Adaptive evolution to thermal stress underpins climate resilience in a cosmopolitan arthropod

Reviewer #1 (Public review):

Summary:

In this manuscript, Lei and co-workers aim to uncover the genetic underpinnings of thermal adaptation across three strains of the diamondback moth (Plutella xylostella) through experimental evolution over three years under three different thermal regimes. They identify systematic differences in trait responses (e.g., survival, fecundity), metabolic profiles, gene expression, and in the amino acid sequence of the PxSODC gene, among others. These results suggest that the diamondback moth has a strong potential for rapid physiological adaptation to different thermal regimes. Overall, this is a comprehensive and generally well-executed study that addresses an important question in the face of ongoing climate change.

Strengths:

The authors employ multiple approaches to identify signatures of thermal adaptation across the three strains, such as trait performance comparisons, metabolomics, transcriptomics, and amino acid sequence comparisons. All these different angles form a convincing picture of the underlying factors that underpin thermal adaptation in this experimental system. The manuscript is also generally well written and easy to understand.

Weaknesses:

I am unable to judge the validity of all aspects of this work; I will focus only on areas within my core expertise.

(1) The authors identify pathways that are enriched in different strain comparisons (Figure 3E), but do not provide a detailed interpretation of these results. It would be great if the authors could explain in more detail how the physiological processes of a cold-adapted strain of this species may differ from those of a warmer-adapted strain.

(2) The authors reconstruct a phylogenetic tree of the PxSODC gene using the neighbor-joining algorithm. The limitations of this algorithm have been known for many years now, especially for sequences separated by long evolutionary distances. According to Wang et al. (2016), the last common ancestor of the species shown in Figure S4C occurred 392-350 million years ago. Given this, I would strongly recommend that the authors infer a phylogenetic tree using model-based methods, such as those implemented in RAxML-NG or IQ-TREE. Also, in the absence of a valid outgroup sequence, I would show the gene tree as unrooted or rooted based on the corresponding species tree.

(3) There is a key piece of the puzzle that is currently missing: the structural mechanism behind the mutational effects described in this study (e.g., Figure 5). The authors could leverage AlphaFold to generate structural models of different mutants and conduct molecular dynamics simulations to examine their conformational dynamics.

References:

Wang, Yh., Engel, M., Rafael, J. et al. Fossil record of stem groups employed in evaluating the chronogram of insects (Arthropoda: Hexapoda). Sci Rep 6, 38939 (2016). https://doi.org/10.1038/srep38939

Reviewer #2 (Public review):

Summary:

In this paper, the authors set out to better understand the genetic mechanisms underlying thermal adaptation in insects. They experimentally evolved diamondback moth (Plutella xylostella) populations - a pest species with a wide distribution - under both hot (12h:12h 32{degree sign}C/27{degree sign}C) and cold (15{degree sign}C/10{degree sign}C) thermal conditions, and conducted phenotypic assays and metabolic and transcriptomic profiling to analyze how populations changed to deal with this thermal stress compared to the nonevolved ancestral population (constant 26{degree sign}C). Phenotypic assays showed that evolved hot populations had increased survival at high temperatures (42-43{degree sign}C) while evolved cold populations had lower freezing points compared to the ancestral population. When measured at the constant 26{degree sign}C conditions, metabolic and transcriptomic profiles of 3rd instar larvae from the evolved population were distinctive from the ancestral population, with a set of overlapping metabolic and transcriptomic pathways that were significantly differentially expressed in both hot and cold evolved populations compared to the ancestral. The authors narrowed down this set of candidate genes further by focusing on genes with high expression levels overall, whose expression profile was correlated with differentially expressed metabolites, and that contained mutants in both hot and cold strains. From this set, they chose the PxSODC gene for further functional validation, as it has previously been shown to be involved in the response of insects to abiotic stress with its antioxidative role in cellular defense. At the constant 26{degree sign}C, this gene showed lower expression across development in evolved strains compared to the ancestral population, while it showed similar expression patterns under thermal stress. Knockdown of PxSODC resulted in decreased survival rates at high temperatures and higher freezing points compared to the ancestral population. Based on this validation, the authors hypothesize that the non-synonymous mutation in the PxSODC gene that they found in the cold and hot evolved populations might alter the conformation of the PxSODC protein, increasing enzyme capacity. Their experimental evolution experiment furthermore indicates the capacity of the pest species, the diamondback moth, to adapt to a wide range of temperatures, providing insights into its capacity for global dispersal.

Strengths:

(1) The authors did a tremendous amount of work to characterize the mechanisms underlying thermal adaptation in the diamondback moth, artificially selecting populations for three years in the lab and characterizing how they evolved as a result at different biological levels: from phenotypes in different life stages, to larval metabolites and gene transcription, to functionally validating how one of the resulting gene candidates influences the capacity to deal with thermal stress.

(2) The paper identifies and provides further evidence for candidate genetic mechanisms that might be particularly important for thermal adaptation in insects, including lipid metabolism, oxidoreductase activity, and DNA methylation. It is furthermore interesting that the authors found similar mechanisms to be involved in both the adaptation to cold and hot environments. Their functional validation of some of the genes involved in these mechanisms is very useful to understand how these genes might be causally involved in insect thermal adaptation.

(3) The paper also has applied value: the diamondback moth is a pest species with a wide distribution, so understanding its adaptive capacity to different thermal environments is important for predicting the prevalence and potential further range expansion of this species under future climate change.

Weaknesses:

(1) The paper in its current form is hard to digest and would benefit from improved clarification of the storyline, as well as a tighter integration between the phenotypic, omics, and functional validation data. Currently, it is not always clear what the relevance is of all the reported results, nor why certain decisions were made, or how all the different methods the authors used fit together. For example, the authors functionally validated a second gene, PxDnmt1, but it is unclear why this particular gene was chosen, nor how it relates to their selection regimes when looking at the results obtained with the phenotyping and omics data collection. Seeing how much work the authors did, this makes the paper overwhelming and difficult to read.

(2) The authors at times stretch their results too far, as the ecological relevance of their study design and results is not clear, limiting the generalizability and value of the results for understanding species' adaptive potential under climate change. For example, the selection regimes used present the minimum and maximum known temperatures at which the species can survive and develop, but it is unclear how the temperatures relate to the natural environment of the source population, to what extent wild populations might experience these temperatures, and whether they would experience them at the extended duration used (12h at max/min temperature). Moreover, I wonder whether the comparisons made would identify the genes that matter under natural conditions, as unevolved populations were kept under constant conditions compared to 12h:12h temperature regimes for the evolved populations, and the metabolic and transcriptomic profiling was done under a constant favorable 26{degree sign}C rather than under thermal stress in a, as far as I can tell, randomly chosen life stage (larval stage).

(3) The paper in its current form does not adequately describe the statistical analyses underlying the results, nor do the authors share their code, making it very hard to judge whether the analyses used are appropriate and the results trustworthy. I have concerns about the inappropriate use of t-tests, the lack of correcting for confounding variables, and the need for multiple testing corrections.

Author Response:

Public Review:

We thank you and the reviewers for the thoughtful and constructive comments. The feedback helps us strengthen the manuscript substantially, and we plan to address the key points in the revised version as follows.

Reviewer #1 (Public review):

First, in response to the request for a clearer biological interpretation of the pathway enrichment results, we will expand the Discussion to more directly integrate these findings with the observed life-history divergence between strains.

Second, we agree with the concern regarding the phylogenetic inference of PxSODC. We will therefore re-infer the phylogeny using a model-based Maximum Likelihood approach implemented in IQ-TREE, and, in the absence of an appropriate outgroup, the revised tree will be presented as unrooted.

Third, to address the suggestion for a structural explanation of the mutational effects, we will add new structural analyses using AlphaFold modeling and 100 ns molecular dynamics simulations of the wild-type and mutant PxSODC proteins across three physiologically relevant temperatures.

Reviewer #2 (Public review):

First, we will restructured the Results and streamlined the presentation to better emphasize the central narrative. Extensive descriptive datasets will be moved to the Supplementary Materials, and the rationale linking different analytical layers will be stated more explicitly.

Second, we will also revise the manuscript to better frame the ecological relevance and limitations of the experimental design. Specifically, we will clarify that the thermal selection regimes were chosen to reflect ecologically relevant extremes for the source population from subtropical Fuzhou, where summer and winter temperatures can approach the ranges used in the experiment. We will further explain that the cycling temperature treatments were designed to approximate severe but naturally occurring diurnal fluctuations.

Third, in response to concerns about statistical rigor and reproducibility, we will substantially expanded the statistical methods throughout the manuscript. The revised version will provide a clearer description of the analyses used for each dataset, including sample sizes, comparison structure, and statistical thresholds. We will also clarify the application of multiple-testing correction for both transcriptomic and metabolomic analyses, specified the criteria used in network analyses, and more clearly distinguished the statistical approaches used for pairwise versus multi-group comparisons.