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Abstract: A number of horizontal gene transfers (HGTs) have been identified in the spider mite Tetranychus urticae, a chelicerate herbivore. However, the genome of this mite species has at present not been thoroughly mined for the presence of HGT genes. Here, we performed a systematic screen for HGT genes in the T. urticae genome using the h‐index metric. Our results not only validated previously identified HGT genes but also uncovered 25 novel HGT genes. In addition to HGT genes with a predicted biochemical function in carbohydrate, lipid and folate metabolism, we also identified the horizontal transfer of a ketopantoate hydroxymethyltransferase and a pantoate β‐alanine ligase gene. In plants and bacteria, both genes are essential for vitamin B5 biosynthesis and their presence in the mite genome strongly suggests that spider mites, similar to Bemisia tabaci and nematodes, can synthesize their own vitamin B5. We further show that HGT genes were physically embedded within the mite genome and were expressed in different life stages. By screening chelicerate genomes and transcriptomes, we were able to estimate the evolutionary histories of these HGTs during chelicerate evolution. Our study suggests that HGT has made a significant and underestimated impact on the metabolic repertoire of plant‐feeding spider mites. [ABSTRACT FROM AUTHOR]

Bifenazate is a recently developed acaricide that is mainly used to control spider mites on a variety of crops. Although first thought to be a neurotoxin, genetic evidence obtained from bifenazate resistant Tetranychus urticae strains suggested an alternative mode of action as a Qo pocket inhibitor of the mitochondrial complex III. In this study, we reveal how bifenazate resistance in strains of Panonychus citri is maternally inherited and can confer cross-resistance to the known Qo inhibitor acequinocyl. The mitochondrial genome of P. citri was sequenced and Qo pocket mutations were shown to be linked with the resistant trait. Parallel evolution of cytochrome b mediated bifenazate resistance corroborates the alternative mode of action and yet again illustrates that care should be taken when employing Qo inhibitors as crop protection compounds. [ABSTRACT FROM AUTHOR]

Macrolophus pygmaeus is a heteropteran predator that is widely used in European glasshouses for the biological control of whiteflies, aphids, thrips and spider mites. We have demonstrated that the insect is infected with the endosymbiotic bacterium Wolbachia pipientis. Several gene fragments of the endosymbiont were sequenced and subsequently used for phylogenetic analysis, revealing that it belongs to the Wolbachia supergroup B. The endosymbiont was visualized within the ovarioles using immunolocalization. Tetracycline treatments were used to cure M. pygmaeus from its infection. Although a completely cured line could not be obtained by this approach, the application of a constant antibiotic pressure over 13 generations resulted in a line with a significantly reduced Wolbachia concentration. Crosses performed with this tetracycline-treated line revealed that the endosymbiont causes severe cytoplasmic incompatibility. This is the first report of a reproductive effect induced by Wolbachia in an economically important heteropteran predator that may have vital implications for its commercial production and use in biological control. [ABSTRACT FROM AUTHOR]

Abstract Background The apparent scarcity of available sequence data has greatly impeded evolutionary studies in Acari (mites and ticks). This subclass encompasses over 48,000 species and forms the largest group within the Arachnida. Although mitochondrial genomes are widely utilised for phylogenetic and population genetic studies, only 20 mitochondrial genomes of Acari have been determined, of which only one belongs to the diverse order of the Sarcoptiformes. In this study, we describe the mitochondrial genome of the European house dust mite Dermatophagoides pteronyssinus, the most important member of this largely neglected group. Results The mitochondrial genome of D. pteronyssinus is a circular DNA molecule of 14,203 bp. It contains the complete set of 37 genes (13 protein coding genes, 2 rRNA genes and 22 tRNA genes), usually present in metazoan mitochondrial genomes. The mitochondrial gene order differs considerably from that of other Acari mitochondrial genomes. Compared to the mitochondrial genome of Limulus polyphemus, considered as the ancestral arthropod pattern, only 11 of the 38 gene boundaries are conserved. The majority strand has a 72.6% AT-content but a GC-skew of 0.194. This skew is the reverse of that normally observed for typical animal mitochondrial genomes. A microsatellite was detected in a large non-coding region (286 bp), which probably functions as the control region. Almost all tRNA genes lack a T-arm, provoking the formation of canonical cloverleaf tRNA-structures, and both rRNA genes are considerably reduced in size. Finally, the genomic sequence was used to perform a phylogenetic study. Both maximum likelihood and Bayesian inference analysis clustered D. pteronyssinus with Steganacarus magnus, forming a sistergroup of the Trombidiformes. Conclusion Although the mitochondrial genome of D. pteronyssinus shares different features with previously characterised Acari mitochondrial genomes, it is unique in many ways. Gene order is extremely rearranged and represents a new pattern within the Acari. Both tRNAs and rRNAs are truncated, corroborating the theory of the functional co-evolution of these molecules. Furthermore, the strong and reversed GC- and AT-skews suggest the inversion of the control region as an evolutionary event. Finally, phylogenetic analysis using concatenated mt gene sequences succeeded in recovering Acari relationships concordant with traditional views of phylogeny of Acari.

Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests., Competing Interests: Declaration of interests The authors declare that a priority claim for the above-described method has been submitted to the European Patent Office., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the β-adrenergic-like octopamine receptor (Octβ2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octβ2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octβ2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mitochondrial electron transfer inhibitors at complex II (METI-II), also referred to as succinate dehydrogenase inhibitors (SDHI), represent a recently developed class of acaricides encompassing cyflumetofen, cyenopyrafen, pyflubumide and cyetpyrafen. Despite their novelty, resistance has already developed in the target pest, Tetranychus urticae. In this study a new mutation, H146Q in a highly conserved region of subunit B of complex II, was identified in a T. urticae population resistant to all METI-IIs. In contrast to previously described mutations, H146Q is located outside the ubiquinone binding site of complex II. Marker-assisted backcrossing of this mutation in a susceptible genetic background validated its association with resistance to cyflumetofen and pyflubumide, but not cyenopyrafen or cyetpyrafen. Biochemical assays and the construction of inhibition curves with isolated mitochondria corroborated this selectivity. In addition, phenotypic effects of H146Q, together with the previously described H258L, were further examined via CRISPR/Cas9 gene editing. Although both mutations were successfully introduced into a susceptible T. urticae population, the H146Q gene editing event was only recovered in individuals already harboring the I260V mutation, known to confer resistance towards cyflumetofen. The combination of H146Q + I260V conferred high resistance levels to all METI-II acaricides with LC 50 values over 5000 mg a.i./L for cyflumetofen and pyflubumide. Similarly, the introduction of H258L via gene editing resulted in high resistance levels to all tested acaricides, with extreme LC 50 values (>5000 mg a.i./L) for cyenopyrafen and cyetpyrafen, but lower resistance levels for pyflubumide and cyflumetofen. Together, these findings indicate that different mutations result in a different cross-resistance spectrum, probably also reflecting subtle differences in the binding mode of complex II acaricides., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate plant defense responses. Here, we present a set of proteins identified in an artificial diet on which B. tabaci was salivating. We subsequently studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of an induced- reactive oxygen species (ROS) response in Nicotiana benthamiana. In addition, G4 was able to suppress ROS production in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). G4 localized predominantly in the endoplasmic reticulum in N. benthamiana leaves and colocalized with two identified target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato reduced whitefly fecundity up to 40%, demonstrating that the protein is involved in susceptibility to B. tabaci . Together, our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with ROS production and via an interaction with tomato susceptibility protein MIPDB141. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool has proven its use in genetic studies for various arthropods. However, most transformation protocols rely on microinjection of CRISPR/Cas9 components into embryos, a method which is challenging for many species. Alternatively, injections can be performed on adult females, but transformation efficiencies can be very low as was shown for the two-spotted spider mite, Tetranychus urticae, a minute but important chelicerate pest on many crops. In this study, we explored different CRISPR/Cas9 formulations to optimize a maternal injection protocol for T. urticae. We observed a strong synergy between branched amphipathic peptide capsules and saponins, resulting in a significant increase of CRISPR/Cas9 knock-out efficiency, exceeding 20%. This CRISPR/Cas9 formulation, termed SYNCAS, was used to knock-out different T. urticae genes - phytoene desaturase, CYP384A1 and Antennapedia - but also allowed to develop a co-CRISPR strategy and facilitated the generation of T. urticae knock-in mutants. In addition, SYNCAS was successfully applied to knock-out white and white-like genes in the western flower thrips, Frankliniella occidentalis. The SYNCAS method allows routine genome editing in these species and can be a game changer for genetic research in other hard to transform arthropods., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dicofol has been widely used to control phytophagous mites. Although dicofol is chemically related to DDT, its mode of action has remained elusive. Here, we mapped dicofol resistance in the spider mite Tetranychus urticae to two genomic regions. Each region harbored a glutamate-gated chloride channel (GluCl) gene that contained a mutation-G314D or G326E-known to confer resistance against the unrelated acaricide abamectin. Using electrophysiology assays we showed that dicofol and other diphenylcarbinol acaricides-bromopropylate and chlorobenzilate-induce persistent currents in Xenopus oocytes expressing wild-type T. urticae GluCl3 receptors and potentiate glutamate responses. In contrast, the G326E substitution abolished the agonistic activity of all three compounds. Assays with the wild-type Drosophila GluClα revealed that this receptor was unresponsive to dicofol. Homology modeling combined with ligand-docking confirmed the specificity of electrophysiology assays. Altogether, this work elucidates the mode of action of diphenylcarbinols as mite-specific agonists of GluCl., (© 2023. The Author(s).)

The Arachnida subclass of Acari comprises many harmful pests that threaten agriculture as well as animal health, including herbivorous spider mites, the bee parasite Varroa, the poultry mite Dermanyssus and several species of ticks. Especially in agriculture, acaricides are often used intensively to minimize the damage they inflict, promoting the development of resistance. Beneficial predatory mites used in biological control are also subjected to acaricide selection in the field. The development and use of new genetic and genomic tools such as genome and transcriptome sequencing, bulked segregant analysis (QTL mapping), and reverse genetics via RNAi or CRISPR/Cas9, have greatly increased our understanding of the molecular genetic mechanisms of resistance in Acari, especially in the spider mite Tetranychus urticae which emerged as a model species. These new techniques allowed to uncover and validate new resistance mutations in a larger range of species. In addition, they provided an impetus to start elucidating more challenging questions on mechanisms of gene regulation of detoxification associated with resistance., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pesticide resistance represents a clear and trackable case of adaptive evolution with a strong societal impact. Understanding the factors associated with the evolution and spread of resistance is imperative to develop sustainable crop management strategies. The two-spotted spider mite Tetranychus urticae, a major crop pest with worldwide distribution and a polyphagous lifestyle, has evolved resistance to most classes of pesticides. Tetranychus urticae exists as either a green- or a red-coloured morph. However, the extent of genetic divergence and reproductive compatibility vary across populations of these colour morphs, complicating their taxonomic resolution at the species level. Here, we studied patterns of genetic differentiation and barriers to gene flow within and between morphs of T. urticae in order to understand the factors that influence the spread of resistance mutations across its populations. We derived multiple iso-female lines from Tetranychus populations collected from agricultural crops. We generated genomic and morphological data, characterized their bacterial communities and performed controlled crosses. Despite morphological similarities, we found large genomic differentiation between the morphs. This pattern was reflected in the incomplete, but strong postzygotic incompatibility in crosses between colour morphs, while crosses within morphs from different geographical locations were largely compatible. In addition, our results suggest recent/on-going gene flow between green-coloured T. urticae and T. turkestani. By screening the sequences of 10 resistance genes, we found evidence for multiple independent origins and for single evolutionary origins of target-site resistance mutations. Our results indicate that target-site mutations mostly evolve independently in populations on different geographical locations, and that these mutations can spread due to incomplete barriers to gene flow within and between populations., (© 2023 John Wiley & Sons Ltd.)

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying., Competing Interests: Declaration of Competing Interest The authors declare no competing of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pesticide resistance relies on a myriad of mechanisms, ranging from single mutations to a complex and polygenic architecture, and it involves mechanisms such as target-site insensitivity, metabolic detoxification, or a combination of these, with either additive or synergistic effects. Several resistance mechanisms against abamectin, a macrocyclic lactone widely used in crop protection, have been reported in the cosmopolitan pest Tetranychus urticae . However, it has been shown that a single mechanism cannot account for the high levels of abamectin resistance found across different mite populations. Here, we used experimental evolution combined with bulked segregant analyses to map quantitative trait loci (QTL) associated with abamectin resistance in two genetically unrelated populations of T. urticae . In these two independent QTL mapping experiments, three and four QTLs were identified, of which three were shared between experiments. Shared QTLs contained genes encoding subunits of the glutamate-gated chloride channel (GluCl) and harboured previously reported mutations, including G314D in GluCl1 and G326E in GluCl3 , but also novel resistance candidate loci, including DNA helicases and chemosensory receptors. Surprisingly, the fourth QTL, present only in only one of the experiments and thus unique for one resistant parental line, revealed a non-functional variant of GluCl2 , suggesting gene knock-out as resistance mechanism. Our study uncovers the complex basis of abamectin resistance, and it highlights the intraspecific diversity of genetic mechanisms underlying resistance in a cosmopolitan pest., Competing Interests: The authors declare that they have no conflict of interest related to this manuscript., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Background: Afoxolaner is a novel representative of the isoxazolines, a class of ectoparasiticides which has been commercialized for the control of tick and flea infestations in dogs. In this study, the biological efficacy of afoxolaner against the two-spotted spider mite Tetranychus urticae was evaluated. Furthermore, as isoxazolines are known inhibitors of γ-aminobutyric acid-gated chloride channels (GABACls), the molecular mode of action of afoxolaner on T. urticae GABACls (TuRdls) was studied using functional expression in Xenopus oocytes followed by two-electrode voltage-clamp (TEVC) electrophysiology, and results were compared with inhibition by fluralaner, fipronil and endosulfan. To examine the influence of known GABACl resistance mutations, H301A, I305T and A350T substitutions in TuRdl1 and a S301A substitution in TuRdl2 were introduced., Results: Bioasassays revealed excellent efficacy of afoxolaner against all developmental stages and no cross-resistance was found in a panel of strains resistant to most currently used acaricides. Laboratory selection over a period of 3 years did not result in resistance. TEVC revealed clear antagonistic activity of afoxolaner and fluralaner for all homomeric TuRdl1/2/3 channels. The introduction of single, double or triple mutations to TuRdl1 and TuRdl2 did not lower channel sensitivity. By contrast, both endosulfan and fipronil had minimal antagonistic activities against TuRdl1/2/3, and channels carrying single mutations, whereas the sensitivity of double and triple TuRdl1 mutants was significantly increased., Conclusions: Our results demonstrate that afoxolaner is a potent antagonist of GABACls of T. urticae and has a powerful mode of action to control spider mites. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Pyrethroids are widely applied insecticides in agriculture, but their frequent use has provoked many cases of resistance, in which mutations in the voltage-gated sodium channel (VGSC), the pyrethroid target-site, were shown to play a major role. However, for the spider mite Tetranychus urticae , it has also been shown that increased detoxification contributes to resistance against the pyrethroid bifenthrin. Here, we performed QTL-mapping to identify the genomic loci underlying bifenthrin resistance in T. urticae . Two loci on chromosome 1 were identified, with the VGSC gene being located near the second QTL and harboring the well-known L1024V mutation. In addition, the presence of an L925M mutation in the VGSC of a highly bifenthrin-resistant strain and its loss in its derived, susceptible, inbred line indicated the importance of target-site mutations in bifenthrin resistance. Further, RNAseq experiments revealed that genes encoding detoxification enzymes, including carboxyl/choline esterases (CCEs), cytochrome P450 monooxygenases and UDP-glycosyl transferases (UGTs), were overexpressed in resistant strains. Toxicity bioassays with bifenthrin (ester pyrethroid) and etofenprox (non-ester pyrethroid) also indicated a possible role for CCEs in bifenthrin resistance. A selection of CCEs and UGTs were therefore functionally expressed, and CCEinc18 was shown to metabolize bifenthrin, while teturUGT10 could glycosylate bifenthrin-alcohol. To conclude, our findings suggest that both target-site and metabolic mechanisms underlie bifenthrin resistance in T. urticae , and these might synergize high levels of resistance.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is the most economically important mite pest in agricultural areas and chemical acaricides are widely used to control T. urticae populations. Cyflumetofen is a recently introduced acaricide that inhibits the mitochondrial electron transport chain at complex II (succinate dehydrogenase, SDH), which represents the most recently developed mode of action for mite control worldwide. In the present study, started upon the launch of cyflumetofen in Turkey, a five-year survey was performed to monitor cyflumetofen susceptibility in 28 T. urticae populations collected from agricultural fields across the country. The first resistance case that might cause control failure in practical field conditions was uncovered in 2019, three years after the registration of cyflumetofen. In addition, an extremely resistant population (1722-fold resistance) was also detected towards the end of 2019. Cyflumetofen resistance did not decrease in the laboratory after relaxation of selection pressure for over one year in field-collected populations, suggesting the absence of a fitness cost associated with resistance in these populations. Next to phenotypic resistance, metabolic and physiological mechanisms underlying the decreased susceptibility were also investigated. Synergism assays showed the involvement of P450 monooxygenases in cyflumetofen resistance. Downregulation of carboxylesterases as resistance mechanism, is underpinned by the fact that pre-treatment with esterase inhibitor DEF decreased cyflumetofen toxicity in field-collected strains. Furthermore, a novel H258L substitution in the subunit B of complex II was uncovered in a field population. In silico modeling of the new mutation suggested that the mutation might indeed influence toxicity to complex II inhibitors cyenopyrafen and pyflubumide, but most likely not cyflumetofen. However, further studies are needed to uncover the exact role of this mutation in resistance to this new class of complex II inhibitors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Generalist herbivores such as the two-spotted spider mite Tetranychus urticae thrive on a wide variety of plants and can rapidly adapt to novel hosts. What traits enable polyphagous herbivores to cope with the diversity of secondary metabolites in their variable plant diet is unclear. Genome sequencing of T. urticae revealed the presence of 17 genes that code for secreted proteins with strong homology to "intradiol ring cleavage dioxygenases (DOGs)" from bacteria and fungi, and phylogenetic analyses show that they have been acquired by horizontal gene transfer from fungi. In bacteria and fungi, DOGs have been well characterized and cleave aromatic rings in catecholic compounds between adjacent hydroxyl groups. Such compounds are found in high amounts in solanaceous plants like tomato, where they protect against herbivory. To better understand the role of this gene family in spider mites, we used a multi-disciplinary approach to functionally characterize the various T. urticae DOG genes., Results: We confirmed that DOG genes were present in the T. urticae genome and performed a phylogenetic reconstruction using transcriptomic and genomic data to advance our understanding of the evolutionary history of spider mite DOG genes. We found that DOG expression differed between mites from different plant hosts and was induced in response to jasmonic acid defense signaling. In consonance with a presumed role in detoxification, expression was localized in the mite's gut region. Silencing selected DOGs expression by dsRNA injection reduced the mites' survival rate on tomato, further supporting a role in mitigating the plant defense response. Recombinant purified DOGs displayed a broad substrate promiscuity, cleaving a surprisingly wide array of aromatic plant metabolites, greatly exceeding the metabolic capacity of previously characterized microbial DOGs., Conclusion: Our findings suggest that the laterally acquired spider mite DOGs function as detoxification enzymes in the gut, disarming plant metabolites before they reach toxic levels. We provide experimental evidence to support the hypothesis that this proliferated gene family in T. urticae is causally linked to its ability to feed on an extremely wide range of host plants., (© 2022. The Author(s).)

The organotin acaricide fenbutatin oxide (FBO) - an inhibitor of mitochondrial ATP-synthase - has been one of the most extensively used acaricides for the control of spider mites, and is still in use today. Resistance against FBO has evolved in many regions around the world but only few studies have investigated the molecular and genetic mechanisms of resistance to organotin acaricides. Here, we found that FBO resistance is polygenic in two genetically distant, highly resistant strains of the spider mite Tetranychus urticae, MAR-AB and MR-VL. To identify the loci underlying FBO resistance, two independent bulked segregant analysis (BSA) based QTL mapping experiments, BSA MAR-AB and BSA MR-VL, were performed. Two QTLs on chromosome 1 were associated with FBO resistance in each mapping experiment. At the second QTL of BSA MAR-AB, several cytochrome P450 monooxygenase (CYP) genes were located, including CYP392E4, CYP392E6 and CYP392E11, the latter being overexpressed in MAR-AB. Synergism tests further implied a role for CYPs in FBO resistance. Subunit c of mitochondrial ATP-synthase was located near the first QTL of both mapping experiments and harbored a unique V89A mutation enriched in the resistant parents and selected BSA populations. Marker-assisted introgression into a susceptible strain demonstrated a moderate but significant effect of the V89A mutation on toxicity of organotin acaricides. The impact of the mutation on organotin inhibition of ATP synthase was also functionally confirmed by ATPase assays on mitochondrial preparations. To conclude, our findings suggest that FBO resistance in the spider mite T. urticae is a complex interplay between CYP-mediated detoxification and target-site resistance., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Tetranychus urticae is a polyphagous spider mite that can feed on more than 1100 plant species including cyanogenic plants. The herbivore genome contains a horizontally acquired gene tetur10g01570 (TuCAS) that was previously shown to participate in cyanide detoxification. To understand the structure and determine the function of TuCAS in T. urticae, crystal structures of the protein with lysine conjugated pyridoxal phosphate (PLP) were determined. These structures reveal extensive TuCAS homology with the β-substituted alanine synthase family, and they show that this enzyme utilizes a similar chemical mechanism involving a stable α-aminoacrylate intermediate in β-cyanoalanine and cysteine synthesis. We demonstrate that TuCAS is more efficient in the synthesis of β-cyanoalanine, which is a product of the detoxification reaction between cysteine and cyanide, than in the biosynthesis of cysteine. Also, the enzyme carries additional enzymatic activities that were not previously described. We show that TuCAS can detoxify cyanide using O-acetyl-L-serine as a substrate, leading to the direct formation of β-cyanoalanine. Moreover, it catalyzes the reaction between the TuCAS-bound α-aminoacrylate intermediate and aromatic compounds with a thiol group. In addition, we have tested several compounds as TuCAS inhibitors. Overall, this study identifies additional functions for TuCAS and provides new molecular insight into the xenobiotic metabolism of T. urticae., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In the present study, the acaricide susceptibility status of Hyalomma marginatum in Morocco was investigated in the Rabat-Sale-Kenitra region using the Larval Packet Test. The overall LC 50 value for diazinon and amitraz was 115 ppm (95% CI: [104; 125]) and 22 ppm (95% CI: [21; 23]), respectively. The LC 50 values varied significantly between the nine sampled locations (P<0.001) ranging from 75 ppm (95% CI: [65; 84]) in Ouelmes to 179 ppm (95% CI: [139; 201]) in Jorf El Melha for diazinon and from 18 ppm (95% CI: [15; 21]) in Skhirat to 28 ppm (95% CI: [24; 31]) in Ouelmes for amitraz. Sequencing of the target-site of diazinon, acetylcholinesterase 1 (AChE1), indicated that previously reported resistance mutations in AChE1 were absent in ticks from Jorf El Melha surviving 500 ppm diazinon. This study is the first report on the H. marginatum susceptibilty status to the most frequently used acaricides in Morocco and indicates that acaricide tick resistance is emerging., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Pyridaben is a mitochondrial electron transport complex I inhibitor. The H110R mutation in the PSST subunit has been reported as a major factor in pyridaben resistance in the two-spotted spider mite, Tetranychus urticae. However, backcross experiments revealed that the mutant PSST alone conferred only moderate resistance. In contrast, inhibition of cytochrome P450 (CYP) markedly reduces resistance levels in a number of highly resistant strains. It was reported previously that maternal factors contributed to the inheritance of pyridaben resistance in the egg stage, but the underlying mechanisms have yet to be elucidated. Here, we studied the combined effects of the PSST H110R mutation and candidate CYPs, as metabolic resistance factors, on pyridaben resistance in T. urticae. We found that the maternal effects of inheritance of resistance in the egg stage were associated with CYP activity. Analysis of differential gene expression by RNA-seq identified CYP392A3 as a candidate causal factor for the high resistance level. Congenic strains, where the alleles of both PSST and CYP392A3 were derived from a resistant strain (RR&#95;i; i = 1 or 2) and a susceptible strain (SS&#95;i) in a common susceptible genetic background, were constructed by marker-assisted backcrossing. RR&#95;i showed upregulation of CYP392A3 and high resistance levels (LC 50  > 10,000 mg L -1 ), while SS&#95;i had LC 50  < 10 mg L -1 . To disentangle the individual effects of PSST and CYP392A3 alleles, we also attempted to uncouple these genes in RR&#95;i. We conclude that given the variation in LC 50 values and expression levels of CYP392A3 in the congenic and uncoupled strains, it is likely that the high pyridaben resistance levels are due to a synergistic or cumulative effect of the combination of mutant PSST and associated CYPs, including CYP392A3, but other yet to be discovered factors cannot be excluded., (Copyright © 2021 Elsevier Inc. All rights reserved.)

True fruit flies (Tephritidae) are among the most destructive agricultural pests in the world, attacking a wide range of fruits and vegetables. The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous species but, being widely established in the Mediterranean region, is not considered as a EU quarantine pest. Hence, it is important to discriminate Ceratitis capitata from non-EU tephritid species, present in imported fruit and vegetables, as non-EU species have a quarantine status. However, morphological identification of tephritid larvae, the most frequently intercepted stage in non-EU produce, is difficult and an easy-to-use molecular diagnostic tool would be helpful for rapid species identification. Therefore, a loop-mediated isothermal amplification (LAMP) method was developed for C. capitata and non-EU tephritids Ceratitis cosyra group1 and Ceratitis species from the FARQ complex, C. fasciventris, C. anonae, C. rosa and C. quilicii . LAMP assays were run with DNA from ILVO collected specimens and DNA samples collected during previous research surveys. LAMP primers were species-specific, with LAMP amplification occurring within 45 minutes for the targeted species. In addition, LAMP assays were successful for all C. capitata life stages or a limited amount of tissue. To conclude, the LAMP assays developed in this study were able to distinguish C. capitata from non-EU Tephritidae species and could be a useful tool for the rapid identification of C. capitata ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s). Published by Elsevier B.V.)

The poultry red mite, Dermanyssus gallinae, is the most important blood sucking ectoparasite of egg laying hens and causes economic losses in poultry farms worldwide. Although various management methods exist, the control of poultry red mites (PRMs) mainly relies on acaricides such as pyrethroids and organophosphates (OPs) in many regions of the world. However, repeated use of these synthetic chemicals has resulted in resistance development causing control failure of PRM. In this study, we investigated acaricide resistance mechanisms of Turkish PRM populations. First, we obtained the COI sequence from 30 PRM populations from different regions in Turkey and identified four different COI haplotypes. Toxicity assays showed that four field-collected PRM populations were highly resistant to the pyrethroid alpha-cypermethrin, with resistance ratios (RRs) varying between 100- and 400-fold, while two of these populations had a RR of more than 24-fold against the OP acaricide phoxim. Biochemical assays showed a relatively higher activity of glutathione-S-transferases and carboxyl-cholinesterases, two well-known classes of detoxification enzymes, in one of these resistant populations. In addition, we also screened for mutations in the gene encoding the voltage-gated sodium channel (vgsc) and acetylcholinesterase 1 (ace-1), the target-site of pyrethroids and OPs, respectively. In all but two PRM populations, at least one vgsc mutation was detected. A total of four target-site mutations, previously associated with pyrethroid resistance, M918T, T929I, F1534L, F1538L were found in domain II and III of the VGSC. The T929I mutation was present in the vgsc of almost all PRM populations, while the other mutations were only found at low frequency. The G119S/A mutation in ace-1, previously associated with OP resistance, was found in PRM for the first time and present in fourteen populations. Last, both alive and dead PRMs were genotyped after pesticide exposure and supported the possible role of target-site mutations, T929I and G119S, in alpha-cypermethrin and phoxim resistance, respectively. To conclude, our study provides a current overview of resistance levels and resistance mutations in Turkish PRM populations and might aid in the design of an effective resistance management program of PRM in Turkey., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tick-borne pathogens cause the majority of diseases in the cattle population in Morocco. In this study, ticks were collected from cattle in the Rabat-Sale-Kenitra region of Morocco and identified morphologically, while tick-borne pathogens were detected in cattle blood samples via polymerase chain reaction assay and sequencing. A total of 3394 adult ixodid ticks were collected from cattle and identified as eight different tick species representing two genera, Hyalomma and Rhipicephalus . The collected ticks consisted of Hyalomma marginatum , Hyalomma anatolicum excavatum , Rhipicephalus sanguineus sensu lato , Rhipicephalus bursa , Hyalomma detritum , Hyalomma lusitanicum , Hyalomma dromedarii , and Hyalomma impeltatum . The overall prevalence of tick-borne pathogens in blood samples was 63.8%, with 29.3% positive for Babesia / Theileria spp., 51.2% for Anaplasma / Ehrlichia spp., and none of the samples positive for Rickettsia spp. Sequencing results revealed the presence of Theileria annulata , Babesia bovis , Anaplasma marginale , Theileria buffeli , Theileria orientalis , Babesia occultans , Anaplasma phagocytophilum , Anaplasma capra , Anaplasma platys , Anaplasma bovis , Ehrlichia minasensis , and one isolate of an unknown bovine Anaplasma sp. Crossbreeds, females, older age, and high tick infestation were the most important risk factors for the abundance of tick-borne pathogens, which occurred most frequently in Jorf El Melha, Sidi Yahya Zaer, Ait Ichou, and Arbaoua locations.

Background: Brine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors., Results and Discussion: Here, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions., Conclusions: A relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research., (© 2021. The Author(s).)

Background: The cys-loop ligand-gated ion channels, including the glutamate-gated chloride channel (GluCl) and GABA-gated chloride channel (Rdl) are important targets for drugs and pesticides. The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite Tetranychus urticae, an economically important crop pest. However, abamectin resistance has been reported for multiple T. urticae populations worldwide, and in several cases was associated with the mutations G314D in GluCl1 and G326E in GluCl3. Recently, an additional I321T mutation in GluCl3 was identified in several abamectin resistant T. urticae field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength., Results: The GluCl3 I321T mutation was introgressed into a T. urticae susceptible background by marker-assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50-fold. Next, we used CRISPR-Cas9 to introduce I321T, G314D and G326E in the orthologous Drosophila GluCl. Genome modified flies expressing GluCl I321T were threefold less susceptible to abamectin, while CRISPRed GluCl G314D and G326E flies were lethal. Last, functional analysis in Xenopus oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three T. urticae Rdls are affected by abamectin., Conclusion: Three different techniques were used to characterize the role of I321T in GluCl3 in abamectin resistance and, combining all results, our analysis suggests that the I321T mutation has a complex role in abamectin resistance. Given the reported subtle effect, additional synergistic factors in resistance warrant more investigation. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Varroa destructor is one of the main problems in modern beekeeping. Highly selective acaricides with low toxicity to bees are used internationally to control this mite. One of the key acaricides is the organophosphorus (OP) proinsecticide coumaphos, that becomes toxic after enzymatic activation inside Varroa We show here that mites from the island Andros (AN-CR) exhibit high levels of coumaphos resistance. Resistance is not mediated by decreased coumaphos uptake, target-site resistance, or increased detoxification. Reduced proinsecticide activation by a cytochrome P450 enzyme was the main resistance mechanism, a powerful and rarely encountered evolutionary solution to insecticide selection pressure. After treatment with sublethal doses of [ 14 C] coumaphos, susceptible mite extracts had substantial amounts of coroxon, the activated metabolite of coumaphos, while resistant mites had only trace amounts. This indicates a suppression of the P450 (CYP)-mediated activation step in the AN-CR mites. Bioassays with coroxon to bypass the activation step showed that resistance was dramatically reduced. There are 26 CYPs present in the V. destructor genome. Transcriptome analysis revealed overexpression in resistant mites of CYP4DP24 and underexpression of CYP3012A6 and CYP4EP4 RNA interference of CYP4EP4 in the susceptible population, to mimic underexpression seen in the resistant mites, prevented coumaphos activation and decreased coumaphos toxicity., Competing Interests: The authors declare no competing interest.

Background: Tetranychus urticae is a notorious crop pest with a worldwide distribution that has developed resistance to a wide range of acaricides. Here, we investigated the resistance levels of a T. urticae population collected from an ornamental greenhouse in Peloponnese, Greece, and analyzed its resistance mechanisms at the molecular level., Results: Toxicological assays showed resistance against compounds with different modes of action, with resistance ratios of: 89-fold for abamectin; > 1000-fold for clofentezine; > 5000-fold for etoxazole; 27-fold for fenpyroximate and pyridaben; 20- and 36-fold for spirodiclofen and spirotetramat, respectively; and 116- and > 500-fold for cyenopyrafen and cyflumetofen, respectively. Bioassays with synergists indicated the involvement of detoxification enzymes in resistance to abamectin, but not to cyflumetofen and spirodiclofen. RNA sequencing (RNA-seq) analysis showed significant over-expression of several genes encoding detoxification enzymes such as cytochrome P450 monooxygenases and UDP-glycosyltransferases, which have been previously associated with acaricide resistance. Known target-site resistance mutations were identified in acetyl-choline esterase, chitin synthase 1 and NDUFS7/psst, but putative novel resistance mutations were also discovered in targets such as glutamate-gated chloride channel subunit 3. Interestingly, target-site resistance mutations against pyrethroids or bifenazate were not identified, possibly indicating a recent reduced selection pressure in Greece, as well as a possible opportunity to rotate these chemistries., Conclusion: We identified and characterized a striking case of multiple acaricide resistance in a field population of T. urticae. Exceptionally strong resistance phenotypes, with accumulation of multiple resistance mutations and over-expression of P450s and other detoxification genes in the same field population are reported., (© 2020 Society of Chemical Industry.)

Cytochrome P450 monooxygenases (P450s) play a key role in the detoxification of phytochemicals in arthropod herbivores. We present here an overview of recent progress in understanding the breadth and specificity of gene expression plasticity of P450s in response to phytochemicals. We discuss experimental setups and new findings in mechanisms of P450 regulation. Whole genome transcriptomic analysis of arthropod herbivores, either after direct administration of phytochemicals or after host plant shifts, allowed to integrate various levels of chemical complexity and lead to the unbiased identification of responsive P450 genes. However, despite progress in identification of inducible P450s, the link between induction and metabolism is still largely unexplored, and to what extent the overall response is biologically functional should be further investigated. In the near future, such studies will be more straightforward as forward and reverse genetic tools become more readily available., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The European red mite, Panonychus ulmi (Koch), is one of the most important apple orchard pests worldwide. Fenpyroximate, a mitochondrial electron transport inhibitor of complex I (METI-I), is a commonly used acaricide to control this pest. In this study, we determined fenpyroximate resistance levels for 11 P. ulmi populations from Iran and a spirodiclofen-resistant strain from Germany (PSR-TK). The LC 50 values ranged between 121.8 and 5713.9 mg a.i. L -1 and the highest resistance ratio (RR) was 47-fold for the Padena population. PBO, TPP and DEM synergist ratios (SRs) were the highest for the PSR-TK (SR = 6.7), Shahin Dej (SR = 6.1) and Semirom3 (SR = 3.6) populations, respectively. In vitro enzyme activity measurements also showed that there was a higher glutathione S-transferases (GSTs) activity in the PSR-TK and Shahin Dej population compared to the most susceptible populations, whereas the esterase and P450 monooxygenase activity were not significantly higher in the resistant populations. Last, we screened all populations for the presence of two mutations previously associated with METI-I resistance in spider mites but none of these mutations could be detected. To conclude, moderate to high levels of fenpyroximate resistance were observed in P. ulmi populations from Iran, with increased detoxification most likely underlying fenpyroximate resistance.

The P450 family (CYP genes) of arthropods encodes diverse enzymes involved in the metabolism of foreign compounds and in essential endocrine or ecophysiological functions. The P450 sequences (CYPome) from 40 arthropod species were manually curated, including 31 complete CYPomes, and a maximum likelihood phylogeny of nearly 3000 sequences is presented. Arthropod CYPomes are assembled from members of six CYP clans of variable size, the CYP2, CYP3, CYP4 and mitochondrial clans, as well as the CYP20 and CYP16 clans that are not found in Neoptera. CYPome sizes vary from two dozen genes in some parasitic species to over 200 in species as diverse as collembolans or ticks. CYPomes are comprised of few CYP families with many genes and many CYP families with few genes, and this distribution is the result of dynamic birth and death processes. Lineage-specific expansions or blooms are found throughout the phylogeny and often result in genomic clusters that appear to form a reservoir of catalytic diversity maintained as heritable units. Among the many P450s with physiological functions, six CYP families are involved in ecdysteroid metabolism. However, five so-called Halloween genes are not universally represented and do not constitute the unique pathway of ecdysteroid biosynthesis. The diversity of arthropod CYPomes has only partially been uncovered to date and many P450s with physiological functions regulating the synthesis and degradation of endogenous signal molecules (including ecdysteroids) and semiochemicals (including pheromones and defense chemicals) remain to be discovered. Sequence diversity of arthropod P450s is extreme, and P450 sequences lacking the universally conserved Cys ligand to the heme have evolved several times. A better understanding of P450 evolution is needed to discern the relative contributions of stochastic processes and adaptive processes in shaping the size and diversity of CYPomes., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The intensive use of pesticides is a common practice for the management of the two-spotted spider mite, Tetranychus urticae, in greenhouses and field farms of Ethiopia. However, incidence of resistance and possible resistance mechanisms in T. urticae populations from Ethiopia have not yet been studied. Here, we assessed the toxicity of various acaricides-bifenazate, abamectin, emamectin benzoate, profenofos, fenbutatin oxide, fenpyroximate, amitraz and chlorfenapyr-on T. urticae populations sampled from six flower greenhouse farms, three strawberry greenhouse farms, one field-grown vegetable farm and two wild populations. In parallel, all populations were screened for known target-site mutations. All tested populations were fully susceptible to bifenazate, abamectin, emamectin benzoate and profenofos, but resistant against fenbutatin oxide and fenpyroximate. Four populations showed considerable levels of resistance against amitraz and one population was resistant to chlorfenapyr. Several target-site mutations were identified in the tested populations, including G119S, A201S, T280A, G328A and F331W/C/Y in acetylcholinesterase and the F1538I and L1024V mutation in the voltage-gated sodium channel. The F1538I mutation was found in eight out of 12 populations, whereas the L1024V mutation was only found in two populations. The H92R mutation in the PSST subunit of complex I and the I1017F mutation in chitin synthase 1 was detected in half of the tested populations. The G326E and I321T mutations in the glutamate-gated chloride channel 3 were also detected, but more rarely, whereas mitochondrial cytochrome b mutations were not detected. The current study revealed multiple resistance patterns in Ethiopian T. urticae populations and together with the wide presence of target-site mutations, calls for the wise use of acaricides in the management of T. urticae in Ethiopia.

The tomato russet mite, Aculops lycopersici, is among the smallest animals on earth. It is a worldwide pest on tomato and can potently suppress the host's natural resistance. We sequenced its genome, the first of an eriophyoid, and explored whether there are genomic features associated with the mite's minute size and lifestyle. At only 32.5 Mb, the genome is the smallest yet reported for any arthropod and, reminiscent of microbial eukaryotes, exceptionally streamlined. It has few transposable elements, tiny intergenic regions, and is remarkably intron-poor, as more than 80% of coding genes are intronless. Furthermore, in accordance with ecological specialization theory, this defense-suppressing herbivore has extremely reduced environmental response gene families such as those involved in chemoreception and detoxification. Other losses associate with this species' highly derived body plan. Our findings accelerate the understanding of evolutionary forces underpinning metazoan life at the limits of small physical and genome size., Competing Interests: RG, WD, JG, SR, NW, JT, JA, EP, SL, RF, YV, TV, RC No competing interests declared, MK Reviewing editor, eLife, (© 2020, Greenhalgh et al.)