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Abstract Genetically modified plants are one of the tactics used in integrated pest management - IPM. There is great concern about the impact of these plants on non-target organisms. On the other hand, there is little information in the literature on the effects of transgenics (Bacillus thuringiensis) Bt on populations of phytophagous mites, and the physiological responses that this attack promotes on plants. The objective of this work was to evaluate the biology of the T. ludeni mite in Bt cotton, expressing the Cry1F and Cry1Ac proteins. To evaluate the behavior of food and oviposition preference of the T. ludeni with Bt cotton and isohybrid. Verify if the physiological stress caused by T. ludeni’s attack is differentiated in Bt cotton. The mites were reared in Bt cotton and isohybrid, in a total of 40 replicates in the completely randomized design and the biological cycle was evaluated. The food preference and oviposition analysis were done with 10 replicates, with choice. The physiological stress was evaluated through chlorophyll fluorescence, under greenhouse conditions. The data of the T. ludeni biology were analyzed by Student's t-test, for food and oviposition preference the chi-square test was performed. Regression models were fitted for the fluorescence parameters. The model identity test was used to evaluate the differences between Bt and isohybrid treatments. Cry1F and Cry1Ac proteins have not affected the biology of T. ludeni. The photosynthetic parameters in Bt cotton plants were less influenced by T. ludeni infestation. Resumo O uso de plantas geneticamente modificadas é uma das táticas utilizadas no manejo integrado de pragas - MIP. Observa-se grande preocupação com o impacto dessas plantas sobre organismos não alvos. Por outro lado, existe pouca informação na literatura sobre efeitos dos transgênicos (Bacillus thuringiensis) Bt em populações de ácaros fitófagos, e as respostas fisiológicas que esse ataque promove nas plantas. Objetivou-se com esse trabalho avaliar a biologia do ácaro T. ludeni em algodoeiro Bt, expressando as proteínas Cry1F e Cry1Ac. Avaliar se há comportamento de preferência alimentar e postura de T. ludeni em relação ao algodoeiro Bt e seu iso-híbrido. E verificar se o estresse fisiológico causado pelo ataque de T. ludeni é diferenciado em algodoeiro Bt. Os ácaros foram criados em algodoeiro Bt e iso-híbrido, em um total de 40 repetições no delineamento inteiramente casualizado, onde foi avaliado o ciclo biológico. A análise de preferência alimentar e de posturas foi feita com 10 repetições, com escolha. O estresse fisiológico foi avaliando através da fluorescência da clorofila, em casa de vegetação. Os dados da biologia de T. ludeni foram analisados pelo teste t Student, para preferência alimentar e postura foi realizado o teste qui-quadrado. Para os parâmetros da fluorescência, foram ajustados modelos de regressão. Para testar as diferenças entre Bt e iso-híbrido foi utilizado o teste de identidade de modelos. As proteínas Cry1F e Cry1Ac não afetaram a biologia de T. ludeni. Os parâmetros fotossintéticos em plantas de algodoeiro Bt foram menos influenciados pela infestação de T. ludeni.

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

Pesticide resistance arises rapidly in arthropod herbivores, as can host plant adaptation, and both are significant problems in agriculture. These traits have been challenging to study as both are often polygenic and many arthropods are genetically intractable. Here, we examined the genetic architecture of pesticide resistance and host plant adaptation in the two-spotted spider mite, Tetranychus urticae, a global agricultural pest. We show that the short generation time and high fecundity of T. urticae can be readily exploited in experimental evolution designs for high-resolution mapping of quantitative traits. As revealed by selection with spirodiclofen, an acetyl-CoA carboxylase inhibitor, in populations from a cross between a spirodiclofen-resistant and a spirodiclofen-susceptible strain, and which also differed in performance on tomato, we found that a limited number of loci could explain quantitative resistance to this compound. These were resolved to narrow genomic intervals, suggesting specific candidate genes, including acetyl-CoA carboxylase itself, clustered and copy variable cytochrome P450 genes, and NADPH cytochrome P450 reductase, which encodes a redox partner for cytochrome P450s. For performance on tomato, candidate genomic regions for response to selection were distinct from those responding to the synthetic compound and were consistent with a more polygenic architecture. In accomplishing this work, we exploited the continuous nature of allele frequency changes across experimental populations to resolve the existing fragmented T. urticae draft genome to pseudochromosomes. This improved assembly was indispensable for our analyses, as it will be for future research with this model herbivore that is exceptionally amenable to genetic studies.

Summary Unicellular and multicellular tomato trichomes function as mechanical and chemical barriers against herbivores. Auxin treatment increased the formation of II, V and VI type trichomes in tomato leaves. The auxin response factor gene SlARF4, which was highly expressed in II, V and VI type trichomes, positively regulated the auxin‐induced formation of II, V and VI type trichomes in the tomato leaves. SlARF4 overexpression plants with high densities of these trichomes exhibited tolerance to spider mites. Two R2R3 MYB genes, SlTHM1 and SlMYB52, were directly targeted and inhibited by SlARF4. SlTHM1 was specifically expressed in II and VI type trichomes and negatively regulated the auxin‐induced formation of II and VI type trichomes in the tomato leaves. SlTHM1 down‐regulation plants with high densities of II and VI type trichomes also showed tolerance to spider mites. SlMYB52 was specifically expressed in V type trichomes and negatively regulated the auxin‐induced formation of V type trichome in the tomato leaves. The regulation of SlARF4 on the formation of II, V and VI type trichomes depended on SlTHM1 and SlMYB52, which directly targeted cyclin gene SlCycB2 and increased its expression. In conclusion, our data indicates that the R2R3 MYB‐dependent auxin signalling pathway regulates the formation of II, V and VI type trichomes in tomato leaves. Our study provides an effective method for improving the tolerance of tomato to spider mites.

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.

Most molecular-genetic studies of plant defense responses to arthropod herbivores have focused on insects. However, plant-feeding mites are also pests of diverse plants, and mites induce different patterns of damage to plant tissues than do well-studied insects (e.g. lepidopteran larvae or aphids). The two-spotted spider mite (Tetranychus urticae) is among the most significant mite pests in agriculture, feeding on a staggering number of plant hosts. To understand the interactions between spider mite and a plant at the molecular level, we examined reciprocal genome-wide responses of mites and its host Arabidopsis (Arabidopsis thaliana). Despite differences in feeding guilds, we found that transcriptional responses of Arabidopsis to mite herbivory resembled those observed for lepidopteran herbivores. Mutant analysis of induced plant defense pathways showed functionally that only a subset of induced programs, including jasmonic acid signaling and biosynthesis of indole glucosinolates, are central to Arabidopsis’s defense to mite herbivory. On the herbivore side, indole glucosinolates dramatically increased mite mortality and development times. We identified an indole glucosinolate dose-dependent increase in the number of differentially expressed mite genes belonging to pathways associated with detoxification of xenobiotics. This demonstrates that spider mite is sensitive to Arabidopsis defenses that have also been associated with the deterrence of insect herbivores that are very distantly related to chelicerates. Our findings provide molecular insights into the nature of, and response to, herbivory for a representative of a major class of arthropod herbivores.

Keto-carotenoids contribute to many important traits in animals, including vision and coloration. In a great number of animal species, keto-carotenoids are endogenously produced from carotenoids by carotenoid ketolases. Despite the ubiquity and functional importance of keto-carotenoids in animals, the underlying genetic architectures of their production have remained enigmatic. The body and eye colorations of spider mites (Arthropoda: Chelicerata) are determined by β-carotene and keto-carotenoid derivatives. Here, we focus on a carotenoid pigment mutant of the spider mite Tetranychus kanzawai that , as shown by chromatography, lost the ability to produce keto-carotenoids. We employed bulked segregant analysis and linked the causal locus to a single narrow genomic interval. The causal mutation was fine-mapped to a minimal candidate region that held only one complete gene, the cytochrome P450 monooxygenase CYP384A1 , of the CYP3 clan. Using a number of genomic approaches, we revealed that an inactivating deletion in the fourth exon of CYP384A1 caused the aberrant pigmentation. Phylogenetic analysis indicated that CYP384A1 is orthologous across mite species of the ancient Trombidiformes order where carotenoids typify eye and body coloration, suggesting a deeply conserved function of CYP384A1 as a carotenoid ketolase. Previously, CYP2J19, a cytochrome P450 of the CYP2 clan, has been identified as a carotenoid ketolase in birds and turtles. Our study shows that selection for endogenous production of keto-carotenoids led to convergent evolution, whereby cytochrome P450s were independently co-opted in vertebrate and invertebrate animal lineages.

Background GC16 is a novel pesticide with acaricidal properties against the spider mite Tetranychus pueraricola (Ehara & Gotoh). Its physiological mechanisms have been described previously, but its molecular mechanisms of action remain unclear. Thus, we aimed to explore the acaricidal mechanisms of GC16 through transcriptomic and proteomic analyses. The results were verified using transmission electron microscopy (TEM), immunofluorescence assay, and western blotting. Results Transcriptomic and proteomic analyses revealed 2717 differentially expressed genes (DEGs) and 374 differentially expressed proteins (DEPs) between the GC16-treated and control mites. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEGs and DEPs were enriched in the autophagy pathway. TEM showed that the number of autophagosomes and autolysosomes was higher in the GC16-treated mites than in the control mites. Immunofluorescence assay and western blot results consistently indicated that GC16 treatment significantly enhanced the relative expression of the autophagy protein LC3 in insect Sf9 cells. The intracellular calcium concentration in the GC16-treated Sf9 cells was 2.30 times higher than that in the control cells, suggesting that GC16 disrupted calcium homeostasis and potentially acted as a calcium-driven nerve agent. Conclusions Autophagy is involved in the toxicity of GC16 against T. pueraricola and may be activated by elevated Ca 2+ levels. This study reveals the molecular insecticidal mechanisms of GC16 and provides rationale for the field application of GC16 to control pest mites.

Using PCR to distinguish closely related species can be difficult because they may have very similar genomes. Advances in bioinformatics make it possible to design PCR primers that are species-specific. In this study, we developed a bioinformatics method for extracting species-specific primer candidate sequences (i.e., unpaired primers that were specific to a single species) from RNA-Seq data sets of 19 species of spider mites (Acari, Tetranychidae). Using k -mer counting, we obtained between 257 and 48,621 species-specific unpaired primer candidates for the 19 species. We then manually obtained a second primer that was also species-specific. The primer pairs were then confirmed to work in the target species and not to work in the non-target species. Finally, species-specific primer pairs were obtained for 17 of the 19 species tested. Such species-specific primers may be used for practical species discrimination by optimizing multiplex PCR. Our primer design method is expected to be applicable to other taxa.

The extreme adaptation potential of the generalist herbivore Tetranychus urticae (the two-spotted spider mite) to pesticides as well as diverse host plants has been associated with clade-specific gene expansions in known detoxifying enzyme families, and with extensive and rapid transcriptional responses. However, how this broad transcriptional potential is regulated remains largely unknown. Using a parental/F1 design in which four inbred strains were crossed to a common inbred strain, we assessed the genetic basis and inheritance of gene expression variation in T . urticae . Mirroring known phenotypic variation in the progenitor strains of the inbreds, we confirmed that the inbred strains we created were genetically distinct, varied markedly in pesticide resistance, and also captured variation in host plant fitness as is commonly observed in this species. By examining differences in gene expression between parents and allele-specific expression in F1s, we found that variation in RNA abundance was more often explained in trans as compared to cis , with the former associated with dominance in inheritance. Strikingly, in a gene ontology analysis, detoxification genes of the cytochrome P450 monooxygenase (CYP) family, as well as dioxygenases (DOGs) acquired from horizontal gene transfer from fungi, were specifically enriched at the extremes of trans -driven up- and downregulation. In particular, multiple CYPs and DOGs with broad substrate-specificities for pesticides or plant specialized compounds were exceptionally highly upregulated as a result of trans -regulatory variation, or in some cases synergism of cis and trans , in the most multi-pesticide resistant strains. Collectively, our findings highlight the potential importance of trans -driven expression variation in genes associated with xenobiotic metabolism and host plant use for rapid adaptation in T . urticae , and also suggests modular control of these genes, a regulatory architecture that might ameliorate negative pleiotropic effects.