Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites

Veneti et al_Heredity_raw data_30may2012

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Multiple Rescue Factors Within a Wolbachia Strain

Wolbachia-induced cytoplasmic incompatibility (CI) is expressed when infected males are crossed with either uninfected females or females infected with Wolbachia of different CI specificity. In diploid insects, CI results in embryonic mortality, apparently due to the the loss of the paternal set of chromosomes, usually during the first mitotic division. The molecular basis of CI has not been determined yet; however, several lines of evidence suggest that Wolbachia exhibits two distinct sex-dependent functions: in males, Wolbachia somehow “imprints” the paternal chromosomes during spermatogenesis (mod function), whereas in females, the presence of the same Wolbachia strain(s) is able to restore embryonic viability (resc function). On the basis of the ability of Wolbachia to induce the modification and/or rescue functions in a given host, each bacterial strain can be classified as belonging in one of the four following categories: mod+ resc+, mod− resc+, mod− resc−, and mod+ resc−. A so-called “suicide” mod+ resc− strain has not been found in nature yet. Here, a combination of embryonic cytoplasmic injections and introgression experiments was used to transfer nine evolutionary, distantly related Wolbachia strains (wYak, wTei, wSan, wRi, wMel, wHa, wAu, wNo, and wMa) into the same host background, that of Drosophila simulans (STCP strain), a highly permissive host for CI expression. We initially characterized the modification and rescue properties of the Wolbachia strains wYak, wTei, and wSan, naturally present in the yakuba complex, upon their transfer into D. simulans. Confocal microscopy and multilocus sequencing typing (MLST) analysis were also employed for the evaluation of the CI properties. We also tested the compatibility relationships of wYak, wTei, and wSan with all other Wolbachia infections. So far, the cytoplasmic incompatibility properties of different Wolbachia variants are explained assuming a single pair of modification and rescue factors specific to each variant. This study shows that a given Wolbachia variant can possess multiple rescue determinants corresponding to different CI systems. In addition, our results: (a) suggest that wTei appears to behave in D. simulans as a suicide mod+ resc− strain, (b) unravel unique CI properties, and (c) provide a framework to understand the diversity and the evolution of new CI-compatibility types

Veneti et al_Heredity_raw data_30may2012

Raw data associated with all tables presented in the manuscrip

Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis

Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro