Thomas Van Leeuwen

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.

The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.

A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae

Plants produce a wide range of allelochemicals to defend against herbivore attack, and generalist herbivores have evolved mechanisms to avoid, sequester, or detoxify a broad spectrum of natural defense compounds. Successful arthropod pests have also developed resistance to diverse classes of pesticides and this adaptation is of critical importance to agriculture. To test whether mechanisms to overcome plant defenses predispose the development of pesticide resistance, we examined adaptation of the generalist two-spotted spider mite, Tetranychus urticae, to host plant transfer and pesticides. T. urticae is an extreme polyphagous pest with more than 1,100 documented hosts and has an extraordinary ability to develop pesticide resistance. When mites from a pesticide-susceptible strain propagated on bean were adapted to a challenging host (tomato), transcriptional responses increased over time with ∼7.5% of genes differentially expressed after five generations. Whereas many genes with altered expression belonged to known detoxification families (like P450 monooxygenases), new gene families not previously associated with detoxification in other herbivores showed a striking response, including ring-splitting dioxygenase genes acquired by horizontal gene transfer. Strikingly, transcriptional profiles of tomato-adapted mites resembled those of multipesticide-resistant strains, and adaptation to tomato decreased the susceptibility to unrelated pesticide classes. Our findings suggest key roles for both an expanded environmental response gene repertoire and transcriptional regulation in the life history of generalist herbivores. They also support a model whereby selection for the ability to mount a broad response to the diverse defense chemistry of plants predisposes the evolution of pesticide resistance in generalists.

Mitochondrial heteroplasmy and the evolution of insecticide resistance: Non-Mendelian inheritance in action

Genes encoded by mitochondrial DNA (mtDNA) exist in large numbers per cell but can be selected very rapidly as a result of unequal partitioning of mtDNA between germ cells during embryogenesis. However, empirical studies of this “bottlenecking” effect are rare because of the apparent scarcity of heteroplasmic individuals possessing more than one mtDNA haplotype. Here, we report an example of insecticide resistance in an arthropod pest (Tetranychus urticae) being controlled by mtDNA and on its inheritance in a heteroplasmic mite strain. Resistance to the insecticide bifenazate is highly correlated with remarkable mutations in cytochrome b, a mitochondrially encoded protein in the respiratory pathway. Four sites in the Qo site that are absolutely conserved across fungi, protozoa, plants, and animals are mutated in resistant mite strains. Despite the unusual nature of these mutations, resistant mites showed no fitness costs in the absence of insecticide. Partially resistant strains, consisting of heteroplasmic individuals, transmit their resistant and susceptible haplotypes to progeny in highly variable ratios consistent with a sampling bottleneck of ≈180 copies. Insecticide selection on heteroplasmic individuals favors those carrying resistant haplotypes at a frequency of 60% or more. This combination of factors enables very rapid evolution and accounts for mutations being fixed in most field-collected resistant strains. The results provide a rare insight into non-Mendelian mechanisms of mitochondrial inheritance and evolution, relevant to anticipating and understanding the development of other mitochondrially encoded adaptations in arthropods. They also provide strong evidence of cytochrome b being the target site for bifenazate in spider mites.

Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods

Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods.

Genetic analysis and cross-resistance spectrum of a laboratory-selected chlorfenapyr resistant strain of two-spotted spider mite (Acari: Tetranychidae)

A laboratory susceptible strain of Tetranychus urticae was selected with chlorfenapyr resulting in a resistant strain. After 12 cycles of exposure, the resistance ratio (RR) calculated from the LC50s of susceptible and selected strain was 580. The resistant strain was screened with 16 currently used acaricides for cross-resistance. Cross-resistance was detected with amitraz (RR = 19.1), bifenthrin (RR = 1.3), bromopropylate (RR = 7.5), clofentezine (RR = 29.6) and dimethoate (RR = 17.6). No cross-resistance was detected with the new molecules acequinocyl, bifenazate and spirodiclofen. Mortality caused by chlorfenapyr in the F1 progeny from reciprocal crosses between both strains indicated that the mode of inheritance was incomplete recessive. Mortality in F2 progeny indicated that the resistance was under the control of more than one gene. Synergist experiments with S,S,S-tributylphosphorotrithioate (DEF), piperonylbutoxide (PBO) and diethylmaleate (DEM), which are inhibitors of esterases, monooxygenases and glutathion-S-transferases respectively, suggested a major role of esterases in the resistance to chlorfenapyr.

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution

Significance Aedes albopictus is a highly adaptive species that thrives worldwide in tropical and temperate zones. From its origin in Asia, it has established itself on every continent except Antarctica. This expansion, coupled with its ability to vector the epidemic human diseases dengue and Chikungunya fevers, make it a significant global public health threat. A complete genome sequence and transcriptome data were obtained for the Ae. albopictus Foshan strain, a colony derived from mosquitoes from its historical origin. The large genome (1,967 Mb) comprises an abundance of repetitive DNA classes and expansions of the numbers of gene family members involved in insecticide resistance, diapause, sex determination, immunity, and olfaction. This large genome repertory and plasticity may contribute to its success as an invasive species. The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

A gene horizontally transferred from bacteria protects arthropods from host plant cyanide poisoning

Cyanogenic glucosides are among the most widespread defense chemicals of plants. Upon plant tissue disruption, these glucosides are hydrolyzed to a reactive hydroxynitrile that releases toxic hydrogen cyanide (HCN). Yet many mite and lepidopteran species can thrive on plants defended by cyanogenic glucosides. The nature of the enzyme known to detoxify HCN to β-cyanoalanine in arthropods has remained enigmatic. Here we identify this enzyme by transcriptome analysis and functional expression. Phylogenetic analysis showed that the gene is a member of the cysteine synthase family horizontally transferred from bacteria to phytophagous mites and Lepidoptera. The recombinant mite enzyme had both β-cyanoalanine synthase and cysteine synthase activity but enzyme kinetics showed that cyanide detoxification activity was strongly favored. Our results therefore suggest that an ancient horizontal transfer of a gene originally involved in sulfur amino acid biosynthesis in bacteria was co-opted by herbivorous arthropods to detoxify plant produced cyanide. DOI: http://dx.doi.org/10.7554/eLife.02365.001

Genetic and biochemical analysis of a laboratory‐selected spirodiclofen‐resistant strain of Tetranychus urticae Koch (Acari: Tetranychidae)

BACKGROUND Spirodiclofen is a selective, non-systemic acaricide from the new chemical class of tetronic acid derivatives. In order to develop strategies to minimise resistance in the field, a laboratory-selected spirodiclofen-resistant strain of the two-spotted spider mite, Tetranychus urticae Koch, was used to determine genetic, toxicological, biochemical and cross-resistance data. RESULTS Selecting for spirodiclofen resistance in the laboratory yielded a strain (SR-VP) with a resistance ratio of 274, determined on the larval stage. The egg stage remained far more susceptible. No cross-resistance was found against other established acaricides, except for spiromesifen. Based on synergist experiments and enzyme assays, it appeared that especially P450 monooxygenases, but also esterases and glutathione-S-transferases, could be involved in the metabolic detoxification of spirodiclofen. Genetic analysis showed that the resistance is inherited as an intermediate trait under control of more than one gene. CONCLUSIONS Resistance to spirodiclofen exceeded by far the recommended field rate. A good acaricide resistance management programme is necessary to prevent fast resistance build-up in the field. Spirodiclofen can be used in alternation with most established acaricides, except for other tetronic acid derivatives. Without selection pressure, resistance tends to be unstable and can decrease in the presence of susceptible individuals owing to the intermediate, polygenic inheritance mode.

Acaricide resistance and resistance mechanisms in Tetranychus urticae populations from rose greenhouses in the Netherlands

BACKGROUND Spider mites are important crop pests that rapidly develop resistance to acaricides. To investigate whether acaricide resistance is a threat to greenhouse rose culture in the Netherlands, the susceptibility of 15 strains of Tetranychus urticae was tested to several currently used acaricides, and resistance mechanisms were investigated. RESULTS Although the observed levels of susceptibility differed between strains and acaricides, resistance was detected in most strains. The activity of detoxifying enzymes was significantly increased in most field-collected strains, and a number of amino acid substitutions known to be involved in resistance were detected. CONCLUSIONS Resistance levels to traditional acaricides such as bifenthrin and abamectin were prominent, and might result in control failure under field conditions. Resistance to more recently registered compounds was detected in several populations. Resistance levels were generally unstable in the laboratory without selection pressure. The toxicological, biochemical and genetic data in this study will be essential in devising an efficient resistant management for Dutch rose culture.