Mite performance on tea leaves and sensitivity to green tea catechins.

(a) Adult females of the Kanzawa spider mite (KSM) Tetranychus kanzawai and the two-spotted spider mite (TSSM) Tetranychus urticae. Scale bars indicate 200 µm. (b) Feeding damage on leaf discs (10 mm in diameter) of the tea plant Camellia sinensis by tea-adapted KSM, non-adapted KSM, and TSSM. Each leaf disc was fed by 15 adult females at 25°C for 1 day. Control indicates mite-free leaf disc. (c) Survival and (d) fecundity of adult females of tea-adapted KSM (n = 74), non-adapted KSM (n = 74), and TSSM (n = 75) on leaves of tea seedlings at 25 °C for 10 days. In each population, mites that had grown to the teleiochrysalid stage on leaves of kidney bean Phaseolus vulgaris and molted to adults within 2 h were transferred to a single tea leaf (14 to 15 mites/leaf) and the numbers of survivors and eggs-laid were counted daily. Data were collected from 5 independent experimental runs using different single leaves. Mites that escaped from the leaves during the experiment were excluded from the data. Different letters indicate a significant difference (p < 0.001) in the survival and fecundity among mite populations by log-rank test with Bonferroni correction and Tukey’s HSD test, respectively. (e) The HPLC chromatogram of green tea catechins in leaves of tea seedlings used in the mite performance test. The tea leaf disc (10 mm in diameter) was homogenized in liquid nitrogen using a homogenizer pestle. Green tea catechins were extracted with 50% (v/v) acetonitrile in water using a tube tumbler rotator for 1 h at room temperature. The extract (5 µL) was analyzed by HPLC at a flow rate of 1 mL/min and a detection wavelength of 280 nm. Red arrowheads and a gray arrowhead indicate green tea catechins and caffeine, respectively. The numbers in parentheses following the compound names indicate the retention time for each peak. (f) Dose–response relationships between catechin levels and survivorship of tea-adapted KSM, non-adapted KSM, and TSSM. Newly-molted adult females from each population were placed on a Kimwipe piece soaked in different concentrations (ppm, w/v) of an aqueous solution of a catechin mixture at 25 °C for 1 day. The catechin mixture consisted of (−)-epigallocatechin gallate (EGCg), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECg), and (−)-epicatechin (EC). The number of survivors was counted at 3 days after recovery from soaking in the catechin solution. Data were collected from 3 to 5 independent experimental runs (n = 25 to 45) and are presented with a regression line and a 95% confidence band. (g) Chemo-orientation behavior to green tea catechins in tea-adapted KSM, non-adapted KSM, and TSSM. One single adult female from each population was transferred onto a glass plate (9.0 × 9.0 mm). One half (4.5 × 9.0 mm) of the glass plate was coated with the 10% (w/v) catechin mixture, 10 mM fenpropathrin kwon as a mite repellent substance (positive control), or their solvent consisting of 0.1% (w/v) brilliant blue FCF and 0.1% (v/v) Tween 20 in methanol (negative control), and the other half (4.5 × 9.0 mm) was coated with the solvent. To distinguish the contents of the coating, the top two corners of the back of the glass plate were marked. The mite movement on the glass plate was recorded with a digital camera at 29.97 fps at 25 °C. The red curves indicate the trajectory of the mite’s locomotion for 10 min. The numerical value at the bottom right of each image indicates a preference index, which quantifies the mite repellency and attractiveness of a test compound, ranging from −1 (complete localization on one side coated with the solvent) to +1 (complete localization on the other side). (h) Feeding preference for green tea catechins in tea-adapted KSM, non-adapted KSM, and TSSM. Kidney bean leaf discs (10 mm in diameter) were coated with the 10% (w/v) catechin mixture, 10 mM fenpropathrin (positive control) in methanol, or their solvent methanol (negative control) and were covered with stretched Parafilm that prevented direct contact of mite legs with treated compounds on the surface of the leaf disc. Adult females from each population were transferred onto leaf discs (20 mites/disc) and allowed to feed by penetrating the stretched Parafilm with their stylets at 25 °C for 24 h. Data are presented as the mean ± SE and collected from 3 to 7 independent experimental runs and compared using Dunnett’s test.

Green tea catechins in tea leaves and their toxicity to spider mites.

(a) The amount of green tea catechins in fresh tea leaves. Extracts from the leaves soaked in 50% (v/v) acetonitrile in water were analyzed by HPLC. Green tea catechins were quantified using calibration curves generated with the standard solutions of (−)-epigallocatechin gallate (EGCg), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECg), and (−)-epicatechin (EC). Data were collected from 5 independent experimental runs and are presented as the mean ± SE. (b) Catechin dose–response curves of survival of tea-adapted KSM and TSSM soaked in different concentrations (ppm, w/v) of an aqueous solution of EGCg, EGC, ECg, or EC at 25 °C for 24 h. Data were collected from 3 to 5 independent experimental runs using newly-molted adult females (n = 28 to 51) and are presented with a regression curve and a 95% confidence interval. The number of survivors was counted at 3 days after recovery from the soaking treatment. The dotted lines indicate the average amounts of each catechin contained in fresh tea leaves.

Gene expression patterns at the mRNA and protein levels in spider mites in response to consumption of tea or bean leaves revealed a specific high expression of the horizontally transferred gene TkDOG15 in tea-adapted KSM fed on tea leaves.

Principal component analysis of (a) transcriptome and (b) proteome of tea-adapted KSM, non-adapted KSM, and TSSM fed on tea or kidney bean leaves at 25 °C for 24 h. Data were collected from 2 (transcriptome) and 3 (proteome) independent experimental runs with 100 to 150 adult females each. (c) Comparative transcriptomics and proteomics revealed 32 mRNAs and 29 proteins that were highly expressed in tea-adapted KSM fed on tea leaves compared to tea-adapted KSM fed on bean leaves, non-adapted KSM fed on tea leaves, and TSSM fed on tea leaves. Among them, TkDOG15 was the only gene that was commonly upregulated at both mRNA and protein levels in tea-adapted KSM fed on tea leaves. For the DOG15 gene, (d) mRNA and (e) protein expression levels in tea-adapted KSM, non-adapted KSM, and TSSM fed on tea and kidney bean leaves. Expression levels were analyzed from the transcriptome and proteome data. Data are presented as the mean ± SE and different letters indicate a significant difference between treatments by Tukey’s HSD test. (f) Survival of tea-adapted KSM on tea leaves (15 mites/leaf) for 10 days after RNAi-mediated silencing of the TkDOG15 gene. Mites that had grown to the teleiochrysalid stage on kidney bean leaves and molted to adults within 2 h were soaked in dsRNA for the TkDOG15 gene (dsTkDOG15) and a 382-bp fragment of the intergenic region (dsNC, negative control, genomic coordinates: scaffold 12, position 1690614–1690995) for 24 h. Soaked mites were then transferred to a single tea leaf and the number of survivors was counted daily. Mites that escaped from the leaf during the experiment were excluded from the data. Data were collected from 5 independent experimental runs. Kaplan-Meier survival curves were compared using the log-rank test. (g) Expression of the TkDOG15 gene in adult females of tea-adapted KSM relative to that of the PR49 gene at 4 days after the RNAi treatment with dsTkDOG15 and dsNC. Data are presented as the mean ± SE and compared using Student’s t-test.

Comparisons of DOG15 structure and enzymatic activity between KSM and TSSM.

(a) Amino acid sequences of DOG15 in tea-adapted KSM, non-adapted KSM, and TSSM. The sequence of TuDOG15 (tetur20g01790) was obtained from the ORCAE database (https://bioinformatics.psb.ugent.be/orcae/overview/Tetur), and that of tea-adapted and non-adapted KSM was obtained from each RNA-seq reads. These sequences were aligned using the MUSCLE algorithm. The amino acid sequences of tea-adapted and non-adapted KSM were completely matched, but amino acid substitutions were confirmed at Q127A and T203A (red star) in KSM and TSSM. The active sites based on the two tyrosine (Y111 and Y202) and histidine (H208 and H210) residues were matched in KSM and TSSM (black dot). (b) AlphaFold2 model of TkDOG15 showing differing residues between TkDOG154 (A127 in pink; A203 in yellow) and TuDOG15 (Q127 and T203; not shown). Two tyrosine and two histidine residues coordinating iron cofactor (orange) are displayed in active site. (c) Relative enzymatic activity to green tea catechins in TkDOG15 and TuDOG15. Relative enzymatic activity was calculated by multiplying the specific activity of DOG15 for each catechin by the abundance of DOG15 in tea-adapted KSM and TSSM on tea plants (Figure 3e), respectively. Data are presented as the mean ± SD and compared using Student’s t-test.

Primers for real time qRT-PCR and dsRNA synthesis.