Adaptation of an herbivorous arthropod to green tea plants by overcoming catechin defenses

Reviewer #1 (Public review):

Summary:

This study investigates the molecular mechanisms allowing the KSM mite to infest tea plants, a host that is toxic to the closely related TSSM mite due to high concentrations of phenolic catechins. The authors utilize a comparative approach involving tea-adapted KSM, non-adapted KSM, and TSSM to assess behavioral avoidance and physiological tolerance to catechins. The main finding is that tea-adapted KSM possesses a specific detoxification mechanism mediated by an enzyme, TkDOG15, which was acquired via horizontal gene transfer. The study demonstrates that adaptation is a two-step process: (1) structural refinement of the TkDOG15 enzyme through amino acid substitutions that enhance enzymatic efficiency against catechins, and (2) significant transcriptional upregulation of this gene in response to tea feeding. This enzymatic adaptation allows the mites to cleave and detoxify tea catechins, enabling survival on a toxic host plant.

Strengths:

A multiomics approach (transcriptomics and proteomics) provided a compelling cross-validation of its findings. Functional bioassays, such as RNAi and recombinant enzyme assays, demonstrated that the adapted mite has higher activity against catechins via TkDOG15. Other methodologies, like feeding assay using a parafilm-covered leaf disc, were effective in avoiding contact chemosensation.

Comments on revised version.

The authors have satisfied all previous concerns through necessary text revisions and clarified discussions. The manuscript is now well-balanced and scientifically sound.

Reviewer #2 (Public review):

Summary:

The fascinating topic of the host range of arthropods, including insects, and the detoxification of host secondary metabolites has been elucidated through studies of the host specificity of two closely related species. The discovery that key genes were acquired from fungi through horizontal gene transfer (HGT) is particularly significant.

Strengths:

(1) The discovery that the TkDOG15 enzyme, acquired through HGT from fungi, plays a key role in the detoxification of green tea catechins in the Kanzawa mite, revealing a new mechanism of plant-herbivore interactions, is highly encouraging.

(2) The verification of this finding through various experiments, including behavioral, toxicological, transcriptomic, and proteomic analyses, RNAi-based gene function analysis, and recombinant enzyme activity assays, is also highly commendable.

(3) By proposing a two-step model in which amino acid substitutions and expression regulation of a specific enzyme gene (TkDOG15) enable host adaptive evolution, this study contributes significantly to our understanding of the evolutionary mechanisms of speciation and plant defense overcoming.

Comments on revised version.

I believe the manuscript has been significantly refined since the initial draft was submitted.

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment:

This important study provides mechanistic evidence that tea-adapted two-spotted spider mite overcomes green tea catechin defenses via the horizontally transferred dioxygenase TkDOG15, supporting a two-step adaptation model, combining enzyme refinement and inducible upregulation. The evidence is convincing because multi-omics signals converge with functional validation (RNAi knockdown and recombinant enzyme assays) and well-controlled behavioral/toxicity assays to link TkDOG15 activity and expression to survival and feeding on tea.

We thank the editors and reviewers for this positive assessment of the importance of our study and the strength of the evidence. We would like to point out one factual correction. The assessment describes the tea-adapted mite as the "two-spotted spider mite" (TSSM, Tetranychus urticae), but the species adapted to tea in this study is the Kanzawa spider mite (KSM, Tetranychus kanzawai). We would suggest revising "tea-adapted two-spotted spider mite" to "tea-adapted spider mite" or "tea-adapted Kanzawa spider mite" accordingly.

Reviewer #1 (Public review):

Summary:

This study investigates the molecular mechanisms allowing the KSM mite to infest tea plants, a host that is toxic to the closely related TSSM mite due to high concentrations of phenolic catechins. The authors utilize a comparative approach involving tea-adapted KSM, non-adapted KSM, and TSSM to assess behavioral avoidance and physiological tolerance to catechins. The main finding is that tea-adapted KSM possesses a specific detoxification mechanism mediated by an enzyme, TkDOG15, which was acquired via horizontal gene transfer. The study demonstrates that adaptation is a two-step process: (1) structural refinement of the TkDOG15 enzyme through amino acid substitutions that enhance enzymatic efficiency against catechins, and (2) significant transcriptional upregulation of this gene in response to tea feeding. This enzymatic adaptation allows the mites to cleave and detoxify tea catechins, enabling survival on a toxic host plant.

Strengths:

A multiomics approach (transcriptomics and proteomics) provided a compelling crossvalidation of its findings. Functional bioassays, such as RNAi and recombinant enzyme assays, demonstrated that the adapted mite has higher activity against catechins via TkDOG15. Other methodologies, like feeding assay using a parafilm-covered leaf disc, were effective in avoiding contact chemosensation.

Weaknesses:

Although TkDOG15 is assumed to "detoxify" catechins by ring cleavage, the study doesn't identify or characterize the breakdown metabolic products. If the metabolites are indeed non-toxic compared to the parent catechins, that would strengthen the detoxification hypothesis. Also, the transcriptomic and proteomic analyses identified other potential detoxification enzymes, such as CCEs, UGTs, and ABC (Supplementary Tables 3-1 & 3-2), which were also upregulated. The manuscript focuses almost exclusively on TkDOG15, potentially overlooking a multigenic adaptation mechanism, where these other enzymes might play synergistic roles, although it was mentioned in the discussion section.

Reviewer #1 (Recommendations for the authors):

There is no need for additional experiments, but I suggest revising the discussion section to mention the weaknesses pointed out above.

We thank the reviewer for the positive assessment and helpful suggestions. We have revised the Discussion (L276-283) to address both points as limitations. First, we now note that we did not characterize the products of TkDOG15-mediated catechin cleavage, and that confirming their reduced toxicity relative to the parent catechins would further support its detoxification role. We note this as a direction for future work. Second, we note that DOG15 in KSM on tea was the only enzyme upregulated at both the mRNA and protein levels, whereas the CCEs, UGTs, ABC transporter, and other DOGs were enriched in only one dataset. We now state that tea adaptation in KSM may be multigenic, with these enzymes potentially acting synergistically with DOG15 and warranting functional validation.

Minor corrections below:

(1) Figure 1a: For better readability, I recommend adding "KSM" and "TSSM" to the two pictures, respectively.

Done.

(2) L165: tetur20g01790 refers to a TSSM gene, while TkDOG15 refers to a TSM protein. Revise it accordingly. (Same for L442 and L481).

The reviewer is correct that tetur20g01790 is the TSSM gene ID. As the KSM genome is not yet available, we identified the TkDOG15 gene, the KSM ortholog of tetur20g01790, by de novo assembly of our RNA-seq reads. We have revised L169 and L455 accordingly.

(3) L264: Supplemental Table 3-2.

Done (L271).

Reviewer #2 (Public review):

Summary:

The fascinating topic of the host range of arthropods, including insects, and the detoxification of host secondary metabolites has been elucidated through studies of the host specificity of two closely related species. The discovery that key genes were acquired from fungi through horizontal gene transfer (HGT) is particularly significant.

Strengths:

(1) The discovery that the TkDOG15 enzyme, acquired through HGT from fungi, plays a key role in the detoxification of green tea catechins in the Kanzawa mite, revealing a new mechanism of plant-herbivore interactions, is highly encouraging.

(2) The verification of this finding through various experiments, including behavioral, toxicological, transcriptomic, and proteomic analyses, RNAi-based gene function analysis, and recombinant enzyme activity assays, is also highly commendable.

(3) By proposing a two-step model in which amino acid substitutions and expression regulation of a specific enzyme gene (TkDOG15) enable host adaptive evolution, this study contributes significantly to our understanding of the evolutionary mechanisms of speciation and plant defense overcoming.

Weaknesses:

While transcriptome/proteome analyses reported changes in the expression of other detoxification-related enzymes, including CCEs, UGTs, ABC transporters, DOG1, DOG4, and DOG7, it is regrettable that the contribution of each enzyme, including its interaction with TkDOG15 and the functional analysis of each enzyme within the overall catechin detoxification system, was not investigated.

We thank the reviewer for the encouraging assessment and this comment. We agree that the contributions of the other detoxification-related enzymes, including their interaction with DOG15, remain to be investigated. As this point overlaps with a comment from Reviewer 1, we have revised the Discussion (L276-283) to note that DOG15 was the only enzyme upregulated at both the mRNA and protein levels, whereas the CCEs, UGTs, ABC transporter, and other DOGs were enriched in only one dataset. We now state that tea adaptation in KSM may be multigenic, with these enzymes potentially acting synergistically with DOG15, and that the functional analysis of their individual and combined contributions warrants future work.

Reviewer #2 (Recommendations for the authors):

The manuscript titled "Adaptation of an Herbivorous Arthropod to Green Tea Plants by Overcoming Catechin Defenses" presents a well-designed, mechanistically insightful study that advances our understanding of herbivore adaptation to plant chemical defenses. The work is scientifically sound and of potential interest to a broad readership in chemical ecology and evolutionary biology.

However, before the manuscript can be considered for acceptance, the authors must adequately address the comments outlined below regarding clarity, presentation, and interpretation across the manuscript.

We thank the reviewer for the positive evaluation of our study. We have carefully addressed each of the specific comments below regarding clarity, presentation, and interpretation, and we believe these revisions have substantially improved the manuscript.

Specific comments on each section:

(1) Abstract

(a) The authors are encouraged to add a concise concluding sentence summarizing the broader significance of the study and indicating potential future research directions or limitations, which would strengthen the impact of the abstract.

We have added a concluding sentence to the Abstract summarizing the broader significance of the study and indicating future directions (L38-40).

(b) The authors may consider adding representative quantitative results to the abstract, as this would enhance clarity and increase the impact and interpretability of the study for readers.

We have added representative quantitative results to the Abstract. Specifically, we now state that the mRNA and protein levels of DOG15 in tea-adapted T. kanzawai are up to 31.6 and 12.1 times higher, respectively, than in T. urticae fed on tea plants (L30-32). For consistency, we now refer to the gene as "DOG15" throughout the Abstract (L29, L30, and L36).

(2) Introduction

(a) While the paragraph is informative, it reads more like a summary of the main results than a statement of study objectives. The authors are encouraged to reframe this section to explicitly define the study's aims and hypotheses.

We have reframed the final paragraph of the Introduction to explicitly state the study's aims and hypotheses rather than to summarize the results (L73-81).

(b) The authors should avoid excessive citation of multiple references for a single thematic statement when one key reference is sufficient. Where appropriate, inclusion of more recent literature is encouraged.

We have reduced multiple citations for single statements to the most representative references: Cabrera et al. (2006) for the health benefits of catechins (L46) and Grbić et al. (2011) and Dermauw et al. (2013) for the DOG gene count (L66-67).

(3) Materials and Methods

(a) The Materials and Methods section is comprehensive and technically sound; however, its length and density reduce overall clarity. The authors are encouraged to streamline descriptions of standard or well-established protocols and rely on appropriate citations where possible.

We agree that clarity can be improved by removing redundancy. The Materials and Methods are intentionally detailed to allow independent replication of our protocols, so we have retained this detail and instead removed the overlapping methodological descriptions from the figure captions, where the same information was repeated (see our response to comment 6a).

(b) Greater consistency is needed in reporting biological and technical replicates across different experiments (e.g., performance assays, transcriptomics, proteomics, and enzymatic activity assays) to enhance reproducibility.

We have standardized the reporting of replicates across all experiments to the format "x independent experimental runs (n = y per run)." Throughout the manuscript, "independent experimental runs" denotes biological replicates, with technical replicates specified separately where applicable (three technical replicates for qRT-PCR).

(c) The authors should provide brief justification for key methodological parameters, such as catechin concentrations, exclusion criteria in behavioral assays, and thresholds used for defining DEGs and DEPs, to improve transparency and interoperability.

We have added brief justifications for the three parameters. 1) The catechin concentration range was chosen to encompass the individual catechin levels measured in fresh tea leaves (L340-341). 2) In the behavioral assays, inactive mites were excluded because their movement was insufficient to determine chemo-orientation behavior, and escaped mites were excluded because they did not complete the assay (L365-367). 3) The thresholds for DEGs and DEPs follow criteria commonly applied in mite transcriptomic studies (Vidal-Quist et al., 2025, newly added to the references) (L414-416) and are consistent with our previous spider mite proteomic analysis (Arai et al., 2025) (L444-445).

(4) Results

(a) While significant differences in survival and fecundity are reported, briefly indicating the magnitude of these differences (e.g., percentage or fold change) would improve clarity and strengthen the presentation (Lines 91-96).

We have added the magnitude of the differences (L94-97). The revised text now states that after 10 days, almost 90% of tea-adapted KSM survived, compared with about 5% of non-adapted KSM and 33% of TSSM, and that tea-adapted KSM laid up to about 2 eggs/surviving female daily, whereas the other two populations laid almost no eggs.

(b) The final sentences include interpretative and concluding statements regarding catechins as key metabolites and mite adaptation. These statements would be more appropriate for the Discussion section rather than the Results (Lines 127-130). Follow the same for the rest of the Results section also.

Following the reviewer's suggestion, we have removed the interpretive and concluding statements from the end of the Results section, so that it now reports only the observations (L129-130). The interpretation regarding the multiple modes of action of catechins and the insensitivity of tea-adapted KSM is already presented in the Discussion (L206-213 and Conclusions), so we did not duplicate it there. We also reviewed the remaining Results subsections and confirmed that they report the experimental observations and their direct conclusions without broader interpretation.

(c) The comparison among catechin classes is clear; however, briefly listing the mean concentrations of each catechin (as shown in Figure 2a) in the text would improve readability without duplicating the figure (Lines 135-139).

We have added the approximate mean concentration of each catechin to the text (L136-137).

(d) Please clarify in the Results whether the same exposure concentration and duration were applied for all catechins and mite species, or explicitly direct readers to the Methods section (Lines 141-142).

We have clarified in the Results section that all four catechins were tested at the same concentration series (0, 10, 10², 10³, 10⁴, and 10⁵ ppm) and the same exposure duration (24 h) for both mite populations (L143).

(e) The phrase "lower sensitivity" should be explicitly linked to LC₅₀ estimates to ensure that the basis of comparison is immediately clear to readers (Lines 143-144).

Following the reviewer's suggestion, we have linked the sensitivity comparison to the LC₅₀ values (L143-147). The comparison is now stated relative to TSSM based on the LC₅₀ estimates, and for ECg and EC we note that the LC₅₀ of tea-adapted KSM exceeded the highest concentration tested.

(f) This section clearly identifies TkDOG15 as a key gene underlying tea adaptation in KSM; however, the authors are encouraged to briefly clarify the criteria used to define "highly enriched" mRNAs and proteins (e.g., fold-change and statistical thresholds) in the Results text or by explicitly directing readers to the Methods. This would improve transparency and facilitate interpretation of the multi-omics comparisons (Lines 147-173).

We have added the criteria used to define the enriched mRNAs and proteins (log2 fold change ≥ 1 with adjusted p-value < 0.05 for mRNA and p-value < 0.05 for protein) and referred readers to the Materials and Methods (L159-160).

(g) The enzymatic comparison between TkDOG15 and TuDOG15 is well presented; however, the authors are encouraged to briefly discuss whether the two amino acid substitutions (Q127A and T203A) were individually or jointly responsible for the increased catalytic efficiency, or to acknowledge this as a limitation and potential direction for future functional studies (Lines 176-190).

We have added a brief discussion of whether the two substitutions (Q127A and T203A) act individually or jointly (L254-257). We note that T203A is adjacent to the active-site residue Y202 and may contribute more directly to catalytic efficiency, and we acknowledge that dissecting their individual contributions by site-directed mutagenesis is a direction for future work.

(5) Discussion

(a) The authors appropriately acknowledge that the molecular basis of chemosensory insensitivity and the contribution of additional detoxification enzymes remain unresolved. To further improve clarity, these statements could be explicitly framed as hypotheses or future research directions to clearly distinguish them from experimentally supported mechanisms (Lines 205-208; 266-270).

We have reframed the statements on chemosensory insensitivity (L209-213) and the contribution of additional detoxification enzymes (L272-274) as hypotheses and future directions, distinguishing them from the experimentally supported mechanisms.

(b) While DOG15 is convincingly identified as a key contributor to tea adaptation, a brief clarification of its relative importance compared with other upregulated detoxification enzymes would strengthen interpretative balance, even if the roles of these enzymes remain unresolved (Lines 259-265).

DOG15 was the only enzyme upregulated at both the mRNA and protein levels (Figure 3d,e), and the only enzyme functionally validated in this study, by RNAi silencing (Figure 3f) and recombinant enzyme assays (Figure 4c). We have established that DOG15 contributes to tea adaptation, but because the other upregulated enzymes were not functionally tested, their relative contributions cannot be determined at this stage. As we note in the Discussion, tea adaptation in KSM may be multigenic, with these enzymes potentially acting synergistically with DOG15 (L277-283). We therefore did not add further text, to avoid duplication.

(c) The discussion linking host plant adaptation to reproductive isolation and ecological speciation is interesting and well contextualized; however, these evolutionary implications should be slightly tempered or explicitly framed as potential long-term outcomes beyond the immediate scope of the present study (Lines 271-281).

We have tempered the evolutionary implications (L292-294). The revised sentence now frames the link to reproductive isolation and ecological speciation as a potential outcome over longer evolutionary timescales rather than a direct finding of the present study.

(6) Figure captions

(a) The figure captions (Figures 1-4) are exceptionally detailed and, in several places, repeat methodological information already described in the Materials and Methods. The authors are encouraged to shorten the captions by retaining only information necessary to interpret the figures, while referring readers to the Methods for experimental details.

We have shortened the figure captions (Figures 1-4) by removing methodological details that are described in the Materials and Methods, retaining only the information needed to interpret each figure. Where appropriate, readers are now referred to the Materials and Methods or to Supplemental Figure 1-2 for the full experimental procedures.

(b) Several captions contain long, multi-sentence descriptions that may hinder readability. The authors may consider simplifying the wording, grouping related panels more concisely, and removing procedural details (e.g., extraction conditions, exposure durations, and instrument settings) to improve clarity and visual accessibility.

As described in our response to comment 6a, we have simplified the figure captions by removing procedural details such as extraction conditions, exposure durations, and instrument settings, and by grouping related panels more concisely. These details are retained in the Materials and Methods.

(c) In Figure 1, the panel labels (a-h) do not appear in a clear sequential order. For consistency with the other figures and to improve readability, the authors should ensure that panel lettering is arranged in a logical, sequential order throughout the manuscript.

We appreciate the reviewer's attention to panel ordering. In the current layout, the panel lettering follows the order in which the panels are first cited in the text. Arranging the panels in a strict left-to-right, top-to-bottom sequence would require reducing the size of several panels, including the HPLC chromatogram in panel (e) and the survival and fecundity time courses in panels (c) and (d), which would compromise their readability. We have therefore retained the current arrangement, in which related panels are grouped together and the larger panels are kept at a legible size. We hope the reviewer finds this acceptable.