Introduction

Low temperature is a key factor impacting the growth and survival of invertebrates, and cold tolerance is critical to the ongoing growth, development and distribution of insect populations1. Insects have evolved several methods to adapt to temperature fluctuations, including rapid cold hardening (RCH), which is an adaptive response to cold stress2. RCH was firstly described by Lee et al3. and is a phenomenon where insects rapidly improve their cold tolerance in response to short-term exposure to cold temperatures. RCH is widespread among arthropods and allows insects to track thermal fluctuations and quickly optimize cold tolerance2,4,5,6. In combination with long-term thermal acclimation, RCH preserves essential ecological functions needed for insects to survive sublethal cold stress and contributes to increased coldness tolerance in the field7.

The underlying mechanisms of RCH include cell membrane modifications, transcriptional regulation of related genes and proteins, and regulation of selected cellular signals8. RCH can also induce the synthesis of cryoprotectants in some insects6,9, which also occurs during cold shock. The lipids related to cold tolerance include palmitic, linoleic and oleic acid, which are essential for energy storage, heat preservation and cold tolerance10,11 In insects, the increase in unsaturated fatty acids prevents the crystallization of lipids in cell membranes. In a prior study, the concentration of unsaturated fatty acids in Lipoptena cerviwas significantly higher in late autumn than in the summer12. Organisms adjust the composition of their cell membrane to maintain membrane fluidity at different temperatures13, and this is an important cold tolerance mechanism in insects14. Cell membranes in Drosophila melanogasterwere shown to undergo structural reorganization during RCH, the content of linoleic acid in membranes raised and resulted in increased fluidity15. A similar phenomenon was also observed in Sarcophaga crassipalpis16. At the molecular level, insects respond to cold shock by regulating the transcription of genes encoding heat shock proteins (HSPs), antifreeze proteins, and aquaporins (AQP)17. The upregulation of genes encoding metabolic enzymes, such as those involved in glycerol and trehalose synthesis, was also observed during RCH in some insects18,19,20,21. However, it is important to note that RCH in some insects does not involve obvious changes in transcription; for example, gene expression assays after acclimation did not result in the synthesis of new gene products during RCH in D. melanogaster22.

Liriomyza trifoliiis a pest of vegetables and flowers23, and its introduction to China has caused significant damage to a variety of horticultural plants. L. trifoliiinvaded China in 2005 and rapidly spread to provinces in southeastern China24. Due to global warming, fluctuations in climate have increased and are predicted to continue. During the temperature fluctuations in early spring and late autumn, RCH plays an important role in the survival of overwintering insects; consequently, studies on cold tolerance have become more prominent in insect ecology, physiology and genetics25. We previously reported that L. trifoliiadults exposed to a 4 h mild acclimation period at temperatures less than 5℃ showed enhanced resistance to cold; furthermore, survival at low temperatures for 2 h increased from 20–63%26. In addition, our previous results showed that the transcriptional response is correlated with RCH in the pupal stage of the two Liriomyza species, but more transcriptional changes were identified in L. sativae than in L. trifolii27. Therefore, the objective of this study was to use transcriptomic and metabolomic techniques to identify the underlying mechanism(s) for RCH in L. trifolii adults and to more fully understand thermal plasticity in insects.

Results

Biochemical changes in L. trifolii elicited by RCH

Palmitic (16:0), palmitoleic (16:1n-7) and linolenic (18:3n-3) acids were the dominant phospholipid fatty acids in L. trifolii (Table 1). Collectively, these three fatty acids represented more than 80% of the total fatty acids in L. trifolii reared at 25℃. Peaks of lesser abundance include stearic (18:0) and linoleic (18:2n-6) acids. The proportion of selected fatty acids changed in response to different temperature treatments (Table 1). Palmitoleic acid levels increased significantly, whereas other fatty acids either decreased or remained the same after L. trifolii adults were exposed to RCH-inducing conditions (5℃ for 4 h). The ratio of unsaturated vs. saturated fatty acids (UFA/SFA) increased slightly after exposure to 5℃ for 4 h. After a 2 h cold shock following rapid cold hardening (RCHCS), the proportion of linolenic acid (18:3n-3) increased significantly from 24.8 to 33.62%, while palmitic acid (16:0) and palmitoleic acid (16:1n-7) levels were significantly lower than the 4 h hardening group. When the RCHCS and CS groups were compared, the differences in fatty acid composition were not significant; however, the UFA/SFA ratio after CS was significantly higher than that after the RCHCS treatment.

Table 1 Fatty acid composition and content between different groups.
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After a 4 h acclimation period, the glucose content increased significantly as compared with the control group. The glucose content in the RCHCS group was significantly higher than the CS group (F3,8 = 22.10, p < 0.05) (Fig. 1A). Changes in trehalose content correlated with changes in glucose (F3,8 = 67.68, p < 0.05) (Fig. 1B). The glycerol content increased significantly relative to the control after a 4 h acclimation period, but decreased significantly after 2 h of cold shock. The glycerol content in the CS group was significantly higher than the RCHCS group where adults were exposed to cold stress after acclimation (F3,8 = 169.81, p < 0.05) (Fig. 1C).

Fig. 1
figure 1

Changes in the sugar and glycerol content. A: glucose, B: trehalose, C: glycerol (CK: without any temperature treatment; RCH: exposed to 5℃ for 4 h; RCHCS: acclimated at 5℃ for 4 h before exposed to discriminating temperature for 2 h; CS: exposed to discriminating temperature for 2 h without acclimation. Different letters above the bars show significant difference at 0.05 levels).

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RNA-seq analysis of RCH in L. trifolii

RNA-seq was utilized to quantify L. trifolii gene expression in response to RCH treatments, and 106.28 Gb of clean sequence was obtained. The GC content was 39.03–39.82%, and the Q30 values were ≥ 90%. Under the conditions of FDR ≤ 0.05 and fold change ≥ 2, unigenes in Control group were compared to CS, RCH and RCHCS groups to screen for differentially expressed unigenes. Pairwise analysis of differentially expressed genes (DEGs) between groups revealed that 463 genes were differentially expressed, including 219 and 144 that were upregulated and downregulated, respectively (Table 2; Fig. S1). Fifty-three unigenes were differentially expressed after cold shock, including 29 and 24 that were upregulated and downregulated, respectively. In the RCH vs. RCHCS comparison, 396 DEGs were identified including 333 and 63 upregulated and downregulated unigenes, respectively (Table 2; Fig. S1).

Table 2 The number of differential genes between groups.
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When the control (CK) was compared with the CS group, six KEGG pathways were significantly enriched including the neuroactive ligand receptor interaction pathway and the hippo signal pathway (Fig. 2A and Fig. S2). Among the differentially expressed unigenes in the CK vs. RCH comparison, 65 mapped to KEGG pathways and 20 pathways were significantly enriched (FDR ≤ 0.05); these included cytochrome P450, apoptosis, lysosome and other pathways (Fig. 2B and Fig. S2). When DEGs in CS and RCHCS were compared, 61 mapped to KEGG pathways and 20 pathways were significantly enriched (FDR ≤ 0.05). DEGs were primarily enriched in MAPK signaling, fatty acid metabolism, unsaturated fatty acid synthesis and cytochrome P450 (Fig. 2C and Fig. S2).

Fig. 2
figure 2

KEGG analysis of DEGs. A: CK vs. CS; B: CK vs. RCH; C: CS VS RCHCS (CK: without any temperature treatment; RCH: exposed to 5℃ for 4 h; RCHCS: acclimated at 5℃ for 4 h before exposed to discriminating temperature for 2 h; CS: exposed to discriminating temperature for 2 h without acclimation).

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Eight DEGs were randomly selected for qRT-PCR analysis to verify the accuracy of transcriptome data (Fig. 3). The expression of eigh DEGs was almost consistent with the RNA-seq data, which supports the reliability of the data.

Fig. 3
figure 3

Real-time quantitative verification of differentially expressed genes.

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Correlations between biochemical analyses and gene expression

Relationships between the level of unsaturated fatty acids and selected unigenes were analyzed (Fig. 4). The unsaturated degree of fatty acids showed a positive correlation with expression of the selected unigene (Gene ID: c25696.graph_c0) which was annotated as Elovl (elongation of very-long-chain fatty acids). With the exception of glucose (F = 1.15, p > 0.05 Gene ID: c32826.graph_c0), the other cryoprotectants (trehalose and glycerol) had significant positive correlations with related unigenes (trehalose: F = 111.9, p < 0.01, gene ID: c31968.graph_c2; glycerol: F = 191.1, p = 0.0052, gene ID: c31420.graph_c0). The genes associated with trehalose and glycerol were related to trehalose transmembrane transporter and pancreatic triacylglycerol, respectively (Fig. 5). The expression of relevant genes in the correlation analysis was shown in Table S2.

Fig. 4
figure 4

Correlation between unsaturated rate of fatty acid and expression of related unigene in L. trifolii. Different colors represent different treatments. Gray represents control, blue represents RCH, green represents RCHCS, red represents CS. (CK: without any temperature treatment; RCH: exposed to 5℃ for 4 h; RCHCS: acclimated at 5℃ for 4 h before exposed to discriminating temperature for 2 h; CS: exposed to discriminating temperature for 2 h without acclimation).

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Fig. 5
figure 5

Correlation between cryoprotectants and selected unigenes in L. trifolii.A: Glucose; B: Trehalose; C: Glycerol. Different colors represent different treatments. Gray represents control, blue represents RCH, green represents RCHCS, red represents CS. (CK: without any temperature treatment; RCH: exposed to 5℃ for 4 h; RCHCS: acclimated at 5℃ for 4 h before exposed to discriminating temperature for 2 h; CS: exposed to discriminating temperature for 2 h without acclimation).

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Discussion

Rapid cold hardening improves insect cold tolerance and helps pests deal with low temperature stress28; therefore, the RCH process provides useful predictive information on whether insect overwintering will be successful. Studies have shown that L. sativae and L. huidobrensis, which are closely related to L. trifolii, have the capability to undergo RCH29. In this study, the underlying mechanism of RCH in L. trifolii was studied from both molecular and biochemical perspectives.

One biochemical change associated with RCH is modification of cell membranes. Organisms adjust the composition of membranes to maintain fluidity during different temperatures, and this is recognized as an important component of cold tolerance13. Fat deposits supply and store energy, which can increase cold tolerance in insects and protect tissues from damage30. In this study, changes in the concentration of fatty acids were detected after exposure to different cold treatments. Our results provide evidence that cell membranes are restructured during RCH in leafminers. The proportion of palmitoleic acid (16:1n-7) increased significantly at the expense of linolenic acid (18:3n-3) and stearic acid (18:0). This is consistent with previous research which showed a positive relationship between cold tolerance and palmitoleic acid abundance in D. melanogaster31. Interestingly, a significant increase in the ratio of UFA to SFA was not observed in D. melanogasterundergoing RCH16. In L. trifolii, RCH failed to increase the UFA/SFA ratio due to the concomitant rise in linolenic acid (18:3n-3) levels.

The synthesis of cryoprotectants is a strategy that poikilotherms use to improve cold tolerance after cold acclimation32. When exposed to cold stress, most insects accumulate antifreeze substances such as carbohydrates to maintain body functions and increase cold tolerance. In D. melanogaster, RCH increased both glucose and trehalose levels31. Trehalose is a principle component of insect blood sugar and functions as a cryoprotectant18,19,20,21,33. In the event of dehydration due to heat or cold shock, trehalose protects cellular membranes and stabilizes protein structure34. In this study, glucose, trehalose and glycerol increased significantly during RCH, indicating that L. trifolii produces multiple cryoprotectants in response to cold stress. Interestingly, when the RCHCS and CS treatments were compared, glucose and trehalose content showed a greater increase after the RCHCS treatment, whereas glycerol content decreased. These results indicate that differences in trehalose, glucose and glycerol content varies depending on whether cold stress is direct (e.g. cold shock) or indirect (after cold acclimation). Therefore, the improvement of cold tolerance in L. trifolii after short-term acclimation is closely related to the accumulation of trehalose and glucose, which are cryoprotectants that function in RCH.

Studies on the mechanistic basis of cold resistance in insects has gradually shifted to the molecular level, and is generally the outcome of various genes and pathways. Transcriptomic sequencing technology provides a powerful tool for understanding the insect response to abiotic stress35. In the present study, the underlying molecular mechanisms that function in rapid cold hardening of L. trifolii were studied by transcriptomic sequencing. Our RNA-seq results illustrated that multiple DEGs, including those encoding DNA binding protein, hormone receptor, epidermal protein, and fat body protein function in RCH. This is similar to the transcriptome of the pupae of the two species (L. trifolii and L. sativae), where genes that were commonly expressed included cytochrome P450, cuticular protein, glucose dehydrogenase, solute carrier family and cationic amino acid transporter27. Among these genes, cuticular protein is perhaps the most important structural protein in insects and functions in epidermal integration, ossification sites, and adaptation to the environment36. In the present study, the DEG encoding cuticular protein was expressed at a very high level in response to direct or indirect exposure to cold temperatures. Furthermore, the gene encoding fatty acid desaturase (FAD) also showed significant differential expression during acclimation. Prior studies have shown that membrane lipid fluidity decreases when the temperatures fall, and this signals FAD to synthesize unsaturated fatty acids to improve cold tolerance37. Therefore, future experiments are planned to integrate studies of the above genes and signal pathways and use them in combination with physiological and biochemical methods to further reveal the mechanisms of RCH in L. trifolii.

Previous study found that that rapid cold hardening did not lead to transcriptional regulation in D. melanogaster or S. crassipalpis38. However, our transcriptomic analysis showed that for L. trifolii, the rapid cold hardening process was highly regulated. Also, the combination of transcriptomic data and biochemical studies resulted in the selection of four related unigenes that were selected for further analyses. For example, the UFA/SFA ratio in this study was positively correlated with Elovlexpression, which has been reported to mediate the biosynthesis of unsaturated fatty acids39. Rapid cold hardening in other insects is known to involve changes at the transcriptional level. For example, genes encoding HSPs were upregulated during rapid cold hardening in the rice water weevil, Lissorhoptrus oryzophilus40. However, in our study, differential expression of genes in the HSP family was not detected in L. trifolii during RCH. Collectively, these findings indicate that insects differ in the underlying molecular mechanisms of rapid cold hardening, which warrants further study.

Liriomyza trifolii is a significant invasive pest that causes considerable damage to horticultural crops and vegetables. In recent years, the frequency of extreme weather events has increased globally. Rapid cold hardening (RCH) is a critical survival strategy for the L. trifolii population, as its cold tolerance varies with temperature fluctuations, leading to changes in pest distribution. Understanding the RCH characteristics of L. trifolii is essential for improving the accuracy of pest forecasting and monitoring. Moreover, studying the rapid cold tolerance of L. trifolii is crucial for gaining insights into the mechanisms of pest adaptation to temperature changes.

Conclusions

In this study, the mechanistic basis of rapid cold hardening in L. trifolii was investigated at the molecular and biochemical level using transcriptomics and liquid chromatography, respectively. Biochemical experiments showed that cryoprotectants and palmitoleic acid (16:1n-7) increased after a short period of cold acclimation, which indicates that antifreeze substances and fatty acid composition may play a role in the rapid cold hardening of L. trifolii. A total of 493 genes were differentially expressed in L. trifolii after 4 h of acclimation; these included 385 DEGs in the CS and RCH groups. KEGG pathway enrichment analysis showed that these DEGs clustered in multiple metabolic pathways, including those involved in the conversion of energy (metabolism of various amino acids, sugars and fatty acids) and MAPK signaling pathways, thus confirming that RCH is mediated at the transcriptional level in L. trifolii.

Materials and methods

Insects

L. trifoliiwas collected in Yangzhou city (32.39°N, 119.42°E), China, in 2020. Insects were fed on kidney beans in the laboratory under controlled environmental conditions at 25 ± 1 ℃, 65 ± 5% relative humidity (RH), and a 16:8 light/dark photoperiod as described by Chen and Kang41. Foliage with tunnels was gathered to facilitate pupation, and the pupae were carefully collected into test tubes using a soft brush, then sealed with gauze until emergence. Both adults and larvae were maintained on beans for mating and oviposition.

Temperature treatments

Based on our previous results, L. trifoliiadults showed the strongest RCH response after a 4 h exposure at 5 ℃; therefore, the temperatures used in this study are identical to those reported previously26. Treatments included the following: RCH group, adults exposed to 5 ℃ for 4 h; CS group, adults exposed to cold shock (-10.6℃) for 2 h; RCHCS group, adults acclimated at 5 ℃ for 4 h and then exposed to cold shock (-10.6 ℃) for 2 h; and Control group, adults maintained at rearing conditions. For each treatment, 90 adults were divided into three replicates were used, and samples were prepared for the determination of fatty acids, cryoprotectants and transcriptome sequencing. Samples were frozen in liquid nitrogen immediately after treatment and were stored at -70 ℃ until the assays were conducted.

Determination of biochemical changes

Fatty acids were extracted as described by Zhang et al42. and analyzed by gas chromatography–mass spectrometry (GC/MS). Samples containing fatty acid methyl esters were loaded into a Trace ISQ GC/MS (Thermofisher, USA), and 1 µl of each sample was subjected to chromatographic analysis. The oven temperature varied from 100 to 200 ℃, and the temperature increment was 5℃/min. Samples were analyzed using a DB-35 MS column (internal diameter, 25 mm) with helium gas as a carrier at 1 ml/min.

Sugar content was measured by high performance liquid chromatography as described previously43. Glycerol was measured by spectrophotometry at 420 nm as described by Zhang et al42. and the concentration was determined by comparing it with a standard curve for glycerol.

Transcriptome sequencing and analysis

Total RNA was extracted from prepared samples of L. trifoliiwith the SV Total RNA isolation system (Promega, USA). RNA purity and integrity were determined as described previously by Chang et al44.. Transcriptome sequencing was provided by Biomarker Technologies Inc. (Beijing, China). Sequencing libraries were generated with the NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA), and RNA-seq data were uploaded to the National Center for Biotechnology Information (NCBI) as accession no. PRJNA782449.

Clean reads were obtained by removing adapter sequences, artificially-introduced bases, and low-quality reads from raw data using Trimmomatic 0.32 (http://www.usadellab.org/cms/index.php?page=trimmomatic) before transcript assembly. Transcriptome assembly was accomplished using Trinity45with min_kmer_cov set at 2; and all other parameters were set at default values. The unigene library was assembled to obtain individual UniGene databases; the general UniGene library was obtained by clustering the database through CD-Hit to facilitate comparison of expression patterns46. Gene function was annotated using on the following databases: NR (NCBI - nonredundant protein sequences); Pfam (Protein Family); COG (Clusters of Orthologous Groups of proteins); Swiss-Prot (curated protein sequence database); KEGG (Kyoto Encyclopedia of Genes and Genomes)47,48 and GO (Gene Ontology).

The fragments per kilobase of transcript sequence per million (FPKM) nucleotides were calculated and read counts were mapped. The assembled transcriptomes of samples were compared with each other. Differential expression analysis was conducted with the DESeq2 R package as described by Varet et al49.. The P values were adjusted with the Benjamini-Hochberg procedure to control false discovery rates (FDR). An FDR < 0.05 and fold-change |FC| ≥ 2 were used to evaluate significant differences in gene expression.

Eight unigenes were selected to verify the sequencing results using quantitative real-time PCR (qRT-PCR). Primers were designed with Primer Premier v. 5.0 (http://www.premierbiosoft.com/primerdesign/) (Table S1). cDNA was synthesized in 20 µl reaction volumes as described by Chang et al44., and Actinwas used as a reference gene50. Relative changes in gene expression were analyzed using the 2−ΔΔCtmethod51. Each PCR reaction included three replicates.

Statistical analysis

One-way ANOVA (Tukey’s multiple comparison) was used to detect significant differences in fatty acid and cryoprotectant levels using SPSS v. 16.0 (SPSS, Chicago, IL, USA). Correlations between cryoprotectants, unsaturated fatty acids and gene expression were analyzed with GraphPad Prism8 (GraphPad Software Inc., San Diego, CA, USA).