Experimental evolution to thermal stress indicates climate resilience in a cosmopolitan arthropod
- State Key Laboratory of Agriculture and Forestry Biosecurity, Institute of Applied Ecology, Fujian Agriculture and Forestry University, China
- Institute of Plant Protection, Fujian Academy of Agricultural Sciences, China
- International Joint Research Laboratory of Ecological Pest Control, Ministry of Education, Fujian Agriculture and Forestry University, China
- Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Fujian Agriculture and Forestry University, China
- Department of Biological Sciences, Brock University, Canada
- Gulbali Institute, Charles Sturt University, Australia
Figures
Figure 1 with 1 supplement
Evolved phenotypic changes in temperature-adapted strains.
(A) Stage-specific thermal tolerance responses (survival rates) of the ancestral and hot strains, with 20 individuals used in each of the six replicates for every treatment. (B) Supercooling and freezing points of pupae for the ancestral and cold strains, with 40 biologically independent samples used in each treatment. Data are presented as mean ± SEM. Statistical analyses are performed using t-tests with significant levels indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001).
-
Figure 1—source data 1
Raw data for stage-specific thermal tolerance and pupal supercooling/freezing points of temperature-adapted strains.
- https://cdn.elifesciences.org/articles/110352/elife-110352-fig1-data1-v1.xls
Figure 1—figure supplement 1
Phenotypic fitness variation in the ancestral, hot, and cold strains of P. xylostella.
The curves show the age-stage survival rate (sxj), age-specific survival rate (lx), female age-specific fecundity (fxj), and population age-specific fecundity (mx) of the ancestral, hot, and cold strains.
Figure 2 with 1 supplement
Metabolomic analysis of 3rd-instar larvae across ancestral, hot, and cold strains (AS, HS, and CS).
(A) Classification of metabolites, with a total of 781 metabolites being identified in different strains. (B) Principal component analysis (PCA) of the 781 metabolites across different strains. PC1 and PC2 represent the first and second principal components, respectively. (C) Volcano plot showing the down-regulated (green dots) and up-regulated (red dots) metabolites based on comparison between HS/CS and AS. (D) Classification of differential metabolites between HS/CS and AS. (E) Venn diagram showing the common and unique differential metabolites in HS and CS as compared to AS. (F) Fold changes and classifications of the common differential metabolites in HS and CS as compared to AS.
Figure 2—figure supplement 1
Metabolomic analysis of the 3rd-instar larvae across the ancestral, hot, and cold strains of P. xylostella.
(A) Inter-sample correlation heat map of the metabolites. Z-score standardized values for each of the metabolites were used in clustering analysis. The color bar indicates an increase in the content of each metabolite, scaling from blue to red. (B) KEGG functional classification of differential metabolites between the HS/CS and AS. (C) Correlation analysis of differential metabolites between the HS/CS and AS. Pearson’s correlation coefficient (r) was used to evaluate the biological correlation between different replicates.
Figure 3 with 1 supplement
Transcriptomic analysis of the 3rd-instar larvae across the ancestral, hot, and cold strains.
(A) Principal component analysis (PCA) of genes across different strains. PC1 and PC2 represent the first and second principal components, respectively. (B) Volcano plots of differential gene expression, showing significantly up-regulated (red dots) and down-regulated (green dots) genes between HS/CS and AS (FDR <0.05, fold change >2). (C) Cluster analysis of the transcriptome. The colors represent the Pearson correlation coefficients between samples, indicating transcriptomic similarity. (D) The number of common or unique differentially expressed genes between HS/CS and AS. (E) KEGG function classification of differentially expressed genes between HS/CS and AS.
Figure 3—figure supplement 1
Transcriptomic analysis of the 3rd-instar larvae across the ancestral, hot, and cold strains of P. xylostella.
(A) qRT-PCR validation of transcriptome data. (B) Soft threshold selection in WGCNA. Left: The soft threshold selection graph. Right: The mean connectivity of genes under different soft thresholds. (C) Correlation heat map showing the association of the 29 modules with common differential metabolites between the hot/cold and ancestral strains.
Figure 4
Weighted gene co-expression network analysis (WGCNA) of transcriptomes for the 3rd-instar larvae across the ancestral, hot, and cold strains (HS, CS, and AS) of P. xylostella.
(A) Hierarchical cluster tree illustrating 29 modules identified by WGCNA. (B) Numerical distribution of genes of different modules as identified by WGCNA clustering. (C) In WGCNA, module M13 shows the highest number of metabolites strongly correlated with genes. (D) Overlap of genes in module M13 from WGCNA with common differentially expressed genes between HS/CS and AS.
Figure 5 with 5 supplements
Role of PxSODC in temperature adaptation of P. xylostella.
(A) Allele frequencies of SNPs in the PxSODC gene amplified by PCR from the ancestral, hot, and cold strains (AS, HS, and CS). The analysis involves ten 4th-instar larvae from each of the strains; the dot (·) indicates identity with the reference base. (B) Frequency of amino acid translations from non-synonymous codon mutations in the PxSODC gene in different strains. (C) Stage-specific survival rates of the ancestral and mutant strains (AS, SODC-MU1, SODC-MU2, and SODC-MU3) under extreme heat conditions. (D) Supercooling and freezing points of the pupae from different strains (AS, SODC-MU1, SODC-MU2, and SODC-MU3). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s test was used for comparison. Six biologically independent samples were used in (C), and significant levels between groups with the same stress duration are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001). A total of 40 biologically independent samples were used in (D), and statistical significance is indicated by different letters (p<0.05).
-
Figure 5—source data 1
Raw data for thermal tolerance phenotypes of PxSODC knockout strains.
- https://cdn.elifesciences.org/articles/110352/elife-110352-fig5-data1-v1.xls
Figure 5—figure supplement 1
Bioinformatic prediction and analysis of the PxSODC gene sequence.
(A) The structure of the PxSODC gene, consisting of three exons and two introns. (B) Secondary structure prediction of PxSODC. Yellow represents strand, pink represents helix, and grey represents coil. (C) Unrooted Maximum Likelihood phylogenetic tree of PxSODC based on amino acid sequences. The tree was inferred using IQ-TREE with 1000 bootstrap replicates, with a blue dot marking the position of P. xylostella.
Figure 5—figure supplement 2
Spatio-temporal expression patterns of the PxSODC gene in the ancestral, hot, and cold strains of P. xylostella.
(A) Stage-specific profiling of the PxSODC expression patterns among different strains in the favorable temperature environment (26 °C), and one-way ANOVA with Tukey’s test was used for comparison (p<0.05). (B) Expression levels of PxSODC in the 3rd-instar larvae of the ancestral and hot strains after 2 hr exposure to different high temperature environments (32°, 34°, 36°, 38°, and 40 °C), and t-test was used for comparison (p<0.05). (C) Expression levels of PxSODC in the 3rd-instar larvae of the ancestral and cold strains after 2 hr exposure to different low temperature environments (12°, 10°, 8°, 6°, and 4 °C), and t-test was used for comparison (p<0.05). Data are presented as mean ± SEM, with n=3 biologically independent samples for each data point.
Figure 5—figure supplement 3
Molecular dynamics simulations of wild-type (WT) and mutant (MU) PxSODC proteins at 15 °C, 26 °C, and 32 °C.
The favorable temperature (26 °C) served as the physiological baseline. (A–C) RMSD time-series trajectories over 100 ns at 15 °C (A), 26 °C (B), and 32 °C (C). (D) Solvent accessible surface area (SASA) across three temperatures, demonstrating the more compact structure of MU at 15°C and 26°C. (E) Number of intramolecular hydrogen bonds across three temperatures. Under cold stress (15 °C), MU actively increased hydrogen bonds compared to the 26 °C baseline, whereas WT lost bonds. Under heat stress (32 °C), MU fully maintained its bond count. Data in (D) and (E) are presented as mean ± SE from 100 ns simulations.
Figure 5—figure supplement 4
CRISPR/Cas9-mediated mutagenesis of the PxSODC gene.
Black underlines indicate the sgRNA target sequence in the second exon; yellow underlines highlight the PAM recognition site; dashes indicate deleted base sequences; and blue letters indicate inserted base sequences.
Figure 5—figure supplement 5
Comparison of the survival rates (sxj and lx) and female fecundity (fx and mx) of P. xylostella strains, showing the role of PxSODC in the temperature adaptation.
(A) Age-stage survival rates (sxj) of the ancestral and mutant strains (AS, SODC-MU1, SODC-MU2, and SODC-MU3) in different temperature environments. (B) Age-specific survival rates (lx), female age-specific fecundity (fx), and population age-specific fecundity (mx) of the ancestral and mutant strains (AS, SODC-MU1, SODC-MU2, and SODC-MU3) in different temperature environments.
Figure 6
SOD expression and activity and superoxide anion (O2-) levels across developmental stages and temperature environments in different strains of P. xylostella.
(A) Expression levels of the SOD genes at different developmental stages of ancestral, hot, and cold strains (AS, HS, and CS) in the favorable temperature environment (26 °C). (B) SOD enzyme activity at different developmental stages of the AS, HS, and CS in the favorable temperature environment. (C) O2- levels at different developmental stages of AS, HS, and CS in the favorable temperature environment. (D) Expression levels of the genes from the SOD family in the 3rd-instar larvae of AS, HS, and CS in the hot (32 °C, 34 °C, 36 °C) and cold (12 °C, 10 °C, 8 °C) environments. (E) SOD enzyme activity in the 3rd-instar larvae of AS, HS, and CS in the extreme temperature environments. (F) O2- levels in the 3rd-instar larvae of AS, HS, and CS in the hot and cold environments. (G) Expression levels of SOD family (excluding the PxSODC gene) at different developmental stages of the ancestral and SODC-MU strains in the favorable temperature environment. (H) SOD enzyme activity at different developmental stages of the ancestral and SODC-MU strains in the favorable temperature environment. (I) O2- levels at different developmental stages of the ancestral and SODC-MU strains in the favorable temperature environment. n=3 biologically independent samples in (A, D, G); within each of the boxes, the numerical value represents log2-fold change of the gene expression level in the treated samples with respect to the control. n=4 biologically independent samples in (B, C, E, F, H, I), with data being presented as mean ± SEM. One-way ANOVA with Tukey’s test was used for comparison in (A, B, C) and (G, H, I) (p<0.05). t-test was used for comparison in (D, E, F) (p<0.05).
-
Figure 6—source data 1
Raw data for SOD enzyme activity and superoxide anion (O₂⁻) levels across strains, developmental stages, and temperature conditions.
- https://cdn.elifesciences.org/articles/110352/elife-110352-fig6-data1-v1.xls
Figure 7 with 2 supplements
Comparison of metabolites and DNA methylation across different strains of P. xylostella.
(A) A Venn diagram showing the metabolites that are consistently different between the ancestral and mutant strains across different developmental stages. (B) Three metabolites with persistent discrepancy across different developmental stages in the ancestral and mutant strains. (C) Expression level of the DNA methyltransferase 1 gene (PxDnmt1) in the ancestral, hot, and cold strains. n=17 biologically independent samples. (D) DNA methyltransferase activity in the ancestral, hot, and cold strains. n=11 biologically independent samples. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s test was used for comparison in (C, D) (p<0.05). (E) Injection of dsDnmt1 significantly reduced the expression level of PxDnmt1 in the ancestral strain of P. xylostella. n=3 biologically independent samples. (F) Silencing of PxDnmt1 decreased 5-methylcytosine (5-mC) content in the female adults and pupae of P. xylostella. n=4 biologically independent samples. (G) Female adults with silenced PxDnmt1 exhibited a significantly decreased critical thermal maximum (CTMax). n=20 biologically independent samples. (H) Pupae with silenced PxDnmt1 displayed elevated supercooling and freezing points. n=20 biologically independent samples. Data are presented as mean ± SEM; unpaired t-test was used for comparison in (E, F, G, H) (p<0.05).
-
Figure 7—source data 1
Raw data for PxDnmt1 expression, DNA methyltransferase activity, and effects of PxDnmt1 silencing on thermal tolerance.
- https://cdn.elifesciences.org/articles/110352/elife-110352-fig7-data1-v1.xls
Figure 7—figure supplement 1
Metabolomic analysis of different developmental stages in the ancestral and mutant strains of P. xylostella.
(A) Classification of metabolites. A total of 608 metabolites were detected in the ancestral and mutant strains (AS and SODC-MU1/SODC-MU2). (B) Principal component analysis (PCA) of the 608 metabolites in the ancestral and mutant strains (AS and SODC-MU1/SODC-MU2). PC1 and PC2 represent the first and second principal components, respectively. (C) Volcano plot showing down-regulated (blue dots) and up-regulated (red dots) metabolites identified in the mutant strains compared to the ancestral strain. (D) Venn diagram showing common and unique differential metabolites between the ancestral and mutant strains at different developmental stages.
Figure 7—figure supplement 2
Differential metabolite classification and functional analysis in the ancestral and mutant strains across different developmental stages of P. xylostella.
(A) Classification of differential metabolites between the ancestral and mutant strains at different developmental stages. (B) KEGG function classification of differential metabolites in the mutant strains compared to the ancestral strain. The vertical axis represents the names of the KEGG pathways, and the horizontal axis indicates the number of differential metabolites annotated to each pathway and their proportion of the total metabolites annotated to that pathway.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Plutella xylostella) | Ancestral strain (AS) | Field collection, Fuzhou, China (26°05'N, 119°18'E) | Collected July 2012; maintained at 26 °C | |
| Strain, strain background (P. xylostella) | Hot strain (HS) | This paper | Evolved at 32 °C/27 °C (12 h/12 h);~75 generations | |
| Strain, strain background (P. xylostella) | Cold strain (CS) | This paper | Evolved at 15 °C/10 °C (12 h/12 h);~15 generations | |
| Genetic reagent (P. xylostella) | SODC-MU1 | This paper | PxSODC knockout;+1 bp insertion; homozygous at G5 | |
| Genetic reagent (P. xylostella) | SODC-MU2 | This paper | PxSODC knockout;+2 bp insertion; homozygous at G5 | |
| Genetic reagent (P. xylostella) | SODC-MU3 | This paper | PxSODC knockout; −1 bp deletion; homozygous at G5 | |
| Strain, strain background (E. coli) | DH5α competent cells | YEASEN, Shanghai, China | Cloning host | |
| Gene (P. xylostella) | PxSODC | P. xylostella genome | Cu/Zn SOD; ORF 633 bp, 210 aa | |
| Gene (P. xylostella) | PxDnmt1 | P. xylostella genome | DNA methyltransferase 1 | |
| Recombinant DNA reagent | pESI-Blunt vector | YEASEN, Shanghai, China | Cloning vector | |
| Recombinant protein | Cas9 protein | GenCrispr, Nanjing, China | 200 ng/μL in injection mixture | |
| Sequence-based reagent | PxSODC sgRNA | This paper | See Supplementary file 7 | |
| Sequence-based reagent | dsDnmt1 | This paper | dsRNA for PxDnmt1 RNAi; see Supplementary file 7 | |
| Sequence-based reagent | dsEGFP | This paper | Control dsRNA; see Supplementary file 7 | |
| Sequence-based reagent | All primers | This paper | Full list in Supplementary file 7 | |
| Commercial assay or kit | Eastep Super Total RNA Extraction Kit | Promega, Shanghai, China | RNA extraction | |
| Commercial assay or kit | Reverse-Transcription System Kit | Promega, Shanghai, China | cDNA synthesis | |
| Commercial assay or kit | 2×Taq Pro Universal qPCR Master Mix | Vazyme, Nanjing, China | qRT-PCR | |
| Commercial assay or kit | Phanta Max Super-Fidelity DNA Polymerase | Vazyme, China | High-fidelity PCR | |
| Commercial assay or kit | Hieff Clone Zero TOPO-Blunt Simple Cloning Kit | YEASEN, Shanghai, China | TA cloning | |
| Commercial assay or kit | TiANamp Genomic DNA Kit | TIANGEN, China | gDNA extraction | |
| Commercial assay or kit | HiScribe T7 Quick High Yield RNA Synthesis Kit | New England Biolabs, USA | sgRNA in vitro transcription | |
| Commercial assay or kit | T7 High Yield RNA Transcription Kit | Vazyme, Nanjing, China | dsRNA synthesis for RNAi | |
| Commercial assay or kit | Gel Extraction Kit | Omega Bio-tek, USA | PCR product purification | |
| Commercial assay or kit | SOD activity assay kit | Comin Biotechnology, China | SOD enzyme activity | |
| Commercial assay or kit | O₂⁻ assay kit | Comin Biotechnology, China | Superoxide anion levels | |
| Commercial assay or kit | 5-mC ELISA kit | Shanghai Enzyme-linked Biotechnology, China | DNA methylation detection | |
| Chemical compound, drug | Ethylene glycol | Sigma-Aldrich | Supercooling/freezing point assay | |
| Software, algorithm | TWOSEX-MSChart | Chi et al., 2020 | Life table analysis; bootstrap 100,000 | |
| Software, algorithm | HISAT2 V2.1.0 | Kim et al., 2019 | RRID:SCR_015530 | RNA-seq alignment |
| Software, algorithm | DESeq2 | Bioconductor | RRID:SCR_015687 | Differential expression analysis |
| Software, algorithm | WGCNA v2.0 | Langfelder and Horvath, 2008 | RRID:SCR_003302 | Co-expression network analysis |
| Software, algorithm | GROMACS 2022.3 | RRID:SCR_014565 | MD simulations; Amber99sb-ildn | |
| Software, algorithm | AlphaFold2 | DeepMind | RRID:SCR_025089 | Protein structure prediction |
| Software, algorithm | IQ-TREE | RRID:SCR_017254 | ML phylogenetics; bootstrap 1,000 | |
| Software, algorithm | SPSS v23.0 | IBM | RRID:SCR_002865 | Statistical analysis |
| Other | Illumina HiSeq4000 | Illumina, San Diego, USA | RNA-seq platform | |
| Other | QuantStudio Real-Time PCR System | Thermo Fisher, USA | qRT-PCR | |
| Other | Olympus SZX16 microinjection system | Olympus, Japan | CRISPR egg injection | |
| Other | Nanoliter 2000 microinjector | World Precision Instruments, USA | dsRNA injection |
Additional files
-
Supplementary file 1
Life table parameters of the ancestral, hot, and cold strains of P. xylostella at the favorable temperature (26 °C).
- https://cdn.elifesciences.org/articles/110352/elife-110352-supp1-v1.docx
-
Supplementary file 2
Candidate genes linked to thermal adaptation in DBM.
- https://cdn.elifesciences.org/articles/110352/elife-110352-supp2-v1.docx
-
Supplementary file 3
Population fitness parameters of the ancestral strain (AS) and SODC-mutant strains of DBM under the constant favorable environment (26 °C).
- https://cdn.elifesciences.org/articles/110352/elife-110352-supp3-v1.docx
-
Supplementary file 4
Population fitness parameters of the ancestral strain (AS) and mutant strains of DBM under the hot environment (32 °C/27 °C:12 hr/12 hr).
- https://cdn.elifesciences.org/articles/110352/elife-110352-supp4-v1.docx
-
Supplementary file 5
Population fitness parameters of the ancestral strain (AS) and mutant strains of DBM under the cold environment (15 °C/10 °C:12 hr/12 hr).
- https://cdn.elifesciences.org/articles/110352/elife-110352-supp5-v1.docx
-
Supplementary file 6
Information on the PxSODC homologous genes identified in the transcriptome.
- https://cdn.elifesciences.org/articles/110352/elife-110352-supp6-v1.docx
-
Supplementary file 7
The primers used in this study.
- https://cdn.elifesciences.org/articles/110352/elife-110352-supp7-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/110352/elife-110352-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Experimental evolution to thermal stress indicates climate resilience in a cosmopolitan arthropod
eLife 15:RP110352.
https://doi.org/10.7554/eLife.110352.3
