented less than 25% of all maize planted in 2021, with a maximum regional adoption of 64% Bt maize in northeastern Spain. Insect resistance management based on the high-dose/refuge strategy has been implemented in the EU since 1998. This has been accompanied by monitoring to enable early detection of resistance. The monitoring data from laboratory bioassays show no decrease in susceptibility to Cry1Ab had occurred in either pest as of 2021. Also, control failures have not been reported, confirming that Bt maize producing Cry1Ab remains effective against both pests. Conditions in the EU preventing approval of new genetically modified crops, including maize producing two or more Bt toxins targeting corn borers, may limit the future effectiveness of resistance management strategies.

Immunodetection of Truncated Forms of the α6 Subunit of the nAChR in the Brain of Spinosad Resistant Ceratitis capitata Phenotypes.

The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad.

Cysteine peptidases and their inhibitors in Tetranychus urticae: a comparative genomic approach.

BACKGROUND: Cysteine peptidases in the two-spotted spider mite Tetranychus urticae are involved in essential physiological processes, including proteolytic digestion. Cystatins and thyropins are inhibitors of cysteine peptidases that modulate their activity, although their function in this species has yet to be investigated. Comparative genomic analyses are powerful tools to obtain advanced knowledge into the presence and evolution of both, peptidases and their inhibitors, and could aid to elucidate issues concerning the function of these proteins. RESULTS: We have performed a genomic comparative analysis of cysteine peptidases and their inhibitors in T. urticae and representative species of different arthropod taxonomic groups. The results indicate: i) clade-specific proliferations are common to C1A papain-like peptidases and for the I25B cystatin family of inhibitors, whereas the C1A inhibitors thyropins are evolutionarily more conserved among arthropod clades; ii) an unprecedented extensive expansion for C13 legumain-like peptidases is found in T. urticae; iii) a sequence-structure analysis of the spider mite cystatins suggests that diversification may be related to an expansion of their inhibitory range; and iv) an in silico transcriptomic analysis shows that most cathepsin B and L cysteine peptidases, legumains and several members of the cystatin family are expressed at a higher rate in T. urticae feeding stages than in embryos. CONCLUSION: Comparative genomics has provided valuable insights on the spider mite cysteine peptidases and their inhibitors. Mite-specific proliferations of C1A and C13 peptidase and I25 cystatin families and their over-expression in feeding stages of mites fit with a putative role in mite's feeding and could have a key role in its broad host feeding range.

Current situation and forecasting of resistance evolution to lambda-cyhalothrin in Spanish medfly populations.

BACKGROUND: The control of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) in Spanish field populations mainly relies on the insecticides lambda-cyhalothrin and spinosad as bait sprays. However, their sustainable used is compromised by the development of lambda-cyhalothrin resistance and the detection of spinosad resistant alleles. In addition, the use of lure-and-kill traps covered with deltamethrin has increased in the last years. It is thus urgent to predict the impact that the combination of both pyrethroids will have in the evolution of lambda-cyhalothrin resistance and how they could be combined with spinosad so as to establish proper resistance management programs. RESULTS: Toxicity bioassays were performed to analyze the current levels of lambda-cyhalothrin resistance in field populations, proving that it has remained stable in the last decade. An evolutionary model was established to explore the weight of selected parameters in the evolution of lambda-cyhalothrin resistance in C. capitata and to forecast resistance development under different resistance management scenarios. Our results highlight the importance of fitness cost and inheritance to fit the experimental results. The analyses predicted that the rotation of lambda-cyhalothrin and spinosad, when deltamethrin traps are also deployed in the field, will slow down the evolution of resistance, especially when cross-resistance between both pyrethroids is considered. CONCLUSION: Lambda-cyhalothrin resistance has not increased in the last decade, probably due to the alternation of this insecticide with spinosad. Our modelling results indicate that the best option to avoid an increase in lambda-cyhalothrin resistant alleles, considering that deltamethrin use is growing, would be to continue combining their use with spinosad. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Expanding the Medfly Virome: Viral Diversity, Prevalence, and sRNA Profiling in Mass-Reared and Field-Derived Medflies.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is an agricultural pest of a wide range of fruits. The advent of high-throughput sequencing has boosted the discovery of RNA viruses infecting insects. In this article, we aim to characterize the RNA virome and viral sRNA profile of medfly. By means of transcriptome mining, we expanded the medfly RNA virome to 13 viruses, including two novel positive ssRNA viruses and the first two novel dsRNA viruses reported for medfly. Our analysis across multiple laboratory-reared and field-collected medfly samples showed the presence of a core RNA virome comprised of Ceratitis capitata iflavirus 2 and Ceratitis capitata negev-like virus 1. Furthermore, field-collected flies showed a higher viral diversity in comparison to the laboratory-reared flies. Based on the small RNA sequencing, we detected small interfering RNAs mapping to all the viruses present in each sample, except for Ceratitis capitata nora virus. Although the identified RNA viruses do not cause obvious symptoms in medflies, the outcome of their interaction may still influence the medfly's fitness and ecology, becoming either a risk or an opportunity for mass-rearing and SIT applications.

A barley cysteine-proteinase inhibitor reduces the performance of two aphid species in artificial diets and transgenic Arabidopsis plants.

Cystatins from plants have been implicated in plant defense towards insects, based on their role as inhibitors of heterologous cysteine-proteinases. We have previously characterized thirteen genes encoding cystatins (HvCPI-1 to HvCPI-13) from barley (Hordeum vulgare), but only HvCPI-1 C68 → G, a variant generated by direct-mutagenesis, has been tested against insects. The aim of this study was to analyze the effects of the whole gene family members of barley cystatins against two aphids, Myzus persicae and Acyrthosiphon pisum. All the cystatins, except HvCPI-7, HvCPI-10 and HvCPI-12, inhibited in vitro the activity of cathepsin L- and/or B-like proteinases, with HvCPI-6 being the most effective inhibitor for both aphid species. When administered in artificial diets, HvCPI-6 was toxic to A. pisum nymphs (LC(50) = 150 µg/ml), whereas no significant mortality was observed on M. persicae nymphs up to 1000 µg/ml. The effects of HvCPI-6 ingestion on A. pisum were correlated with a decrease of cathepsin B- and L-like proteinase activities. In the case of M. persicae, there was an increase of these proteolytic activities, but also of the aminopeptidase-like activity, suggesting that this species is regulating both target and insensitive enzymes to overcome the effects of the cystatin. To further analyze the potential of barley cystatins as insecticidal proteins against aphids, Arabidopsis plants expressing HvCPI-6 were tested against M. persicae. For A. pisum, which does not feed on Arabidopsis, a combined diet-Vicia faba plant bioassay was performed. A significant delay in the development time to reach the adult stage was observed in both species. The present study demonstrates the potential of barley cystatins to interfere with the performance of two aphid species.

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases.

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests.

Contribution of cysteine and serine proteases to proteolytic digestion in an allergy-eliciting house dust mite.

The digestive physiology of house dust mites (HDM) is of interest to understand their allergenicity towards humans since many of their allergens are digestive enzymes and/or are excreted into airborne fecal pellets. The aim of this study is to provide insight on the biochemical basis of proteolytic digestion in Dermatophagoides pteronyssinus, the most widespread HDM species. First, assays using non-specific protein substrates on purified fecal and body extracts determined that body-associated activity is almost exclusively dependent on cysteine proteases, and specifically on major allergen Der p 1. By contrast, cysteine and serine proteases contributed similarly to the activity estimated on fecal extracts. Second, the screening of group-specific peptide-based protease inhibitors followed by ingestion bioassays revealed that the human skin-derived cysteine protease inhibitor cystatin A produces a significant reduction in mite feeding (i.e. excreted guanine), and triggers the overproduction of Der p 1 (3-fold increase by ELISA). Noteworthy, the inhibition of cysteine proteases by cystatin A also resulted in a reduction in three non-target serine protease activities. Further incubation of these extracts with exogenous Der p 1, but not with other commercial cysteine proteases, restored trypsin (Der p 3) and chymotrypsin (Der p 6) activities, indicating that Der p 1 is responsible for their activation in vivo. Finally, the role of serine proteases on the mite's digestive physiology is discussed based on their remarkable activity in fecal extracts and the autocoprophagic behavior reported in mites in this study.

Inheritance, Fitness Cost, and Management of Lambda-Cyhalothrin Resistance in a Laboratory-Selected Strain of Ceratitis capitata (Wiedemann).

The management of the medfly, Ceratitis capitata, in Spanish citrus crops relies mainly on the use of insecticides and the release of sterile males. However, the development of resistance to different insecticides in field populations, including lambda-cyhalothrin, implies a threat for the sustainable control of this pest. The inheritance, fitness cost, and management of lambda-cyhalothrin resistance were examined in the laboratory-selected W-1Kλ strain. We have demonstrated that lambda-cyhalothrin resistance in W-1Kλ is autosomic, completely dominant, and polygenic. In addition, individuals from W-1Kλ showed a lower embryo to pupal viability, a slower developmental time from egg to pupae, and an increase in adults' weight and longevity. We did not find significant trade-offs in the activity of digestive hydrolytic enzymes, with the exception of higher α-amylase activity in W-1Kλ females. A comparative study with different insecticide treatment strategies showed that lambda-cyhalothrin resistance increased when several consecutive treatments with this insecticide were applied. However, the alternation of this insecticide with spinosad was enough to delay the development of resistance. Our results indicate that the rotation of lambda-cyhalothrin with spinosad-a practice already used in some fields-may contribute to prevent the development of resistance.

Early Molecular Responses of Tomato to Combined Moderate Water Stress and Tomato Red Spider Mite Tetranychus evansi Attack.

Interaction between plants and their environment is changing as a consequence of the climate change and global warming, increasing the performance and dispersal of some pest species which become invasive species. Tetranychus evansi also known as the tomato red spider mite, is an invasive species which has been reported to increase its performance when feeding in the tomato cultivar Moneymaker (MM) under water deficit conditions. In order to clarify the underlying molecular events involved, we examined early plant molecular changes occurring on MM during T. evansi infestation alone or in combination with moderate drought stress. Hormonal profiling of MM plants showed an increase in abscisic acid (ABA) levels in drought-stressed plants while salicylic acid (SA) levels were higher in drought-stressed plants infested with T. evansi, indicating that SA is involved in the regulation of plant responses to this stress combination. Changes in the expression of ABA-dependent DREB2, NCED1, and RAB18 genes confirmed the presence of drought-dependent molecular responses in tomato plants and indicated that these responses could be modulated by the tomato red spider mite. Tomato metabolic profiling identified 42 differentially altered compounds produced by T. evansi attack, moderate drought stress, and/or their combination, reinforcing the idea of putative manipulation of tomato plant responses by tomato red spider mite. Altogether, these results indicate that the tomato red spider mite acts modulating plant responses to moderate drought stress by interfering with the ABA and SA hormonal responses, providing new insights into the early events occurring on plant biotic and abiotic stress interaction.

Field detection and predicted evolution of spinosad resistance in Ceratitis capitata.

BACKGROUND: The sustainable control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is compromised by the development of resistance to malathion and lambda-cyhalothrin in Spanish field populations. At present, field populations remain susceptible to spinosad. However, the resistant strain JW-100s has been obtained under laboratory selection with spinosad, and resistance has been associated with the presence of different mutations causing truncated transcripts of the α6 subunit of the nicotinic acetylcholine receptor (nAChRα6). RESULTS: An F1 screen assay followed by the molecular characterization of surviving flies has been used to search for spinosad-resistant alleles in field populations. Two different resistant alleles giving rise to truncated isoforms of Ccα6 have been identified, which corresponds to an estimated allelic frequency of at least 0.0023-0.0046. The fitness values of the resistant nAChRα6 alleles found in the laboratory strain JW-100s were estimated to be 0.4 for RR and 0.2 for SR. Mathematical modelling predicted that spinosad-resistant alleles will rapidly decline over time in field populations if their fitness cost was the same as estimated for laboratory-resistant alleles. However, they are predicted to increase in the field if their fitness cost is lower and resistance management strategies are not implemented. CONCLUSION: Spinosad-resistant alleles have been detected in field populations for the first time. Our modelling simulations indicate that the best option to delay the appearance of spinosad resistance would be its rotation with other insecticides without cross-resistance. The integrated F1 screen/molecular genetic analysis presented here can be used for future monitoring studies. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Bt maize fed-prey mediated effect on fitness and digestive physiology of the ground predator Poecilus cupreus L. (Coleoptera: Carabidae).

We investigated the effects of a Bt maize hybrid on fitness and digestive physiology of the ground-dwelling predator Poecilus cupreus L., as compared with the near-isogenic hybrid. A tritrophic assay revealed that there was a great decline in the detection of Cry1Ab toxin through the trophic chain, the concentration of the toxin being 945, 349 and 37 ng g(-1) of fresh weight in Bt maize leaves, Spodoptera littoralis (Boisduval) larvae and P. cupreus larvae, respectively. Moreover, the toxin was only detected in 8% of the P. cupreus adults collected from fields growing Bt maize. Developmental time of both larvae and pupae of P. cupreus was not adversely affected by the Cry1Ab toxin via fed-prey. To elucidate potential detrimental effects due to a reduction in the quality of the prey, we assessed the digestive proteolytic activities of P. cupreus adults from a laboratory culture and insects collected in commercial Bt and non-Bt maize fields. Field-collected P. cupreus adults had higher proteolytic activities than those reared in the laboratory, whereas no significant differences were found between P. cupreus adults reared on Bt and non-Bt maize fed-S. littoralis or between P. cupreus adults collected in commercial Bt and non-Bt maize fields.

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.

Mechanisms of resistance to malathion in the medfly Ceratitis capitata.

Target site insensitivity and metabolic resistance mediated by esterases have been previously suggested to be involved in resistance to malathion in a field-derived strain (W) of Ceratitis capitata. In the present study, we have obtained the coding sequence for acetylcholinesterase (AChE) gene (Ccace) of C. capitata. An allele of Ccace carrying only a point mutation Gly328Ala (Torpedo numbering) adjacent to the glutamate of the catalytic triad was found in individuals of the W strain. Adult flies homozygotes for this mutant allele showed reduced AChE activity and less sensitivity to inhibition by malaoxon, showing that target site insensitivity is one of the factors of malathion resistance. In addition, all individuals from the resistant W strain showed reduced aliesterase activity, which has been associated with specific malathion resistance in higher Diptera. However, the alphaE7 gene (CcalphaE7), sequenced in susceptible and resistant individuals, did not carry any of the mutations associated with organophosphorus insecticide resistance in other Diptera. Another esterase mechanism, perhaps a carboxylesterase selective for malathion, in addition to mutant AChE, thus contributes to malathion resistance in C. capitata.